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Molecules, Genes & Cells

by: Novella Considine

Molecules, Genes & Cells BIO 111

Marketplace > Davidson College > Biology > BIO 111 > Molecules Genes Cells
Novella Considine
GPA 3.94

Karen Bernd

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Karen Bernd
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Date Created: 10/11/15
Fall Semester 2002 Introductory Biology 111 Cell and Molecular Biology wwwbiodaVidsoneduBiologyCoursesBiol 1 1html Study Guide Revised June 2001 by Dr A M Campbell and Dr K Bernd Davidson College and Dr J Serie Macalester College Table of Contents Prologue 1 Overview of the Biological Sciences 1 Prologue 2 The Cell 5 Unit 1 Cellular Communication 7 System 1 Liver cells deliver glucose 7 System 2 Heart cells pump harder 15 System 3 Nerves tell skeletal muscles to run 25 System 4 How does an egg know when it has been fertilized 34 Other Communication Systems 41 Some Experimental Approaches 44 Unit II Genetics 50 Mitosis 51 Meiosis and gametogenesis 51 Mendelian Genetics 53 Molecular Genetics 60 A Point Mutation can Change Your Life W W W 62 Gene Regulation 66 Structure Function Relationships 68 Linkage 71 RFLPs 74 Chromosome mapping 80 Molecular Causes of Diseases 97 An Article looking at Genetic Link to Alzheimer s 11 3 Unit 111 Bioenergetics 11 4 Why did the US government spray MeXican marijuana with paraquat 1 1 6 Follow up story to paraquat 13 0 Why do vegetarians eat tofu 13 1 Why is Cyanide the terrorist s poison of choice 13 4 Follow up story on poisoned grapes 14 6 14 Why would authorities ask you to update vaccinations after a ood Two Research Questions and Approaches in Bioenergetics 15 0 Unit IV Other Interesting Topics 14 8 Cancer 14 8 AID S 16 3 Genetic Engineering 18 Prologue 2 The Cell Brief Overview Reading Chapter 1 Focused Reading p 35 quotEvolutionary Milestonesquot stop at Controlling internal environmentsquot Chapter 4 The Organization of Cells WWW Focused Reading Virtual Plant Cell Purves6e Chapter 4 Eukaryotic Cell Tour Immuno uorescence Labeling of ER Relative Sizes from glucose to cells and larger To look at images on the world wide web WWW you must use a computer that is connected to the campus network that has Netscape or similar browser and go to the Biology 111 Home Page To reach the Biology 111 Home Page up at the top of the screen in the box labeled Location you must type in the web address called a URL ltwwwbiodavidsoneduBiologyCoursesBiol l l htmlgt You must type this exactly as it appears above except do not include the lt and gt symbols Once you have reached the Biology 111 Home Page all you need to do is click once on the appropriate underlined words to see the focused reading material If you get a message that says Bad URL or the le cannot be found then you have probably made a typo lfyou wait for quite a while and nothing ever appears or you get the message that says the server could not be contacted then the computer that provides this service the web server is too busy with all your classmates and cannot talk with you right now You should try again later in one or two minutes Once you have found this Virtual Cell you will see a cartoon of a plant cell Point and click the mouse and you can see inside the cell We strongly recommend that you use the Hot Spots option and then click on the picture This will allow you to see more and more detail You should also use the search function to select certain organelles Your text has supplemental tutorials and activities online Connections between the text and online material are indicated in the book by hand on a mouse icon Web reading will also include some of these supplemental materials and will be seen in the study guide as purves6e Chapter Title of the material To access this material you must type in the URL ltwwwwhfreemancompurves6egt click on the chapter and then the title that are indicated in the study guide assignment Study Questions 1 The cell theory was one of the first fundamental theories of the biological sciences What are the elements of the cell theory and why is this theory so important In what ways does this theory drive the modern biological sciences 2 A major tenet in the biological sciences is that form follows function Give an example that illustrates this point on the cellular level Be able to explain how this example illustrates the point 3 What are the differences between prokaryotes and eukaryotes Give an example of each type of cell 4 Approximately how big are typical prokaryotic and eukaryotic cells About how big is this What else is this size How much smaller is a cell than say a marble or a bowling ball or a typed period quotquot What are the limiting factors in cell size ie why can t cells be larger than they are Why aren39t they smaller 5 Eukaryotic cells are full of little compartments called organelles Why What is adaptive or useful about having all these little compartments 6 For each organelle or cellular structure described in Chapter 4 A Be able to give a very brief a few words description of its basic functions B Be able to draw and label each organelle or accurately describe its structure Pay close attention to the distinguishing features of the organelle eg the curved stacked cistemae of the Golgi apparatus the small and large subunit structure of the ribosome the double membrane surrounding the nucleus the microtubular core of the cilia and agella etc Study note Online supplement includes ashcards for each chapter that are good for definitions 7 Today s reading includes two animations of the same content Compare Virtual Plant Cell and Purves6e Chapter 4 Eukaryotic Cell Tour What are the strong and weak points of each 8 Be able to describe or make a sketch of the structures of a chloroplast a mitochondrion a nucleus Occasionally there will be places in the study guide where you will find factoids called News Items The information in these news items is not for you memorize nor will it be tested However you might find them very interesting since they contain recent research ndings based on the material you are learning For example News Item Researchers recently identified a giant sulfur bacterium which they called Thiamargarita namibiensis This organism is remarkable because it is a prokaryote but the cells grow to have diameters of 750 um see quotRelative Size website for illustration of how big or small this is While this is still very small when compared to a house prokaryotes have no internal membrane systems and so the exchange of nutrients and waste products must occur by diffusion According to previous thought a prokaryotic cell this large is not possiblebut there it is off the coast of Chile How can this organism survive You can39t change the laws of nature so does it use mechanisms we haven39t seen before Since this organism was only discovered recently much more work must be done to answer questions like these For more information see HN Shulz et al 1999 Science Vol 284 p4935 qTOP Unit 1 Cellular Communication Brief Overview Reading Chapter 23 amp 5 Note Yes three chapters is a lot This reading will be discussed throughout this m We will focus discussion on different sections on different days Brie y reading through all of the material now means that when you get to it as focused reading it is not completely foreign Living beings can be composed of a single cell e g bacteria cyanobacteria and protists such as Paramecium and Chlamydomonas or many cells the latter are called multicellular organisms The human being is an example of a multicellular organism about which much is known Estimates place the number of cells in the human body at 70 trillion If you counted these cells at a rate of one cell per second it would take you over 2 million years to count every cell in the human body With a few exceptions eg the red blood cells each of these individual cells is a living entity with its own complete set of genes and life maintenance equipment Each of these cells maintains its own existence and also makes a vital contribution to the life of the multicellular organism In order for multicellular organisms to function properly their cells must communicate For instance your muscles must contract only when your brain sends a message to contract and not any other time Your salivary glands must secrete a lot of saliva when there is food in your mouth and only a little saliva at other times Your heart rate must increase when you exercise but not when you sleep Unit Ifocuses on how cells communicate with one another in order to coordinate their functions While we will focus most closely on cellular communication in multicellular creatures you should keep in mind that communication is very important to unicellular creatures as well For instance they must swim toward nutrition or sunlight if they are photosynthetic and must be able to sense when conditions are right to reproduce we will see this in the lab In this unit we will consider four examples of cellular communication Each system uses a slightly different communication system and taken together these four systems represent most of the communication systems we understand so far System 1 The Production of Glucose sugar by the Liver During Stress Glucose is the primary sugar used by biological creatures for fuel Humans like other creatures burn oxidize glucose to carbon dioxide and water and use the energy released by this process to perform life s many functions To ensure that cells have enough glucose to burn and therefore enough energy the body maintains a constant supply of glucose in the blood at a level of about 100 mg of glucose per 100 mL of blood However when we are scared or under stress our bodies respond by increasing the blood glucose level to ensure that we have enough fuel to fight or ee from what is scaring us This extra glucose comes from the liver During meals glucose enters the body and the liver takes it out of the blood and stores it for later use To put glucose in storage the liver hooks many glucose molecules together polymerizes them to form a large storage molecule called glycogen Then when glucose is needed later either because you haven t eaten for a while or because you are scared these big glucose 7 polymers will be broken down into individual glucose molecules and the glucose will be dumped into the blood to provide fuel for all of the cells of the body Focused Reading p 3436 quotMacromolecules Giant polymersquot stop at quotProteinsquot p 4346 Carbohydrates stop at end of page Focused WWWebreading Purves6e Chapter 3 Tutorial 31 Carbohydrate section Study Questions 1 What is glucose used for in biological creatures What is glycogen used for What is the relationship between glucose and glycogen 2 What is a polymer What is a monomer Is glucose a polymer or monomer What is glycogen Explain What is a monosaccharide A disaccharide A polysaccharide 3 Be able to recognize a monosaccharide and polysaccharide when you see one drawn You do not have to be able to draw these molecules yourself 4 Glucose molecules are joined to form glycogen by a process called dehydration synthesis or condensation synthesis Glycogen is broken down to form glucose by the process of hydrolysis quotHydroquot means water What does water have to do with these two processes Be able to illustrate both of these reactions including the breaking or forming of bonds and the involvement of water in the process 5 Starch made by plants and glycogen made by animals are formed by joining glucose monomers by alpha glycosidic linkages while cellulose made by plants is made by joining glucose monomers by beta glycosidic linkages What are the differences chemically and what practical significance does this have in your own life During a meal glucose molecules are joined together to form the polymer glycogen in the liver This process is called glycogenesis The quotgenesisquot or creation of glycogen An enzyme catalyzes the formation of each alpha glycosidic bond between each glucose molecule This enzyme is called glycogen synthase The following reading assignment is about the action of enzymes In addition because enzymes are proteins this reading assignment also includes an explanation of proteins and protein structure Also in order to understand how proteins fold you will need to understand about hydrophobic and hydrophilic groups Focused Reading p 2025 quotChemical bondsquot stop at Chemical reactions p 3642 Proteins Polymers of amino acidsquot stop at Carbohydrates p 102112 Enzymes stop at end of chapter Glycogen synthase then lowers the activation energy barrier and allows glucose molecules to be joined together to form glycogen at a reasonable rate at normal body temperatures All chemical reactions in living things that involve the breaking or forming of a covalent bond are catalyzed by enzymes The rate at which enzymes perform their functions can be increased or decreased by 8 allosteric or covalent modulators Thus the rate at which glycogen is synthesized could be increased if the cell increased the rate at which glycogen synthase catalyzes the reaction The enzyme that breaks down glycogen to glucose is glycogen phosphorylase The breakdown of glycogen to glucose is called glycogenolysis The quotlysisquot or degradation of glycogen The rate of this enzyme can also be controlled Therefore the cell can increase or decrease the rate at which glycogen is broken down simply by increasing or decreasing the catalytic rate of glycogen phosphorylase Glycogen synthase and phosphorylase are turned on and off by the process of covalent modulation This process is like allosteric modulation or regulation described on page 136 in your text except that it depends on the process of phosphorylation Phosphorylation is the covalent 0 R Fgto 0 addition of a phosphate group to an enzyme by dehydration synthesis Phosphate groups look like 9 and quotRquot is used to indicate the rest of the molecule R can be anything from a simple hydrogen to an enormous protein In proteins phosphate groups are added onto the side chains added onto an 0H group of certain amino acid residues by standard dehydration synthesis also called a condensation reaction See p35 f1g32a After a phosphate is added by a covalent bond the protein is said to be phosphorylated By looking on page 37 in your text can you gure out which three amino acids are the only ones that can be phosphorylated Hint Which three have an 70H in the side chain Phosphorylation can either turn an enzyme on increase its catalytic rate or turn an enzyme off decrease its rate Regardless of the direction of its action phosphorylation is a kind of switch or signal that changes the rate of an enzyme s activity This is not allosteric modulation which uses weak bonds to regulate the enzyme When you are scared your liver slows the rate of glycogen synthesis and increases the rate of glycogen breakdown Fear causes the phosphorylation of liver enzymes In this example phosphorylation inactivates glycogen synthase and activates glycogen phosphorylase Therefore when these two enzymes are phosphorylated by the cell the rate of glycogen breakdown increases and the rate of synthesis decreases When these two enzymes are dephosphorylated phosphate is removed by the cell this happens when you get calm again the rate of glycogen synthesis increases and the rate of degradation decreases Study Questions 1 What two enzymes are responsible for synthesizing and breaking down glycogen in the liver How is the rate of each enzyme controlled 2 What is glycogenesis What is glycogenolysis Draw a phosphate group and demonstrate how it is added to a protein during the process of phosphorylation Be able to recognize an amino acid and show how it is joined together by a peptide bond to form a dipeptide and nally a protein Proteins have many functions in living things List as many of these functions as you can The many different functions of proteins are possible because these molecules can take many different shapes Explain in chemical terms how proteins form their three dimensional shapes Two proteins with different shapes will have different functions and different amino acid sequences Explain how changing the amino acid sequence of a protein can change its function What is activation energy How do enzymes work What do they do to cause reactions to proceed What don t they do that is what are the limitations of enzymatic catalysis Explain in chemical terms how enzymes can be specific for their substrates What are the biological consequences of enzyme specificity What would the consequences be if enzymes were less specific or not specific at all Explain the catalytic cycle E S gt ES gt P E Using this explanation as background explain how each of the following events would increase the rate of an enzyme catalyzed reaction A increasing the concentration of substrate B increasing the affinity of the enzyme for its substrate C increasing the temperature D increasing the inherent catalytic rate of the enzyme 12 Using a scenario from the social sciences humanities fine arts or your everyday life describe a situation that is analogous to the catalytic cycle Your model is a good one if you can answer questions AD above using this model 13 How are enzymes turned on and off by allosteric modulation 14 The first lab unit explores the effect of environmental conditions on the rate of an enzymatic reaction Write out the reaction we ll be following using the E S gt ES gt P E format What is the enzyme in the reaction 15 Give an example not covered in class of a system in which the control of the rate of an enzyme is important for the proper function of a biological system The enzyme system need not be explained in detail Assume enzymes catalyze all chemical reactions that break and form covalent bonds Use your own experience as a guide and use your imagination We now know how the liver will be able to increase the supply of glucose to the blood but we are missing some very important elements of the system How does the liver quotknowquot that the body is under stress It is sitting quietly in your abdomen it can t see or hear It has to be quottoldquot that stress is occurring The systems which carry this kind of information to body cells are the integrating and controlling systems of the multicellular creature the nervous system and the endocrine system hormones In this case the endocrine system plays a major role in quotinformingquot the liver that the body is under stress and therefore needs more glucose The endocrine system is a system of glands in the body that secrete hormones A hormone is a messenger molecule that is made and secreted released into the blood by an endocrine gland and the hormone travels in the blood to a target organ It binds to the cells of the target organ and causes some change to occur Examples of hormones are insulin lowers the blood sugar level among other things its absence causes diabetes mellitus growth hormone stimulates growth its absence causes dwarfism thyroid hormone increases metabolic rate low levels cause coldness weight gain and lethargy Brief Overview Reading Chapter 41 Focused Reading p 712713 Animal hormones stop at quotSome hormones act In the case we are currently considering the hormone mediating the response of the liver to fear is epinephrine also called adrenaline This hormone is made and secreted by the adrenal gland in response to stress When something stressful happens e g your boss yells at you you are in a car accident you have to give a speech the information about this event enters your brain through your sense organs you hear see touch smell andor feel the stressful event Your brain interprets this event as stressful using memory and some genetic responses such as aversion to pain and your brain then sends a message via a nerve to the adrenal gland Note Interpretation of the event as stressful is an important step in this process Some things are always stressful pain cold dehydration severe hunger etc while other things have to be interpreted as stressful e g social situations threatening words or gestures pressure to succeed etc One way to reduce the physiological response to stress which may be related to such diseases as high blood pressure and cancer is to stop interpreting things as stressful Unfortunately this is not very easy The nerve impulse from the brain reaches the adrenal gland and causes cells to secrete epinephrine The interaction between the nerve and the cells of the adrenal gland is itself an example of intercellular communication We will deal with signaling by the nervous system later Epinephrine enters the blood and goes everywhere to all the cells in the body Even though epinephrine goes everywhere it does not affect every cell of the body It only affects the cells that have epinephrine receptors on their surface These receptors can bind specifically to epinephrine in the same way enzymes bind specifically to their substrates 11 To summarize so far epinephrine is secreted by the adrenal gland when the brain quotdecidesquot that something stressful has happened This hormone goes everywhere in the body but only binds to cells which bear epinephrine receptors on their surfaces like liver cells We also know that the liver will be able to deliver glucose to the blood in response to stress if two of its enzymes glycogen synthase and glycogen phosphorylase can be phosphorylated Somehow then the epinephrine bound to the epinephrine receptors on the liver cells surface has to trigger the phosphorylation of these enzymes inside the cell the process of getting an external signal communicated inside a cell is called signal transduction Signal transduction is usually accomplished through a second messenger system which relays information from the hormone receptor to enzymes inside the cell Before we look at second messenger systems however we have to look more closely at the surface of the cell and how it is constructed Brief Overview Reading Chapter 5 Focused Reading p 7782 Stop at quotMembrane carbohydrates quot p 4951 quotLipids quot Stop at quotCarotenoids and steroidsquot p 3642 Proteins review p 282 quotReceptorsquot Stop at end ofpage p 284 G protein linked receptors Stop at cytoplasmic WWW Reading Crystal Model of a lipid bilayer Fluid Model of a lipid bilayer Receptors are integral membrane proteins with their quotactive sitequot or ligand binding site facing outward for binding with the extracellular ligand NOTE A ligand is any small molecule that binds to a protein at a specific site Hormones are a kind of ligand that binds to binding sites of hormone receptors Cells have in their membrane many copies of a receptor that binds a given hormone In addition each cell has many different kinds of receptors one kind of receptor for every different extracellular signal molecule with which the cell interacts The liver for instance interacts with epinephrine growth hormone thyroid hormone insulin glucagon and many other hormones The cells of the liver then have in their membranes many copies of each of these different receptors which can bind each of these hormones This is part of the quotmosaicquot of the uid mosaic model Study Questions 1 What is a ligand Give some examples 2 Draw a diagram of a phospholipid which illustrates its distinguishing characteristics Do not use the balloon with two tails model found in your textbook come up with a diagram of your own that conveys the important features of the phospholipid molecule 3 Explain in chemical terms why phospholipids are excellent molecular building blocks for membranes 4 Describe the uid mosaic model of membrane structure 5 Describe in chemical terms how an integral membrane protein would differ in amino acid sequence from a soluble protein one that oats freely in the cytoplasm How would an integral membrane protein have to be constructed what types of amino acids would be in what places in the molecule in order to be embedded and oating in the phospholipid bilayer 6 Membrane receptors are one type of integral membrane protein List other types of integral membrane proteins see Fig 51 56 59 513517 Be able to state the function and give a specific example for each type of protein News Item An international team has found that the same ligand estrogen can bind to two different estrogen receptors called alpha and beta When this common ligand binds to alpha it activates gene activation but when it binds to beta it inhibits gene activation Thus the same ligand can give two different signals depending on which receptor is present Science Vol 277 1508 September 1997 gt1 Antiestrogens are used for treating and preventing breast cancer Hypothesize a molecular mechanism to explain how the same ligand could give two different signals such as described above 8TOP We are now ready to put the elements of this story together by introducing the cAMP pronounced cyclic AMP second messenger system which forms the link between the epinephrine receptor and the phosphorylation of liver cell enzymes This system and others like it are called quotsecond messenger systemsquot because they provide a second message to the cell The hormone provides the first message by binding to its receptor on the cell surface The information of this binding is relayed into the cytoplasm through the second messenger system In addition to reading about the cAMP second messenger system you will also read about nucleotides and nucleotide structure since cAMP and ATP and GTP are nucleotides Focused Reading p 4748 quotNucleic acidsquot stop at DNA is a guidequot p 728729 Mechanisms of hormone actionquot stop at quotA cell can havequot Fig 158 p284 1517 p291 1512 p287 p291 Enzyme activities Stop at Different genes WWW Reading ATP Chime Image cAMP Chime Image Purves6e Chapter 15 Tutorial 151 Signal transduction pathway If ever there was a quotdominoquot effect it is the release of glucose in response to fear Here is a summary with a few more details than are given in your book to help you understand what is happening at the molecular level Read this story sentence by sentence Be sure you understand each event before you go on 1 A stressful thing happens and the brain sends a signal to the adrenal gland which secretes epinephrine 2 Epinephrine enters the blood and goes everywhere in the body 3 When epinephrine reaches the liver it binds to epinephrine receptors on the liver cell membrane 4 Binding of ligand with receptor causes the receptor molecule to change its native conformation 5 This change in native conformation reveals a binding site on the cytoplasmic side of the receptor which binds to G protein the stimulatory version of G protein called G5 6 When G protein binds to the epinephrine receptor allosteric modulation The binding causes the G protein to change shape 7 This change in shape causes a GTP binding site in the G protein to log its affinity for GDP and gain affinity for GTP 8 The GDP sitting in the site leaves and GTP binds to the site 9 When GTP binds to the G protein this causes the G protein to change shape again and become able to bind to adenylyl cyclase an integral membrane protein that is an enzyme 10 When the G protein binds to adenylyl cyclase adenylyl cyclase changes shape and this activates an enzymatic site on adenylyl cyclase which binds ATP and converts ATP to cyclic AMP 11 cAMP oats away from adenylyl cyclase and binds to the allosteric modulating site of cAMP dependent protein kinases One particular cAMPdependent protein kinase is Protein Kinase A also known as PKA 12 The cAMPdependent protein kinase becomes activated by this noncovalent allosteric modulation PKA is activated by allosteric modulation caused by binding cAMP 13 The activation PKA causes this protein kinase to phosphoglate an enzyme inthe liver cell called phosphorylase kinase Valuable Hint at no extra charge All kinases phosphorylate The word before quotkinasequot in the enzyme s name usually tells you which molecule the enzyme phosphorylates For instance hexokinase phosphorylates a hexose a 6carbon sugar Phosphofructokinase phosphorylates phosphofructose another 6carbon sugar 14 Phosphorylation activates phosphorylase kinase which phosphorylates glycogen phosphorylase and glycogen synthase 15 The phosphorylation of glycogen phosphorylase activates it thus causing it to break down glycogen to glucose glycogenolysis at a faster rate This releases more glucose into the blood and the blood levels of glucose rise Glycogen synthase is inhibited by phosphorylation Therefore glycogenesis is inhibited in the presence of stress thus helping to keep glucose in its monomeric form Study Questions 1 What are the components of a nucleotide and how are the components of a nucleotide linked together What is the difference between a triphosphonucleotide a 14 diphosphonucleotide and a monophosphonucleotide Give examples of each Chemically how are these nucleotides converted into one another 2 Proteins become activated and inactivated by ligand binding because they change their shape in response to the binding of ligands Identify eve protein in the cAMP second messenger system and describe how ligand binding affects that molecule What is it able to do after ligand binding that it was not able to do before 3 Describe how phosphorylation is used in the cAMP second messenger system Which proteins are phosphorylated and how are they changed by this event 4 The cAMP second messenger system represents an enzyme cascade Why is it called a cascade What is adaptive about such a cascade Why M the system evolve in such a way that the activation of glycogen phosphorylase was directly linked to the epinephrine receptor NOTE There is probably more than one plausible answer to this question Don t stop till you ve really thought about it 5 Be able to describe in chemical terms as described above the entire process of stress induced plasma glucose elevation from the stressful event through elevation of blood glucose levels 6 Choose an example from the social sciences the humanities the fine arts or your everyday experience that is analogous to the cAMP second messenger system Your model is a good one if you can trace the entire pathway outlined in 5 using this analogous system NEWS ITEM A new protein has been identified as a critical player in the metabolism of glycogen PTG Protein Targeting to Glycogen forms a large complex with the opposing enzymes phosphorylase kinase and glycogen synthase Researchers hypothesize that PTG acts as a molecular scaffold bringing the key enzymes into a central location to facilitate their regulation See Printen et al Science Vol 275 14751478 7 March 1997 Now that the cAMP second messenger system has been activated it must be deactivated Otherwise you could not go back to a quotnormalquot state after your stressful encounter You d be permanently wired on a blood sugar high Epinephrine and all hormones and signal molecules binds to its receptor through noncovalent 39 quot ie 39 J r39 39 39 39 quot hydrogen bonds and ionic bonds These bonds are fairly easily broken and as the epinephrine molecule sits in the ligand binding site of the receptor it eventually wiggles free simply because of its constant motion due to kinetic energy Ifthe epinephrine level is still high your brain is still stimulating epinephrine release by the adrenal gland then a new epinephrine molecule will take the old ones place and the receptor will remain activated However when the stress has passed the brain will stop stimulating epinephrine release and the epinephrine levels will fall because the body quickly destroys the free molecule Then when a molecule of epinephrine wiggles free of the epinephrine receptor there will be very few molecules to come in and take its place and the hormone binding site on the receptor will remain unfilled or empty Another way of looking at this is through the quotlaw of mass action from chemistry According to this law if the concentration of reactants increases the reaction rate will increase We can look at the free hormone and its binding site in the following chemical notation H B lt gt HB H the concentration of free hormone in the blood B the concentration of free empty binding sites on the hormone receptors HB the concentration of binding sites containing bound hormone As the concentration of free hormone H increases due to adrenal gland release the rate of the reaction increases and more hormone is bound to receptors Likewise as the hormone concentration decreases the rate of the reverse reaction is increased and more hormone comes free of its receptor ALL HORMONES AND SIGNAL MOLECULES HAVE THIS RELATIONSHIP WITH THEIR RECEPTORS THEREFORE THE STRENGTH OR DEGREE OF SIGNALING DEPENDS ON THE HORMONE CONCENTRATION When the hormone concentration falls the receptor has no hormone bound to the active site This causes the receptor to go back to its original shape In this original shape the receptor cannot bind and activate G protein no hormone bound no G protein activated Also the G protein very slowly in about a minute cleaves its GTP to GDP It removes the last or quotterminalquot phosphate from GTP GTP thus keeps two phosphates and becomes GDP With GDP bound instead of GTP G protein goes back to its original shape and loses its ability to bind to adenylyl cyclase Therefore no new adenylyl cyclases can be activated Also cAMP is degraded to AMP the cyclic connection between the phosphate and the third carbon of the ribose is broken by an enzyme called phosphodiesterase The bond it breaks is a phosphodiester bond quotdiesterquot because it contains two oxygens an ester linkage contains one oxygen and quotphosphoquot because it also contains a phosphate group This reaction is occurring constantly and breaks down cAMP quickly after it is formed The mechanisms that stop the cAMP cascade allow signal transductions to be brief In that way if you want to continue to make extra glucose for the blood your adrenal gland has to continue to release epinephrine in response to input from your brain Your brain therefore has control over the whole process Study Questions 1 Explain why noncovalent bonding between the ligand and the hormone receptor facilitates effective cellular communication What problems would be caused if the hormone bound covalently to its receptor 2 Explain in conceptual or chemical terms the relationship between hormone concentration and signaling strength How does this system work exactly 3 How is the cAMP system stopped once it has started Describe all the mechanisms involved What is adaptive about this immediate inhibition of the system 4 Describe how enzymes are named How can you tell what an enzyme does even though you haven39t ever encountered it before Here are some enzymes to practice on Pyruvate dehydrogenase ribulose bisphosphate carboxylase hint what is a quotcarboxylquot group See page 31 tyrosine kinase DNA polymerase peptidyl transferase aminoacyltRNA synthase phospholipase The cAMP second messenger system was the first one to be discovered It was discovered by Earl Sutherland who won the Nobel Prize for this discovery in 1971 Since then we have learned that many cells use this system for signaling Here are some other examples Secretion of thyroid hormone by the thyroid gland triggered by thyroid stimulating hormone Secretion of cortisol by the adrenal gland triggered by adrenocorticotropic hormone Secretion of progesterone by the ovary triggered by luteinizing hormone Reabsorption of bone triggered by parathyroid hormone Increased heart rate and force of heart contraction triggered by epinephrine Increased water retention by the kidney triggered by antidiuretic hormone Increased triglyceride fat breakdown triggered by epinephrine Learning and memory Mating in Chlamydomonas and you ll see this one in lab qTOP System 2 The Increase in the Force of Contraction of the Heart in Response to Fear Everyone has experienced the quotpoundingquot of the heart in the chest that occurs when one is afraid Edgar Allen Poe frequently referred to this response in order to heighten the sense of fear in his readers In nature fear is an emotion that is produced in response to things that are physically harmful such as predators The physiological response to fear then is to prepare you to ee or fight the thing that is making you afraid In order to do this you have to increase the supply of oxygen to your muscle cells since they will be working especially hard in eeing or fighting You also need more fuel for your muscles to burn for energy This fuel glucose is supplied by the liver as you learned above Blood carries oxygen to the tissues and the only way to increase the oxygen supply is to increase the rate at which blood is delivered to the tissues To do this the heart beats faster and harder It is the mechanism that produces the increase in contraction force that we will consider here NOTE The explanation in this paragraph was teleological The following explanation is causal The force of heart contraction is also controlled by the hormone epinephrine secreted by the adrenal gland in response to fear a kind of stress Epinephrine goes everywhere in the blood including the heart The heart muscle cells bear epinephrine receptors These receptors are called beta adrenergic receptors There are also alpha receptors which do not work through a second messenger system Receptors that bind epinephrine 1adrenaline are called quotadrenergicquot This receptor triggers a cAMP second messenger system in exactly the same fashion as the liver epinephrine receptor does The receptorligand complex activates a G protein which activates adenylyl cyclase which converts ATP to cAMP The cAMP then allosterically activates cAMP dependent protein kinase However this is where the similarities end The cAMPdependent protein kinase of cardiac muscle cells phosphorylates two proteins that we will look at here 17 l Ca2 channels in the plasma membrane and 2 myosin heads We need to look carefully at each of these protein systems beginning with the calcium ion channel in the plasma membrane of heart cells Ion channels are protein molecules that span the membrane are cylindrical in shape and allow the passage of w through the ion channel and thus the cell membrane Each channel is selective for a given ion quotSelectivequot means that they aren t quite as picky about what passes as they would be if they were quotspecificquot So they are pretty good at allowing only one type of ion to pass but not as good as receptors are at binding only one ligand or as speci c as enzymes that bind only one substrate Below is a cut away View of a selective ion channel gte membrane selective ion channel All cells have Ca2 channels Na channels K channels Cl39 channels etc in their plasma membranes Because ions are charged they are extremer hydrophilic and are repelled by the fatty acid tails of the phospholipid molecules in the membrane Therefore THE ONLY WAY AN ION CAN CROSS A MEMBRANE IS WITH THE HELP OF A PROTEIN THAT SPANS THE MEMBRANE The following reading assignments are about ions and ion channels Brief Overview Reading Chapter 2 3 amp 5 Focused reading p 24 quotIons form bonds quot stop at quotPolar and Nonpolar quot p 8586 quotPassive processes of membrane transport stop at quotOsmosis is passive water movement quot p 795 g 451 p88 fig59 WWW Reading Simple Ion Channel RasMol Image 18 Purves6e Ch5 Tutorial 51 Membrane transport passive transport section Ion channels are cylinders with water in the central channel These ion channels allow ions to ow across the plasma membrane down their concentration gradient Ions can only move down their gradient through channels they can only move from an area of high ion concentration toward and area of low ion concentration In the case of the Ca2 channel we are considering Ca2 is in very high concentration on the outside of the cell 10393 M and very low concentration on the inside 10397 M Thus Ca2 moves from the outside of the cell toward the inside of the cell when the Ca2 channel in the membrane is opened Note here that channel proteins cannot quotpumpquot ions up their concentration gradient Therefore this channel cannot move Ca from the inside of the cell toward the outside It can however be open or closed When closed it does not allow the passage of Ca2 toward the inside of the cell When open it w allow this passage Such an ion channel is said to be gated That is it acts as if it has a gate that can be opened to allow ions to ow or closed to stop the ow The Call channel we are currently considering is gated and can be opened or closed The type of gated ion channel we are considering is opened and closed in response to a change in the voltage across the heart muscle cell s plasma membrane These voltage changes occur rhythmically around 80 times per minute and are responsible for producing the normal heart beat A bit later in this unit we will consider how this voltage is created and how an ion channel might respond to changes in voltage For now all you need to know is that ion channels which open and close in response to changes in voltage are called voltage gated channels The Call channel we are considering is a voltagegated channel Other types of channels open and close in response to the binding of a ligand ligand gated channels or stretch stretch mediated channels We will consider ligandgated channels later in this unit While the Ca2 channel we are looking at is voltage gated it is also modi ed by being phosphorylated by cAMPdependent protein kinase When it is phosphorylated it stays open longer than normal thus allowing more Call than normal to enter the heart muscle cell It is this higher concentration of Ca2 inside the cell that produces a more forceful contraction Study Questions 1 What is it about the atomic structure of an ion that makes it charged 2 Ca2 is 10393 M on the outside ofthe cell and 10397 M on the inside How much ofa difference is this In other words what is the magnitude of the Ca2 gradient across the cell membrane If you don t understand molarity read p 2930 in your text NOTE The magnitude of concentration gradients is expressed in terms of the m difference across the membrane e g a 10 fold difference a 30 fold difference that is 10 or 30 times higher on one side than the other Here is an example of why this makes sense Concentration Inside Outside ifference ifference Concentration grithmetic Eold Concentration 1000 mM 900 mM 100 mM 111 times Gradient A Concentration 200 mM 100 mM 100 mM 20 times Gradient B While both concentration gradients have an arithmetic difference of 100 mM the gradient B is actually almost twice the size of A 2 fold versus 111 fold Substances will move almost twice as fast down gradient B as they will down gradient A 3 Describe the chemical structure of an ion channel 4 What do ion channels do Why is this function necessary 5 What does gated mean What is a quot gated channelquot What are the three types of ion channels gates39 6 Choose something from your everyday life that could serve as a good model analogy for a gated channel Explain why this item is a good model for a gated channel You know epinephrine activates the cAMP second messenger system in heart muscle cells called myocardial cells and this causes the Ca channels in the plasma membrane to stay open longer than normal allowing more Ca2 to enter the cell down its concentration gradient We need to look at a few more things before this makes any sense First why is Ca2 in higher concentration outside the cell than inside What creates this concentration gradient and why is it created And secondly how does more cytoplasmic Ca2 help the muscle cell contract with greater force First the Ca2 gradient As you will see throughout this unit Ca2 is widely used as an intracellular signal a signal within the cell Cells keep the intracellular or c oplasmic concentration of Ca2 very low when they are quotat restquot that is not receiving a signal Then if a communication molecule e g a hormone causes an increase in cytoplasmic Ca2 concentration this provides a signal to the cell Low cytoplasmic Ca2 levels means quotdon39t secrete or don t contract or don t pump ions whatever the cell does for a living don t do it High Ca2 levels means quotSecrete Contract Pump ions Do what you doquot This Ca2 signaling system must have two elements present in order for it to work correctly First the cell has to have a way to keep the cytoplasmic Ca2 levels very low under resting conditions Secondly the cell has to have a way to quickly increase the cytoplasmic Ca2 concentration when a signal arrives Because of its signaling role in the cell Ca is frequently called a second messenger or a third messenger though most people don t distinguish between second and third A rapid increase in cytoplasmic Ca2 concentration is allowed by opening of Ca2 ion channels Something happens ligand binding cell stretching or a voltage change which causes the Ca2 channels in the plasma membrane to open thus allowing Ca2 to ood into the cytoplasm The longer the channel stays open the more Call ions enter Note We will talk more about exactly how ion channels are opened and closed later in the unit For now however let s look at how the cell maintains a low level of cytoplasmic Ca2 at rest This is maintained by an active transport system for Call in the cell membrane and in the membrane of the endoplasmic reticulum 20 Focused Reading p 8891 quotActive transport quot stop at quotEndocytosis p 126128 quotATP couples stop at quotEnzymes biologicalquot WWW Reading Immuno uorescence Labeling of the Sarcoplasmic Reticulum Animation of Calcium Pump Purves6e Ch5 Tutorial 51 Membrane Transport Active transport sections Active transport is the m v t of 39 uptheir quot gradient This violates the second law of thermodynamics that everything tends toward maximum randomness or entropy 7 p97 of the text and therefore REQUIRES THE INPUT OF ENERGY FROM THE CELL When the cell burns glucose to carbon dioxide and water energy is given off The cell harvests this energy and stores it in the phosphate bonds of ATP When ATP is converted to ADP the stored energy is released and cellular work can be performed using this energy Very frequently though not always the terminal phosphate released in this reaction is covalently bonded to another molecule e g glucose or a protein The molecule is thereby phosphorylated You have encountered phosphorylation previously in its ability to activate or inactive enzymes by covalent modulation Now you are encountering it again This time phosphorylation will be used to provide the energy required to quotpumpquot ions against their inn gradient It takes one ATP to move two Ca ions into the ER In the myocardial cells there are two sets of Ca2 pumps or active transport systems that remove Ca2 from the cytoplasm One pump is in the plasma membrane and it moves Ca2 from the cytoplasm toward the outside of the cell The other pump is in the membrane of the endoplasmic reticulum and it moves Ca2 from the cytoplasm into the lumen of the ER A lumen is the inside of a tube or hollow ball The lumen of a balloon is the space where the air is the lumen of a garden hose is the space where the water is etc In muscle cells the ER is called the sarcoplasmic reticulum or SR sarco muscle Therefore sometimes the ER and SR are referred to jointly as the SER Lumen o a 1 Both pumps remove Ca2 from the c oplasm either by pumping it outside the cell or into an organelle The latter process is called sequestering Ca2 because the concentration of Ca2 becomes very high in the SER Both pumps cycle by the following mechanism see the diagram on the next page for an illustration of this mechanism which is outlined in steps 1 7 below 1 We begin our study of the Ca2 pump cycle at a random point the pump is dephosphorylated and its Ca2 binding sites are facing the cytoplasm The calcium binding sites have a very high af nity for calcium ions at this point 2 Ca floating in the cytoplasm binds to the binding sites on the pump which causes a conformational change in the pump Even though there is very little Ca2 present in the 21 cytoplasm the few molecules present that bump into the binding sites bind tightly to the pump and stay there The conformational change causes ATP to bind to the ATPbinding site on the pump it had been vacant When ATP binds its terminal phosphate is transferred to the pump phosphorylating it thus ATP becomes ADP This phosphorylation causes the pump to change conformation and present the Ca2 binding sites to the other side of the membrane For pumps in the plasma membrane this ips the Ca2 binding sites outside the cell while the SER pumps now have their Ca2 facing the lumen of the SER Ifthis seems confusing review the Purves6e online tutorial Ch5 tutorial Flipping its Ca2 binding sites to the other side of the membrane causes the Ca2 binding sites to have a lower of the affinity for Call This causes the Ca2 to diffuse off of the Ca2 binding sites and away from the pump The release of Ca2 from the binding sites causes a conformational change in the pump This conformational change causes the pump to become dephosphorylated When the pump becomes dephosphorylated it changes its conformation which makes the Ca2 binding sites ip to the other side of the membrane so they are facing the cytoplasm which results in the binding sites haVing a high affinity for Ca2 again The cycle repeats from step 1 ATPdependent Ca2 pump moving 2 ions across a membrane Outside Cell or SER Lumen 61 ATP ADP 1 ca binding Sites face the w 2 Calcium ion binds to the binding sites This bmdmg causes a conformational cytoplasm and have a high affinity for Ca change in the pump which causes it to bind ATP and become phosphorylated f amp Cytoplasm 4 The phosphorylation ips the 332 Flipping the binding sites causes the binding sites to the other side of binding Sites to lower in affinity the the membrane ions oat free and the pump becomes dephosphorylated Outside Cell or SER Lumen 39AA Finally the ion binding sites have the ion binding sites face the cytoplasm again 23 high affinty for 0310mm again The pump changes conformation again so that This process is called ATP dependent Ca2 transport and the pump is called an ATP dependent Ca2 transporter The role of ATP in the process of transport outlined here is to provide the energy required for the pump to quot ipquot that is open to the opposite side of the membrane the ipping event changes the affinity of the ion binding site The loss or gain of the ion causes changes which allow phosphorylation or dephosphorylation We spend a lot of energy pumping ions It has been estimated that we spend 1020 of all the calories we consume in the active transport of ions While this active transport accomplishes several other things one of its main functions is to facilitate signaling Study Questions lExplain why heart muscle cells and all cells in fact spend energy pumping Ca2 across their membranes Explain how Ca2 is used as a signal in cells 2Explain the mechanism by which Ca2 is pumped across the plasma membrane and the membrane of the SER This process requires ATP for energy How speci cally is ATP involved in this process 3This question provides a slightly different way of looking at the answer you gave in 2 The ATPdependent calcium transporter changes conformation three times during each pump cycle 1 The transporter ips toward the inside and outside of the cell 2 it changes the shapeaffmity of its Ca2 binding sites and 3 it changes the shapeoccupancy of its phosphorylation site What causes each of these changes to occur e g what causes the pump to ip to the outside what causes the affinity of the binding site for Ca to decrease etc Likewise each of these changes in conformation causes something to happen What does each ofthese changes cause eg what happens when the pump ips to the outside What happens when the shape of the phosphorylation site changes 4Again use an analogy to explain the ATPdependent Ca2 pump Try to develop an analogy that models all the aspects of the pump 5Develop an analogy to explain how Ca2 is used as a signal molecule in the cell Make sure your analogy can be used to explain how Ca2 is handled by the cell when it is quotat restquot ie not being signaled qTOP Back to the myocardial cell To summarize so far the brain has interpreted something in the environment as frightening and it has sent a nerve impulse to the adrenal gland to get it to secrete epinephrine Epinephrine levels in the blood and tissue uid have risen and epinephrine has bound to the beta adrenergic receptors on the myocardial cells plasma membranes This has triggered the cAMP second messenger system which has activated cAMPdependent protein kinase that has phosphorylated using ATP as the source of the phosphate the voltagegated Ca2 channel in the cell 24 membrane This phosphorylation has caused this channel to remain open longer than normal Ca2 has moved down its concentration gradient into the cell The resting cell maintained this gradient when cellular proteins actively pumped Ca2 into the extracellular space and inside the SER lumen The phosphorylated Ca2 channel remains open longer than normal allowing more Ca2 than normal to enter the muscle cell So how does this extra Ca2 in the heart muscle cause an increase in contraction strength In order to look at this question we need to look at how muscle cells contract All cells use their cytoskeleton to move But cells which are specialized for contraction have very highly organized r These r are 1 J micro laments described in general on p 7273 and seen in g 477 p836 called act39 and proteins that act as motors called myosin Focused Reading p 835839 Skeletal musclequot stop at Single muscle twitch Note Figures 478 through 4710 WWW Reading Purves6e Ch47Tutorial 471 How the Calcium Pump Fills the SER with Calcium The mechanism used by muscle cells to contract was described by Hodgson and Huxley in the sliding filament theory for which they won the Nobel Prize According to this theory muscles contract when actin and myosin laments slide past one another like this Relaxed Muscle actin myosin filament movement gt lt filament movement Contracted Muscle The actin and myosin protein bers overlap one another They slide past one another to produce contraction This sliding movement is very energetic and requires signi cant amounts of ATP The sliding is produced by the vigorous movement of parts of the myosin lament called myosin heads which form cross bridges These myosin heads bind to the actin and pull then release and reset then bind and pull then release and reset This process is very much like rowing a boat There is a diagram of this process on the next page So what does this contraction process have to do with Ca2 Well here39s the story When the myocardial cell is at rest not contracting the myosin head binding sites on the actin laments are covered by a protein called tropomyosin Unless tropomyosin is moved the myosin head crossbridges cannot form and contraction cannot occur Sitting on the tropomyosin is a second protein called troponin NEWS ITEMS Mutations in troponin and tropomyosin have been shown to be two causes of Familial Hypertrophic Cardiomyopathy an autosomal dominant disorder that causes an enlarged heart disorganized sarcomeres and ultimately death See Thierfelder et al Cell Vol 77 701712 3 June 1994 Mutations in the actin gene have been shown to cause idiopathic dilated cardiomyopathy Science 280 750 1998 cross brid e binding sites ac 39 Xl 39n QED myosin actin MM w a in The myosin heads make crossbridges when they bind to actin Direction of lt Direction of Crossbridge gt Crossbridge Movement x m m K Movement Tit To W till The myosin heads move stroke while bound to the actin This causes the actin filaments to slide together causing the muscle to shorten mmmmmmmmmm The myosin heads then breaktheir crossbridges and swing back to theiroriginal position W MM The crossbridges form again and the cycle repeats When cytosolic Ca levels are low Call is n0t bound to troponin for the same reason that ligands are not bound to proteins when their concentrations are low same concept different example When Ca2 is not bound to troponin troponin has a particular shape that allows tropomyosin to cover the crossbridge binding sites The myosin heads can t bind and contraction cannot occur This all 27 changes when Ca2 levels are high Under these conditions Ca2 binds to troponin This causes troponin to change shape and this change in shape pulls tropomyosin away from the crossbridge binding sites The myosin heads always ready to bind can now bind and contraction continues to occur until Ca2 levels fall causing the tropomyosin to cover over the crossbridge binding sites again Ca2 plays a regulatog role in the strength of cardiac muscle contraction Myocardial cells will not contract atall unless cytoplasmic Ca2 levels rise and tropomyosin is moved out of the way of crossbridge formation So increases in cytosolic Ca2 concentration occur 370 times per minute on average in the heart when you are not frightened this produces the regular heartbeat However epinephrine39s effect on the plasma membrane Ca2 channel allows it to remain open longer allowing more Ca2 than normal into the heart muscle cells Higher Ca2 levels allows more crossbridge binding sites than normal to be exposed This allows crossbridges to be formed more oars pulling in the water means more strength thus the force of the cardiac contraction is increased Finally cAMPdependent protein kinase has an additional action it phosphoglates the myosin 1 These phosphorylated heads are capable of quotrowingquot at a faster rate than when they are not phosphorylated Therefore they can produce more strokes per millisecond Since the limiting factor in this system is the amount of time the crossbridges are formed increasing the stroke rate of the crossbridges increases the amount of movement ie force that can be generated per unit of time Study Questions 1 Using the sliding filament theory explain how muscles contract 2 What role does Ca2 play in muscle contraction 3 How does epinephrine increase the strength of cardiac contraction Explain this in detail as you would for a traditional exam question Then explain it in simple terms as you would to a younger brother or sister 4 You have now encountered many ways that ATP is used in the cell List them and give a brief explanation of each 5 You have now encountered several examples where an event in the cell is triggered by a change in conformation or shape of a protein List all the examples you have encountered and brie y describe the effect of the conformational change in each system 6 quotBeta blockersquot are drugs that block the beta adrenergic receptor so epinephrine cannot bind to the receptor These drugs are used to lower blood pressure and to ease the strain on a weakened heart Explain the mechanism by which beta blockers produce the latter effect 7 In what ways can a cell increase its permeability to a particular ion List all the mechanisms you can think of As always answer this in chemical terms NEWS ITEMS When a muscle is stretched it tends to contract back to it resting size The molecule responsible for the ability to spring back is called titin due to its giant size Titin can be stretched to 4 times its resting length before it must spring back This has been visualized for the first time using some fancy microscopy call optical tweezers 28 which we will talk about later See Erickson Science Vol 276 10901092 16 May 1997 and related research articles An interesting side note Psychophysiologists at Univ Southern California have observed a correlation between low heart rate at rest and aggressive and antisocial personality traits A colleague commented that this was an interesting finding but what do you do with this information Is this a good hypothesis STOP System 3 The Contraction of a Skeletal Muscle in Response to an Impulse from the Nervous System Brief Overview Reading Chapter 5 and 44 The previous two systems we looked at involve a hormone as the communication molecule Now we are going to focus on communication mediated by the other major integrating system the nervous system The nervous system and endocrine system both communicate through chemical messengers The endocrine system uses hormones while the nervous system uses neurotransmitters In both cases the communication molecules are secreted by one cell travel to the target cell and bind to specific receptors in the plasma membrane of the target cell This binding triggers changes in the target cell but unlike hormones neurotransmitters travel only a very short distance to get from the cell that secreted them to their target cell When two neurons are communicating a nerve cell is called a neuron one neuron secretes a neurotransmitter that travels about 01 nm to get to the next neuron quotnmquot means nanometer A nanometer is a billionth of a meter 10399 meter This small gap between neurons across which the neurotransmitter diffuses is called the synaptic cleft and the area where one neuron interacts with another cell is called a synapse The neuron that secretes the neurotransmitter is called the pre synaptic neuron and the one that bears the neurotransmitter receptor and binds the neurotransmitter the target cell is called the post synaptic neuron Here39s a picture and there is also one on page 789 in your text Figure 4414 A Synapse Synaptic Cleft Postsynaptic cell Neuron Muscle or Gland Secretory Vesicles Containing Neurotransmitter smltter Receptors When a neuron is communicating with a muscle cell rather than another neuron the synapse is called a neuromuscular junction The process of communication from the presynaptic neuron to the postsynaptic muscle cell is essentially the same as when two neurons communicate Focused Reading p775776 Neurons are stop at Glial Cells p776777 Neurons generating stop at end of page 777 p785786 Neuron Synapse and Communicationquot stop at quotThe arrivalquot p 789 fig44 13 Just as there are many different hormones there are also many different neurotransmitters The one we will look at here is acetylcholine which is used as the chemical messenger between the nervous svstem and skeletal muscle cells A list of neurotransmitters can be found on page 790 in your text In looking at this system we will begin at the beginning of the process and look at the cellular and chemical events that produce a nerve impulse Nerve impulses are electrical events that is they are caused by the ow of charged particles in this case ions move instead of electrons as is the case for electrical current in power lines The ow of charged particles is called current The force that moves charged particles causing them to ow causing current is voltage or electrical potential Voltage is a separation of charge According to the second law of thermodynamics charged particles will move in such a way that electrical neutrality is produced an equal distribution of positively and negatively charged particles Thus if you separate positive particles from negative particles create concentration gradients of negative and positive charges you have created a voltage a potential force that will compel charged particles to move to correct this imbalance to create electrical neutrality In doing this negatively charged particles will move toward the concentration of positive charge and vice versa until they are completely mixed and the solution is electrically neutral When voltage exists then there is always a negative pole and a positive pole like a battery The negative pole the cathode attracts positively charged ions called cations and the positive pole the anode attracts negatively charged ions called anions The bigger the separation of charge the bigger the voltage the more current will ow between the poles At rest that is when no signal is being sent or received the plasma membranes of all cells including neurons have a voltage across them The outside of the cell is the positive pole and the inside of the cell is the negative pole The separation of charge across the membrane is small with a voltage of only 60 millivolts mV By convention the voltage is given the sign of the pole that is im the cell So a voltage of 60 mV means that the magnitude of charge separation is 60 mV with the inside of the cell negative with respect to the outside 60 mV then is said to be the resting membrane potential This voltage exists in all cells at rest and in neurons when they are not propagating an impulse It is this voltage that allows the creation and propagation of a nervous impulse This voltage is used in other cell types to transmit signals as well But before we look at that we have to look at how this voltage is created in the first place Separation of charge can be thought of as a charge concentration gradient Just as the Ca2 gradient was created by an active transport system so is the membrane voltage Focused Reading p 8891 quotActive Transport stop at quotEndocytosisquot Pay special attention to Fig 512 p90 30 p 777778 quotIon pumps stop at end ofpage 778 p 777 Fig 445 Research Method The ATP dependent N aTKT pump operates exactly like the ATPdependent Ca2 pump we have already considered It is a bit more complicated because it transports two ions in opposite directions across the membrane This pump is therefore called an antiporter two substances opposite directions An antiporter is an example of a cotransporter two substances transported at once in any direction both inward both outward or one in and one out The Ca2 pump on the other hand is called a uniporter because is transports only one substance Even though the NaK pump transports two ions the same rules apply here as they did in the calcium ion pump 1 Phosphorylation and dephosphorylation cause the pump to ip 2 The ip causes a change in the ion binding sites39 affinities 3 The loss or gain of ions into the binding sites causes the pump to be phosphorylated or dephosphorylated 4 The cycle repeats The relationship of K to phosphorylation and site affinity is exactly opposite that of Na since they are being transported in opposite directions across the membrane It is the unegual transport of potassium ion versus sodium ion that CREATES THE RESTING MEMBRANE VOLTAGE The NaK pump transports three sodium ions 3 to the outside of the cell for every two potassium ions 2 it transports to the inside of the cell Therefore the pump separates charge that is it pumps more positive charge to the outside than it does to the inside The pump is therefore said to be electrogenic it generates voltage This makes the outside of the cell membrane slightly positive and the inside slightly negative The magnitude of this charge difference is 60 mV technically 60 mV since the inside is negative Note that the NaK pump has to keep pumping constantly because the membrane has Na and K channels in it that quotleakquot This situation is analogous to bailing a leaking boat You have to keep bailing because the water keeps leaking back into the boat But if you bail as fast as the boat leaks you can stay a oat Likewise at quotrestquot this means no signal is being sent It does not mean the cell is inactive the NalK pump bails as fast as the channels leak so a steady state is maintained In this steady state because of the action of the pump three significant conditions exist 1 There is a concentration gradient for Na across the cell membrane The concentration of Na is very high on the outside of the cell and very low on the inside of the cell This gradient is produced by the NaK pump moving Na from the inside of the cell to the outside 2 There is a concentration gradient for K across the cell membrane The concentration of K is very high on the inside of the cell and very low on the outside of the cell This gradient is produced by the NaK pump moving Kl from the outside of the cell to the inside 3 There is a voltage across the membrane This voltage is produced by a separation of positive charge such that more positive charge is placed on the outside of the cell than on the inside This voltage is produced by the unequal pumping of Na and Kl by the NalK pump Study Questions 1 What is voltage What is current How are these two concepts related 2 Explain the concepts of voltage and current using an analogy 3 Describe the mechanism the ATPdependent NaK pump uses to move ions across the membrane 4 How is the resting membrane potential created What causes the outside of the cell to be positive and the inside to be negative 5 Explain why the NaK pump has to pump ions all the time Use an analogy other than the leaky boat analogy to describe this phenomenon So here the neuron sits with its resting membrane potential at 60 mV and its concentration gradients for Na and K well established The neuron that will ultimately synapse on the muscle cell and get it to move is called a motor neuron It is called quotmotorquot because it causes movement as opposed to a sensory neuron that carries sensation Ifyou want to move your leg you send an impulse from your brain down to the spinal cord In this area sit the motor neurons that communicate with your leg muscles The neurons coming down from the brain synapse on the motor neurons and secrete a neurotransmitter onto their membrane The type of neurotransmitters used at this synapse is not known but probably several different neurotransmitters are involved This causes a change in the motor neurons which causes nerve impulses called action potentials to be transmitted across the motor neuron out to the muscles of the leg Focused Reading p 777781 quotSimple electricalquot stop at quotAction quot When the motor neuron receives the message from the brain the neurotransmitter binds to its receptor in the plasma membrane of the motor neuron This neurotransmitter receptor is physically linked with a Na channel See Fig 4415 on page 786 When the neurotransmitter binds it causes a change in shape surprised in the receptor This change in shape causes the Na channel to open This is an example of a ligand gated channel The binding of a ligand the neurotransmitter causes the opening of the channel When the channel opens Na is free to move quickly down its concentration gradient Because it is in high concentration outside the cell and low concentration inside it quickly moves into the cell When Na moves it carries its positive charge with it Therefore the inside of the cell becomes more 32 and more positive as more and more Nal enters This movement of positive charge causes a change in the membrane voltage At rest it is 60 mV but as sodium enters it becomes 59 58 57 56 etc until it reaches 40 mV 40 mV is called the threshold potential At this point the voltage is sensed by voltage gated Na channels in the neuron s plasma membrane near the recently opened ligandgated channel These voltagegated Na channels change shape when the voltage reaches 55 mV This change in shape causes them to open allowing even more Na to ood into the cell Now the voltage continues to become more positive 40 30 20 etc until the inside actually becomes positive with respect to the outside at 50 mV This is called membrane depolarization because the original poles negative inside and positive outside have been obliterated see left side of fig 448 pg780 There are voltagegated Nal channels all the way along motor neurons from their bodies in the spinal cord to their synaptic terminals in the arm muscles As each area of membrane reaches threshold voltage 40 mV the depolarization is sensed by the neighboring voltagegated Na channels and triggers the channels to open one by one all along the way to the end of the neuron This propagation of a wave of depolarization is called an action potential a quotnerve impulse This will be the command signal to the muscle to contract This process can be diagrammed as follows Propagation of Action Potential Na Along a Neuron Nair Na N2r NaJr Na szr t a M 5 NaJr Entrance of N a 1 Voltagegated Na channels next to into cell causes change depolarized area sense change in Voltage in membrane voltage and open 3 This area then depolarizes the 2 which results in next channel 4 and the next Na is ready depola za OH to enter the cell 5 Do you see why nerve impulses are described as quotwaves of depolarizationquot Study Questions 1 Explain how the resting membrane potential makes the action potential possible Describe this in actual chemical terms and then describe it using an analogy 2 Ligandgated and voltagegated ion channels are both involved in the generation of an action potential Describe the role played by each type of channels 3 Explain how the opening of Na channels in the plasma membrane produces a change in membrane voltage Why is this change called depolarization Note The cell membrane has to be returned to its resting state before it can send another signal through an action potential This membrane repolarization is produced by voltagegated K 33 channels that open after the voltagegated Na channels This causes K to flow down its concentration gradient towards the outside of the cell K carries its positive charge with it and makes the inside of the membrane more negative as it leaves This along with the closing of the Na channels repolarizes the membrane Focused Reading p 78183 Action potentials are stop at Ion channels and p 782 Fig 4411 p 781 Fig 4410 Ifyou can explain these to your nonscience friends you understand action potentials Study Questions 1 What role does K play in an action potential 2 Make a list of the similarities between K and Na in an action potential List the differences between these two ions in an action potential NEWS ITEM A research team from Australia and Germany has cloned a gene that is expressed only in the brain and causes a certain type of epilepsy called benign familial neonatal convulsions The convulsions start about 3 days after birth but usually disappear within a few months The culprit is a mutation in a potassium channel gene that in one family had a 5 base pair insertion which cause the deletion of 300 amino acids at the end of the protein If the channel is not full length then it cannot work properly structure 7 function relationship again Science Vol 279 403 January 1998 3 What would happen to a neuron that contained the mutation described above Eventually the wave of depolarization the action potential reaches the synaptic terminal of the motor neuron This neuron terminates at a skeletal muscle cell in a leg muscle the terminal structure is called a neuromuscular junction This neuromuscular junction looks very much like the synapse you have already encountered where the neuron from the brain told the motor neuron to start its action potential When the action potential reaches the synaptic terminal of the motor neuron it causes the membrane of the presynaptic cell to depolarize just like all the rest of the membrane all the way down from the spinal cord However the synaptic terminal contains voltage gated Ca2 channels in its membrane When the membrane depolarizes these voltagegated Ca2 channels open and Ca2 ows down its chemical concentration gradient into the synaptic terminal NOTE When the cell was quotat restquot the Ca2 gradient was produced by the same plasma membrane Ca2 pump that works in the heart muscle Virtually all cells pump Ca2 out of the cytoplasm using this pump The synaptic terminal of the motor neuron contains secretory vesicles full of the neurotransmitter acetylcholine When Ca2 enters the terminal these vesicles fuse with the plasma membrane and release their contents into the synaptic cleft The process of secretion is an example of exocytosis Focused Reading p9192 stop at Membranes are not simply p92 94 Membranes are dynamic to end of chapter p 91 Fig 514b p 787 fig 4416 WWW Reading Movie of Calcium In ux into a Neuron Purves6e Chapter42 Tutorial 422 Synaptic transmission Exocytosis and endocytosis are mirror images of one another Cells use exocytosis to secrete products eg hormones neurotransmitters cell wall components milk digestive enzymes sweat tears etc Cells use endocytosis to engulf cells and other substances usually for utilization by the engulfing cell Cells engulf bacteria viruses dead cells from one s own body proteins water iron etc Cells also use endocytosis to retrieve membrane added during exocytosis and vice versa This process is called membrane recycling or membrane traf c Some cells sit and secrete constantly Their secretion is said to be constitutive that is it occurs constantly and is not regulated Other cells such as neurons store their secretory product and wait for a signal to secrete this is regulated secretion The signal to secrete is usually a rise in intracellular Ca2 So how does a rise in the level of cytoplasmic Ca2 trigger secretion in most cells and specifically in motor neurons We don t know the complete answer but a story has emerged which is currently very popular Note Results from this field of investigation are so new that investigators have not yet settled on a common set of terms for the molecules involved The terms presented here are from one lab investigating neurons According to this theory there are membrane proteins in secretory vesicles called VAMPs esicle associated membrane protein that can bind membrane proteins in the plasma membrane called syntaxin proteins discovered in aptic vesicles that facilitate vesicle movement taxis movement Both VAMP and syntaxin are integral membrane proteins that means they go through a membrane remember Most of the mass of VAMP is on the c oplasmic side of the vesicle membrane Most of the mass of syntaxin is on the c oplasmic side of the plasma membrane The cytoplasmic portions of VAMP and syntaxin act as 39handles that allow the vesicle to come in contact with the plasma membrane When these two proteins VAMP and syntaxin bind to one another the vesicle and plasma membranes fuse and the contents of the vesicle in this case acetylcholine is secreted into the synaptic cleft The binding of VAMP to syntaxin is facilitated by some additional cytoplasmic proteins called NSF and SNAPs and the entire process is ATPdependent The VAMPsyntaxin cytoplasmic proteins complex falls apart when ATP is hydrolyzed to ADP by an enzymatic site on one of the proteins See figure on next page NEWS ITEMS Release of neurotransmitter has beenvisualized for the first time by using genetic engineering and the lightening bug enzyme luciferase Every time these modified cells secrete neurotransmitters they also produce a small spark of light which can be seen through a microscope This allows researchers to determine how many vesicles fuse with the plasma membrane for any given stimulus See Miesenbock and Rothman Prac Natl Acad Sci USA Vol 94 3402 April 1997 Nicotine that is found in cigarette smoke has been shown to bind to presynaptic acetylcholine receptors and cause a rise in intracellular calcium at the nerve terminus This leads to an increase secretion of other neurotransmitters Science Vol 269 1692 September 1995 However a more recent study has shown that chronic exposure of nicotine can cause two of the three known versions of the acetylcholine receptor to become permanently inactivated The third receptor is still functional which leads to increased neurotransmitter dopamine release and thus the craving for nicotine is sustained Olale etalJ Iquot 1 o and 7 p 39 1 quot 739 r 39 283 675 1997 The protein complex is hypothesized to look something like this a as ma m emb rane secretory vesiol e ngend 0 calcium VAMP A I unknown factors a VAP33 v synaptotagmin syntaxin SNAP25 While high cytoplasmic Ca2 levels clearly trigger this binding and subsequent membrane fusion the mechanism of calcium triggering f i is not 39 39J J J Control is likely to occur through a second protein bound onto the vesicle membrane called synaptotagmin a Ca2 binding protein Investigators hypothesize that when synaptotagmin binds Ca2 it changes shape This change in shape causes a change in shape in the VAMPs which allow them to bind to syntaXin on the inside of the cell membrane thus allowing secretion to occur r In addition to mediating secretion this process of proteinmediated binding followed by membrane fusion is the way substances are transported and speci cally sorted within the cell For instance some cells secrete protein e g insulin and other protein hormones digestive enzymes This protein is made on ribosomes which are quotdockedquot onto the surface of the ER As the protein is made it is translocated moved across39 into the lumen of the ER From there the protein must travel to the Golgi apparatus and then to secretory vesicles for secretion The protein is first concentrated into a specific region of the ER Small vesicles containing the protein then bud off the ER and fuse with the cis face of the Golgi The protein product then transported through the Golgi by budding off each Golgi cistema and fusing with the next cistema until the trans face is reached Then secretory vesicles bud off the trans face ready to release their contents outside the cell upon receiving the appropriate signal The process looks like this new protein ribosome 39 J I u Ca2 ilike complexes syntaxinilike receptor cis Golgi Evidence is building that all of the budding and coalescing processes called vesicular transport are mediated by protein complexes that function like the VAMPsyntaxincytoplasmic protein complex that mediates secretion Thus if one asked how the vesicle that buds off the ER quotknowsquot to fuse with the Golgi and not with a mitochondrion or the nucleus or the plasma membrane the answer is probably that this ER vesicle contains a VAMPlike protein that is specific for that is complementary in structure to a syntaxinlike molecule on the Golgi membrane Thus the transfer of proteins within the endosomal system the system of organelles in the cell that includes the Golgi ER lysosomes phagocytic vesicles and secretory vesiclesreviewed in pages 6466 is probably mediated by specificity of membranebound quotdockingquot proteins NEWS ITEM A protein called syntaxin 5 has been identified as a necessary molecule for the formation of Golgi and the delivery ofvesicles to the Golgi Science Vol 279 696 January 1998 The endomembrane is not just a oneway street for protein synthesis and secretion Vesicles also travel in the other direction and are sometime used 39against us The toxin produced by Shigella dysenteria Shiga toxin enters the cell by endocytosis and causes hemorrhagic colitis Shiga toxin and has recently been shown to travel all the way 39down39 the pathway to the ER before escaping into the cytoplasm and wreaking havoc on the cell F Mallard et al 1998 JCB vol 143 973990 But we digress back to the neuromuscular junction After acetylcholine is secreted into the synaptic cleft it diffuses the very short distance to the postsynaptic membrane of the skeletal muscle cell where it binds to an acetylcholine receptor Acetylcholine receptors are in part ligand gated Na channels Again see page 786 Figure 4415 for an illustration Thus the binding of acetylcholine to the muscle cell membrane triggers an action potential that is spread across the muscle cell membrane in exactly the same way that the action potential was spread down the neuron The mechanism of generation and propagation are the same The action potential that spreads across the muscle cell membrane triggers a rise in guess what intracellular Ca2 levels in the muscle cell this is a very hot area in research today By the same mechanism as in heart muscle this Ca2 binds to troponin causing it to pull tropomyosin away from the crossbridge binding sites on the actin laments Contraction is sustained for as long as cytoplasmic Ca2 levels remain high And cytoplasmic Ca2 levels remain high as long as an action potential is being propagated along the muscle cell membrane And an action potential is propagated as long as acetylcholine is bound to its receptor And acetylcholine is bound to its receptor as long as it is secreted by the presynaptic neuron An enzyme in the synaptic cleft called acetylcholinesterase destroys acetylcholine almost immediately Therefore the presynaptic cell must provide a continual supply of the neurotransmitter if the receptor is to remain activated And the presynaptic neuron secretes acetylcholine as long as action potentials continue to reach the synaptic terminal And action potentials reach the synaptic terminal as long as they are generated at the cell body in the spinal cord which continues as long as the brain is telling you to ee the bear chasing you Now at this point things get a little vague But neuroscientists practice their craft believing that everything even the will to contract is caused by a chemical process Practitioners of neuroscience have a lot of work to do before they truly understand these higher neurological functions One more thing how does the action potential in the muscle cell membrane actually M an increase in cytoplasmic Ca2 levels in the muscle cell We don t know We do know that most of the Ca in this process comes from inside the SR where it has been pumped by the ATPdependent Ca2 pump while the cell was at rest Thus the action potential in the muscle cell membrane must trigger the opening of a Ca channel in the membrane of the SR Ca then is free to ow down its concentration gradient into the cell We do not know the exact 39 whereby the action potential 39 with the Ca2 channel in the SR However recent research has pointed to a protein called triadin which spans the gap between the plasma membrane and the Ca2 channels in the SR triadin spans the triad and we are not referring to 3 cities in NC Therefore triadin is in the right place to bridge the gap but no one has any idea how this could be done See gure 449 pg 981 for a picture of where the T tubule meets the SR Whoever gures it out rst will become very famous since this is the last big mystery about muscle contraction I Study Questions 1 What events are triggered by the arrival of the action potential at the synaptic terminal 2 Describe the process of exocytosis 3 List some ways in which cells use exocytosis and endocytosis How are these two processes used together to ensure that the cell s size does not change 4 Describe the current theory that explains how increased Ca2 concentrations trigger secretion 5 Describe the process by which protein travels from the ER through the Golgi and into secretory vesicles How is this process controlled so that the correct vesicles coalesce with the correct target organelle 6 Muscle cells and neurons are physiologically more similar than one might think In what ways are these cells similar in their chemical responses What types of membrane receptors and channel proteins do both types of cells have In what ways are these two cell types different in their chemistry and responses 7 The action potential in the muscle cell membrane causes a rise in cytoplasmic Ca2 levels Where does this Ca2 come from How does it enter the cytoplasm What must the muscle cell do when it is at rest to ensure that this signaling system will work 8 Outline the entire pathway in chemical terms from wanting to move your arm to actually moving your arm Tell this story using chemical and cellular language as you would for a traditional exam question Then tell it in simpler terms as you would to a younger sister or brother Use as many good analogies as you can System 4 How does a sperm cell tell an egg it has been fertilized We have studied 3 speci c cases of signal transduction l epinephrine bound to a liver cell receptor to tell the cell to put more glucose into the blood 2 epinephrine bound to a heart cell receptor to tell the cell to contract harder and 3 neurotransmitters bound to skeletal muscle receptors to tell the muscles to contract Now we will examine a slightly different situation for signal transduction fertilization One thing is certain once nature develops a good system of moving of secreting of transporting of communicating it keeps using it over and over again The communication system used by an egg to sense fertilization is an ancient one So ancient in fact that it developed before sea urchins frogs fish and mammals diverged from one another during evolution How do we know this Because all animal eggs use the same communication system Odds are that evolution would not have produced the same communication system twice through random mutation and selection Therefore the system probably evolved before the split and all animals took this good idea with them as they diverged into different species This communication system is called the inositol triphosphate second messenger system Focused Reading p 2878 quot Two second messengersquot stop at quotCalcium ions are quot 40 p 288 Figure 1513 The inositol triphosphate 1P3 second messenger system uses the same kind of receptorG proteinenzyme link that the cAMP system does Except this time the enzyme linked to the G protein is not adenylyl cyclase it is phospholipase C Like most enzymes this one tells you what it does It cuts up a phospholipid Let39s talk a little more about phospholipids You know that they make up the cell membrane and you know why They have a hydrophilic quotheadquot that dissolves in the water inside and outside the cell and they have hydrophobic tails that hate water and dissolve in each other in a hydrophobic bilayer To refresh your memory Hydrophilic Hydrophobic quotTailsquot quotHeadquot I H H C O CH Long hydrophobic hyd carbon chain CH2 CH2 CH3 H H C O CH Long hydrophobic hydrocarbon chain CH2 CH2 CH3 There are many different kinds of hydrophilic molecules that can be added to the phosphate on the phospholipid These are added at the quotRquot in the diagram above Regardless of what is added these molecules are all highly polar and many of them are charged which greatly increases the hydrophilic nature of the quotheadquot of the molecule Some examples of molecules that are added to the phosphate group at quotRquot are serine choline and inositol Phospholipids are named according to the molecule added to the phosphate All phospholipids start with quotphosphotidyl blank 1quot and then the name of the added molecule fills in the blank Thus if serine were added the phospholipid would be called phosphotidylserine Ifcholine were added it would be called phosphotidylcholine And if inositol were added it would be called phosphotidylinositol Some phospholipids have inositol bis phosphate added to their phosphate group quotBisquot means quottwoquot So inositol bisphosphate is inositol with two phosphate groups on it When inositol bis phosphate is added to a membrane phospholipid the resulting molecule is called phosphotidylinositol bis phosphate The abbreviation for this molecule is PIPZ This is the substrate molecule for phospholipase C PIPZ can be diagrammed simply as follows 41 Phosphotidylinositol bisphosphate Hydrophobic Tails Inositol bisphosphate PIPZ sits in the inner layer of the plasma membrane s lipid bilayer Like all other phospholipids in this layer it has its hydrophobic tail embedded in the lipid bilayer and its quotheadquot facing the cytoplasmic side of the membrane When phospholipase C is activated by G protein it cuts inositol off of PIPZ in such a way that all the phosphates go with inositol and none remain on the lipid in the membrane The products of this cleavage look like this Inositol tnphosPhate Hydrophobic Tails C lg C I i O GD C Diacylglycerol DAG covalent bond broken by phospholipase C The inositol with the three phosphates is called inositol triphosphate 1P3 This hydrophilic molecule oats away from the membrane into the cytoplasm where it will act as a second messenger The other part of the molecule is called diacylglycerol like triacylglycerol with two instead of three fatty acids chains This is abbreviated DAG DAG stays embedded in the membrane but nonetheless also acts as a second messenger So the cleavage of PIPZ by phospholipase C results in two cleavage products IP3 and DAG both of which will now act as second messengers Study Questions 1 Describe or draw a simple diagram like the ones presented above of atriacylglycerol a generic phospholipid diacylglycerol phosphotidylinositol bisphosphate inositol triphosphate If you need more information about lipids and phospholipids see page 4244 in your text 2 Describe the pathway through which phospholipase C is activated 3 Describe the enzymatic action of phospholipase C What is the substrate for this enzyme and what are the cleavage products of the reaction What general function do these cleavage products have in the cell We have all seen those classic lms of sperm fertilizing an egg the egg is surrounded by hundreds of sperm trying to penetrate the egg s plasma membrane Why is it that only one sperm cell manages to fertilize an egg With all those sperm cells trying to reach the same goal at the same time you would think that at least two might enter the egg at about the same time polyspermy If this were to happen the resulting zygote would be in trouble since it would have 3 haploid genomes 3 copies of each chromosome instead of the normal 2 copies we will cover this later in the Section II An egg with 3 haploid genomes will probably not survive the process of development Evolution is a process of natural selection natural selection allows those with favorable traits or abilities to reproduce This results in advantageous traits or abilities being maintained in the population while less advantageous ones may disappear Natural selection plays a part in all levels of molecular and cellular biology and fertilization is no exception Any egg ie organism that produces this egg that has learned how to permit only one sperm to fertilize it will be more likely to produce a new individual that will have the same selective advantage its mother had which will in turn result in more successful matings for the mother s offspring So the question remains how has evolution natural selection given rise to an egg that permits only one sperm to fertilize it Evolution is not a wasteful process In fact evolution was the first to recognize the importance of recycling We have talked about G proteins that were coupled to receptors which resulted in the production of cAMP as a second messenger To invent a new second messenger system to facilitate signal transduction evolution thought to herself How can I move across the plasma membrane the information that a sperm has arrived while not having to come up with a totally new mechanism The answer is beautiful in its similarity or homology to the cAMP messenger system but with a subtle twist to achieve a very different set of responses Focused Readings p 7677 Animal cells to end of chapter p 73436 quotSexual reproduction quot stop at quotOogenesis quot Figures 423 424 429 p 7534 Fertilization stop at The sperm quot Figures 431 433 434 WWW Readings Immuno uorescence Labeling of ER Movie of Calcium During Fertilization Movie of Flash Animation quotMechanism for 1P3 production and Ca2 ion wavequot Purves6e Ch43 Tutorial 43l Fertilization in the Sea Urchin More links to help you visualize this process They are all part of the same web site quotBill Wasserman s Developmental Biology Webpagequot from Loyola University Once at the site check out the following 39 quot httpwww Inc J J 39 39 39 J v htm Mammalian Oocyte Egg Ovulation 2 Mammalian Egg Sea Urchin Egg Fertilization 4 Acrosome Animation Egg activation 3 Cortical Granule Exocytosis 43 An egg is just like any other cell in many ways It has a plasma membrane a nucleus a Golgi apparatus and an endoplasmic reticulum The egg also has many unique features including the Vitelline layer also called the zona pellucida which is an extracellular matrix analogous to a plant cell wall that contains many copies of a spermbinding protein called ZP3 the third protein discovered in the zona pellucida which functions a receptor ZP3 interacts with bindin proteins on the surface of sperm cells and initiate the acrosomal reaction ZP3 is as specific as any other receptor we have studied it will only bind ligands present on the surface of sperm from the same species as the egg For example rat sperm proteins will not bind to ZP3 on frog eggs Interaction between ZP3 in the vitelline layer and ligands on the sperm head cause the two cells to fuse Many different types of receptors in the sperm s plasma membrane trigger this fusion some are protein kinases and others activate G proteins The bottom line is the sperm is told by its receptors that it is timed to fuse with the egg Over time evolution has selected eggs that have developed two separate mechanisms to prevent polyspermy a fast block an electrical barrier and a slow block a physical barrier 1P3 as a second messenger in fertilization Na on Membrane in ux potential OFI OLYSPERMY changed FAST BLooK p Sperm binds egg receptor 1P ca 2 cortical SLOW BLOCK 3 released 9 reaction 9 OF E produced from ER POLYSPERMY Gprotein h h 139 c p osp o rpase acnvated activated PLC diacylglycerol 7 activation produced protern krnase C CD AG activated PKC 0f 6 N214r HJr increase in exchanger 9 intracellular activated pH Adapted from Developmental Biology third edition by Scott F Gilbert When the plasma membrane of the sperm first fuses with the plasma membrane of the egg there is a change in the membrane potential of the egg cell As we saw in muscles egg cells have a resting potential of about 60 mV with a higher concentration of Na ions outside the cell than inside Fusion of egg and sperm membranes causes Nal channels in the plasma membrane of the egg to open Although the exact gating mechanism for opening these Nal channels is unknown the result is 44 predictable Na ions rush into the egg down their concentration gradient which changes the membrane potential from 60 mV to about 30 mV For unknown reasons this change in polarity prevents sperm from fusing and thus is a fast acting block to polyspermy The second block to polyspermy is where IP3 is used but it is interesting that evolution has selected organisms that have two ways to block polyspermy Can you imagine why you would need a second one given the nature of the rst The second block to polyspermy will create a physical ba1rier to sperm entry rather than an electrical one Ifyou were to examine an unfertilized egg in cross section you would see lots of small vesicles conical granules just below the plasma membrane Inside the cortical granules are proteases and mucopolysaccharides During fertilization these cortical granules to fuse with the plasma membrane of the egg and as we saw in neurotransmitter release the contents of the vesicles is released into the adjacent quot 39 space f i The x f i of cortical granule contents causes two significant events to happen in close succession l the proteases digest the proteins linking the vitelline envelope to the extracellular face of the egg s plasma membrane and probably disrupts the integrity of the unoccupied sperm receptors too 2 the mucopolysaccharides increase the osmotic pressure in the small space between the vitelline envelope and the plasma membrane we ll talk about osmotic pressure later which makes water rush in which like a hydraulic lift causes the vitelline envelope to be pushed away from the plasma membrane By pushing the vitelline envelope away from the egg s plasma membrane a physical barrier has been created to prevent any more sperm from fusing with the egg Now that we know the cortical granules are capable of causing the physical block to polyspermy we are still left wondering how the egg knows when to signal the cortical granules that one sperm has fused with the egg This is where IP3 comes into the picture The sperm plasma membrane proteins interact with the sperm receptor in a manner similar to the cartoon not drawn to scale in the diagram below 45 spermnot drawn tos tale flrst me ssenger plasma membrane of e gg bisrph os phate In Isolat Ion cytoplasm ph 05 pho u dy II no Sll ol IP3 as second messenger endoplasmic reticulum o IP3 rece ptor and lumen of the ER talc Ium ch annel 2 Ca Adapted from Developmental Biology third edition by Scott E Gilbert When the sperm receptor binds its ligand ZP3 the receptor changes shape activates the associated G protein which stimulates phospholipase C which cleaves phosphotidylinositol bis phosphate into two parts IP3 and DAG IP3 is a second messenger that diffuses throughout the cytoplasm where it eventually bumps into the IPS receptor located in the ER membrane The IP3 receptor is a homotetramer made of 4 identical subunits The IP3 receptor has a very high affinity for IP3 and so IP3 binds to its receptor and acts as an allosteric modulator Each subunit has at least three allosteric binding sites one IP3 molecule and two calcium ions all have to bind to each subunit of the receptor Calcium and IP3 modulate the IP3 receptor which is also a ligand gated Ca2 channel causing the normally closed channel to open As we know the ER is a rich source of Ca ions So Ca2 ows down its concentration gradient out of the ER into the cytoplasm This elevates the concentration of Ca ions in the cytoplasm which is the signal required to cause the cortical 46 granules to fuse with the plasma membrane You should re ect upon the number of similarities between this second messenger system and that used by neurons to secrete neurotransmitters As shown in your web reading this Ca signal is propagated as wave from the point of sperm penetration through the entire egg The wave of Ca2 creates a wave of cortical granule exocytosis that results in the entire egg being surrounded by a physical block to polyspermy However the wave of Ca2 is not caused by a wave of 1P3 Instead there is a recently discovered phenomenon called Calcium Induced Calcium Release CICR which is responsible for the wave of Ca The IP3 created by phospholipase C causes just enough Ca2 to be released from the ER to trigger CICR from adjacent Ca2 channels in the ER This is analogous to the way an action potential is propagated in a neuron but this time it is Ca2 instead of Na ions Exactly how this CICR works is an area of intense research But what is clear is that in this system Ca2 has two functions 1 to allow cortical granules to fuse and 2 to spread information to adjacent areas that one sperm has entered the egg However as with all second messengers we need a way to turn off the signal When calcium levels reach a certain level usually 1 10 seconds later calcium ions cause the 1P3 gated channels to close Therefore the same ions that are used to open the channel also act to close it The only difference is the concentration of ions It seems likely that an additional allosteric site exists that has a lower affinity for calcium and this site is used to close the ion channel In summary the sperm binds to its receptor this initiates a chain reaction of enzymes each of which can amplify the original single event which results in the formation of the second messenger of IP IP3 binds to its receptor causing it to open the Ca2 channel so that Ca2 oods into the cytoplasm Ca2 acts the third messenger causing the cortical granules to dump their contents between the plasma membrane and the vitelline envelope causing the vitelline envelope to rise up and create a physical block to additional sperm entering the egg NEWS ITEM Many researchers have tried to determine the identity of the ZP3 receptor and the results are inconclusive So far three proteins bind to ZP3 but it is not clear if all three are necessary Rest assured many people are trying to figure this out since there could be an effective birth control application once we know the complete story See Developmental Biology 53911 Edition by Scott Gilbert Fertilin sperm plasma membrane protein is required for the sperm and egg to adhere A group in Research Triangle Park NC collaborating with one at UC Davis has identified that this protein is necessary for sperm binding AND for sperm motility Without fertilin B the sperm never makes it out of the uterus to the oviduct where the egg is How these two functions are related is not known but it looks like another avenue for reproductive scientists CCho et al 1998 Science vol 281 p 185759 A group of collaborating scientists Massachusetts California France and Mexico has added to the ZP3 story Their work demonstrates that ZP3 is involved in calcium regulation more than one time When ZP3 signaling is initiated it triggers a very quick and transient opening of Ca2 channels If ZP3 signaling continues the pathway activates a sustained Ca2 in ux mechanism and this sustained increase in Ca2 drives the acrosome reaction O39Toole CMB et al 2000 MBC vol 11 p 157184 Study Questions 1 At which steps can the signal cascade be amplified and how does this amplification work 2 Explain to a high school student the molecular events of the slow block to polyspermy 3 Why does an egg need the second and slower block to polyspermy 47 4 Explain how the egg uses a third messenger signal of Ca2 twice 5 In some of your focused reading the text discusses how DAG is used as a second messenger EX 6 Compare and contrast 1 a cardiac muscle s response to epinephrine 2 depolarization leading to a neuron s secretion of neurotransmitters and 3 an egg s response to fertilization 7 List the similarities between a neuron communicating with a muscle and an egg trying to block polyspermy 8 Explain how calcium is used to both open and close the IP3 receptor 9 How does cytoplasmic calcium return to resting levels 10 How can the fertilization signal be deactivated Q NEWS ITEM The molecular events in excitationcontraction of cardiac muscles have been visualized for normal and dysfunctional hearts The key difference seems to be in the degree of CICR in the two situations Science Vol 276 755 May 1997 Many cells use the inositol triphosphate second messenger system Here are a few examples The secretion of a digestive enzymes for carbohydrates by the pancreas pancreatic amylase in response to nervous system simulation The contraction of smooth muscle involuntary muscle in internal organs and blood vessels triggered by acetylcholine The secretion of insulin by pancreas in response to elevated plasma glucose levels The secretion of histamine by mast cells when you have a cold or an allergy The secretion of blood clotting factors by platelets when you are bleeding The response of the immune system to bacterial invasions OTHER COMMUNICATION SYSTEMS It could be true that the majority of cells communicate through the 4 systems you have looked at here the cAMP second messenger system the inositol triphosphate second messenger system membrane voltage changes including action potentials and various method of producing elevated cytoplasmic Ca2 concentrations However we have only scratched the surface in our knowledge of the cell and how it communicates so we will probably discover many additional ways that cells talk to one another Here is a brief summary of some of the other systems of intracellular communication we now know something about The cyclic GMP second messenger system Some cells use a second messenger system very much like the cAMP system where cyclic GMP is used instead of cAMP cGMP is created by the enzyme guanylyl cyclase analogous to adenylyl cyclase which is activated by a G protein system Probably 48 the most well investigated cGMP system is found in the photoreceptors the rod cells in the retina In the dark cGMP is bound to Na channels in the cell membrane keeping them open When light strikes a rod cell cGMP phosphodiesterase is activated thus degrading cGMP to GMP GMP disassociates from the Na channel thus causing it to close The opening and closing of this ligand gated Nal channel causes voltage changes in the rod cell plasma membrane which are propagated toward the brain thus allowing one to sense light NEWS ITEM Viagra the 39wonder drug39 for those suffering from erectile dysfunction is actually a phosphodiesterase inhibitor it inhibits PDES Similar to the CAMP phosphodiesterase we learned about earlier PDE 539s function is to breakdown cGMP into GMP With Viagra around cGMP levels remain high and promote erection Some notsowell known Viagra facts are I it was originally developed to combat angina 2 it is NOT an aphrodisiac and 3 it can cause distorted color vision Why the vision change Not because the users see the world through rosecolored glassesbecause the eye uses cGMP as a 2quotd messenger and Viagra also binds to PDE6 the phosphodiesterase found in the retina Stretch activated ion channels More properly called mechanosensation the transformation of a physical stimulus to an electrochemical signal is mediated by stretchactivated ion channels These ion channels are responsible for our ability to hear feel and maintain our balance These same mechanosensors enable our cells to be aware of their volume These ion channels have been cloned recently and will provide a great deal of understanding to this relatively unexplored area of sensations See Corey and GarciaA overos Science Vol 273 323324 19 July 1996 Gap Junctions Some cells communicate with one another directly without the use of a chemical messenger These cells are actually coupled to one another through proteins in their membranes called gap junctions Gap junctions are like giant ion channels that allow small cytoplasmic molecules to pass directly from the cytoplasm of one cell to the cytoplasm of the adjoining cell Heart muscle cells communicate this way thus allowing the heart to contract as a unit Many other cells communicate in this fashion as well Catalytic Receptors Some receptors are enzymes themselves and are therefore called catalytic receptors An example of such a receptor is the receptor for insulin on muscle and fat tissue When insulin binds to this receptor it changes shape sound familiar and this change in shape increases the enzymatic activity of the cytoplasmic tail of the receptor The receptor then autophosphorylates that is it adds a phosphate to itself Because this part of the molecule phosphorylates it is called a kinase And because it adds the phosphate at a tyrosine residue in its own structure tyrosine is an amino acid it is said to be a tyrosine kinase an enzyme than phosphorylates tyrosine When insulin binds to its receptor it causes a number of changes in the cells that include stimulating the transport of glucose into the cell and stimulating glycogenesis and the synthesis of triacylglycerol Interestingly several genes associated with the development of cancer called oncogenes have been shown to encode defective tyrosine kinase receptors We will cover this in detail in Unit IV For instance the normal receptor allowing response of epidermal cells skin cells to the chemical messenger epidermal growth factor is a receptor with tyrosine kinase activity The cancerous version of these proteins lacks the extracellular binding site for epidermal growth factor but still has the tyrosine kinase part on its cytoplasmic tail Without the binding site the tyrosine kinase is always on thus stimulating too much cell division Eicosinoids These signaling molecules come in three varieties prostaglandins leukotrienes and thromboxanes Prostaglandins mediate pain and in ammation aspirin works by inhibiting the 49 enzyme that produces prostaglandins Leukotrienes mediate some of the immune aspects of in ammation And thromboxanes facilitate blood clotting Note that the eicosinoids are all involved in responses to injury These molecules are actually derivatives of the fatty acid arachidonic acid This makes them very unusual They are produced by the cell membrane of injured or oxygen starved cells and they mediate the in ammation swelling pain and blood clotting associated with 1njury Steroid Hormones The hormones neurotransmitters and sperm cell proteins we have looked at in this unit are all hydrophilic they therefore cannot cross the hydrophobic cell membrane and must remain on the outside of the cell However steroid hormones testosterone estrogen progesterone cortisol and aldosterone are lipids Therefore they are freely soluble in the cell membrane and they cross into the cell Their receptors are oating in the cytoplasm or the nucleus The steroid hormones bind to their receptor which then changes shape The hormonereceptor complex then binds directly to control regions of genes in the 39 and causes these genes to be expressed or stop being expressed These hormones tend to be slow acting and produce longterm changes NEWS ITEM A new family of about 100 genes has been discovered that function as human pheromone receptors These receptors reside in the part of your nose that youmight not know about called the vomeronasal organ This organ is responsible for the perception of odors that we are not conscious of such as pheremones Pheromones are usually fatty acids or steroids and their receptors appear to span the membrane 7 times and are linked to Gproteins see Science Vol 278 79 October 1997 Fatty Acid Based Signal Molecules It turns out that plants are not as helpless as we vertebrates think When corn is attacked by beet armyworm caterpillars the injured plants release a mixture of chemicals called terpenoids which are fatty acids same family as the long tails of phospholipids and DAG These terpenoids are released into the air and attract a parasitic wasp which kills the armyworm caterpillars Terpenoids also stimulate certain genes in the plants to fix the wound created by the caterpillars much the same way we produce scabs to seal wounds from possible infections See summary in Science Vol 276 912 May 1997 Nitric Oxide The cellular and molecular biology community is currently all abuzz about this newly discovered second messenger signaling system Nitric oxide is a g not the same one the dentist gives you that s nitrous oxide This small molecule which lasts only milliseconds inside a cell nonetheless acts as a second messenger and triggers many interesting changes A report in 1996 revealed that the levels of NO plays a role in the degree of symptoms when a person is infected with malaria NEWS ITEM The binding of oxygen to hemoglobin promotes the binding allosteric modulation of nitric oxide to a particular amino acid on the beta chain of hemoglobin When oxygen is released from the hemoglobin molecule the modulated hemoglobin changes shape In this modulated but deoxygenated state hemoglobin can cause blood vessels to become larger in diameter which results in increased blood ow Therefore NO increases the function of hemoglobin from simply a carrier of oxygen to a modulator of blood ow so that areas of low oxygen will receive more blood Science Vol 276 2034 June 1997 Study Questions 1 In general how do cells communicate In answering this veg big question in a manageable way you cannot include very many details although you might want to include a few examples Rather think about what central points you want to make Think about this answer on many levels Explain it to me explain it to a classmate in this class 50 explain it to a Davidson student who has taken no biology since high school explain it to your parents and explain it to a child Use good analogies when appropriate One of the basic tenets in cellular communication is that different cells respond in different ways to the same chemical signal Using systems you have studied in this unit give an example illustrating this point Second messenger systems have been studied extensively by cancer researchers because cancer cells ignore normal messages that tell them to stop dividing Genes associated with the development of cancer are called oncogenes quotoncoquot means cancer as in oncology One set of such genes called the ms genes because they were discovered in a cancer called a at sarcoma code for the production of an abnormal G protein The G protein has a slightly different amino acid sequence than the normal G protein As a result it cannot catalyze the cleavage of GTP to GDP by the G protein Based on what you know about G proteins explain how this abnormal G protein might produce uncontrolled growth in a cancer cell Over 70 different cellular protein kinases have been isolated and identified What do all these kinases have in common Choose three different protein kinases presented in this unit and compare and contrast their functions What turns each of them on What do each of them do In what ways are these processes similar In what ways are they different Myasthenia gravis is a disease which produces a progressive weakening of skeletal muscles and ultimate paralysis It is an autoimmune disease caused by the development of antibodies to the acetylcholine receptor These antibodies bind to the receptor in such a way that they do not activate it but they block the binding site for acetylcholine By the way this is the same mechanism that the drug curare uses to produce paralysis Explain in molecular and cellular terms how this disease causes paralysis What type of paralysis would result from this illness flaccid no contraction possible or rigid muscles permanently contracted Certain types of quotnerve gasquot and pesticides act by blocking the action of acetylcholinesterase in the synaptic clefts and neuromuscular junctions These agents produce paralysis Explain in molecular and cellular terms how these agents produce paralysis What type of paralysis would result from exposure to these agents accid or rigid Explain One of the most deadly poisons known is a toxin produced by the bacterium C lostridz39um botulinum the organism that causes botulism This toxin blocks the release of acetylcholine from nerve endings How do you think this toxin kills you Describe some of the symptoms you think would be produced by this toxin and explain how the blockage of acetylcholine secretion would produce such symptoms Summarize the role played by the cytoskeletal components in the systems you have studied so far 9 While intercellular signaling systems differ in their details they are all based on some common functions that are fundamentally important in all signaling systems What do you think are the three or four phenomena that occur most consistently in cellular signaling systems and upon which cellular signaling is based SOME EXPERIMENTAL APPROACHES The approaches and methods used to investigate the biology of cells and their communication processes are numerous and most are beyond the scope of this course However as a starting point I have chosen a few basic methods upon which many others are based Microscopy The Direct Approach Thanks to the Dutch lens grinders of the 17th century we can see prokaryotic and eukaryotic cells simply by looking through a microscope Because most animal cells are clear as are most of the parts of plant cells only the chloroplasts and chromoplasts are colored cells usually need a little help in order to be seen through the microscope Without this help they would be like small panes of glass present but transparent Several methods are available The simplest is staining the cell to make it colored Other methods allow the microscope to distinguish differences in structures due to their different abilities to defract light For example in phase contrast microscopy we ll see this in lab some structures will appear dark while others will appear light due to differences in defracted light Finally dyes that uoresce when excited by light can be used to label organelles and molecular components of cells These dyes are observed with a uorescence microscope See page 72 and 73 for examples Even with the most expensive optics available the light microscope can only magnify about 1500 times This is enough to allow one to see cells but not enough to allow a clear view of most organelles and cellular inclusions For that you need a source of electromagnetic radiation that has a much shorter wavelength than light In the 1950s engineers perfected the electron microscope which uses electrons instead of light to produce images This method is described on page 71 of your text The transmission electron microscope allows the clear definition of cellular organelles and inclusions such as cortical granules microfilaments etc Viruses can also be seen with this type of microscope Using special methods very large macromolecules can also be visualized e g transport proteins in the cell membrane Some examples are found on pages 60 and 61 For fun ok stop laughing check out the Purves6e Ch4 Activity 43 Know your techniques 15 shows sickled blood cellsiwe ll be discussing the genetic mutation that causes this shape change in the next unit Isolating Living Cells for Experimentation Cell Culture Most plant and animal cells can be kept alive for some time outside the host if they are maintained in conditions that mimic those of the body fluids Cells are placed in culture medium which is a uid designed to provide all the nutrients salts vitamins etc that the host normally provides in the right concentrations and at the right pH Ifyou can get cells to live in cell culture you can do some pretty fancy experiments on them For example if you put muscle cells in culture medium that contains high levels of Ca2 nothing will happen because the living muscle cell can pump Ca2 out of its cytoplasm as fast as it enters However if you then add a Ca ionophore to the medium an ionophore will insert itself into the cell membrane and create an artificial ion channel 52 which cannot be closed the cell will contract This indicates that high levels of intracellular Ca2 trigger muscle contraction By this approach you could determine the concentration of Ca2 necessary to elicit contraction If you wanted to see that the concentration of an ion had actually changed inside a cell you might use an ion sensitive dye that will glow in the dark when it selectively binds to its ion Focused Reading p 206 g 119 research method p313 g 172 p 312313 Gel electrophoresis stop at Recombinant WWW Reading Gel Electrophoresis Methodology Isolation of Organelles Cellular Inclusions and Other Cell Parts Sometimes it is bene cial to isolate part of a cell for study For instance if you are interested in a protein found only in the plasma membrane it may be helpful to isolate the plasma membrane from the rest of the cell Or if you are interested in ribosomes you may wish to isolate them from the rest of the cell All these cell parts are called subcellular fractions and they can be isolated using a method called cell fractionation using a centrifuge or an ultracentrifuge A centrifuge spins samples like a washing machine or the achine they use to train astronauts The potential separation power of spinning is seen in Figure 119 p206 Density gradient centrifugation is used to separate pieces of DNA that have nucleotides that vary slightly in weight Isolation of Proteins bv 39 39 Sieves Quite frequently it is necessary to isolate a single protein from a cell One method that is commonly used to do this is gel electrophoresis In this method a bunch of cells are homogenized to release all proteins The cellular proteins are then usually dissolved in a detergent that covers them with negative charge When these proteins are put in a gel like a slab of Jello and a voltage is placed across the gel one end of the gel is made negative the cathode and the other end is made positive the anode the negatively charged proteins move toward the anode Just like people in a thick forest the smaller they are the quicker they can move through the obstacle course of the gel to get to the anode Thus the smaller proteins move faster than the larger proteins and the proteins of the cell separate by size or molecular weight This method applied to DNA is described on page 13 of your text and is very similar when applied to proteins Ifyou want to study a protein further after it has been isolated gel electrophoresis is not such a good method because detergent is very harsh on proteins and frequently destroys their native conformation during the separation process A better method is one form of chromatography in which proteins are poured over a matrix in a glass tube the tube length can range from 2 inches to 5 feet and the diameter from 025 inches to 3 feet The proteins are not treated to cover them with negative charge as in electrophoresis so they retain their native conformations The proteins enter the matrix and this time the larger proteins get through the matrix rst while the matrix retards the movement of smaller proteins so they come out last This is because the matrix is made of small beads that contain tiny holes or channels which the small proteins are small enough to enter but the large proteins are too big to t into The small proteins spend a lot of time wandering around in these channels and it takes them a long time to get through the entire matrix The large proteins cannot get into the channels so they continue through the tube on the outside of the beads in the 53 space between the matrix particles By taking this alternative route they get to the bottom of the tube rapidly Thus the proteins are separated by size and maintain their native conformations and therefore can be used for further study This type of chromatography can be diagrammed as follows Big Protein Little Protein Proteins In YYYYYVYYY Column Matrix I Glass Tube Proteins Out 1st group of 2nd rou of proteins out g p proteins out qTOP Focused Reading p 3634 quot Antibodies share quot stop at quotT cells quot Fig 1913 WWW Reading Cartoon of Immuno uorescence Method Identification of Proteins wit quot39 139 Abs Since proteins do most of the meaningful work of living creatures it is extremely important to biologists to be able to isolate and identify individual proteins This can be done in a number of ways One commonly used method involves the use of antibodies that bind to proteins with great specificity When a foreign protein is injected into an animal e g rabbit albumin into a mouse or goat insulin into a rabbit the animal39s immune system recognizes this foreign protein and interprets 54 it as a microbial invader The immune system recognizes foreign molecular shapes whether they are harmful or not Thus you can get allergic reactions an immune response to pollen even though pollen can t harm you This immune response to the foreign molecule produces antibodies which are proteins which bind speci cally to the foreign protein called an antigen Antibodies have active sites like enzymes and the antigen is the ligand that binds there The production of antibodies for research can be diagrammed as follows L Mouse Epinephrine Rabbit Epinephrine Receptor Receptor The epinephrine receptors from these two species are slightly different in structure Thus mouse epinephrine will be seen as foreign by a rabbit and an antibody will be produced Mouse Epinephrine Receptor l Rabbit Wait several weeks and then bleed the rabbit Rabbit serum contains antibodies to mouse epinephrine receptor Rabbit antibody against mouse epinephrine receptor Will bind speci cally to this molecule and hopefully to no other Mouse epinephrine mOIecu39es rece pto r So you can raise these speci c antibodies against a protein you might be interested in studying and use the antibody as a probe for that protein since it will bind speci cally to that protein and no other You can probe for proteins in situ which means that they are still in their normal location within the intact cell The identi cation of proteins in situ using antibodies is called immunocytochemistry or immunohistochemistry You can also remove the proteins from the cell separate them by electrophoresis see above and then apply the antibody probe for the protein This method is called an immunoblot or a western blot in the vernacular Identi cation of Speci c Proteins Through the Use of Radiolabelled Ligands This method uses radioactivity to identify specific proteins While there are many variations on this method the basic idea is this You buy or synthesize a ligand that contains a radioactive element For instance if you wanted to study the acetylcholine receptor you would obtain radiolabelled acetylcholine This acetylcholine could contain radioactive hydrogens called tritium or radioactive carbon C or an additional radioactive element such as iodine 125I could be added These radioactive elements are isotopes of the nonradioactive elements Isotopes are described on page 19 of your text These radioligands in this case radiolabelled acetylcholine can be bound to various kinds of cells to determine whether they bear the ligands receptor For instance if you wanted to know if liver cells have acetylcholine receptors in their membranes you would incubate radiolabelled acetylcholine with liver cells Ifthey bind the ligand ie if the cells become radioactive then you can assume if your experiment is properly controlled that they are radioactive because they bound the ligand This should mean that they have acetylcholine receptors If they don t become radioactive then they don t have acetylcholine receptors You can also use this procedure to determine the concentration or density of a receptor in a membrane Therefore you can use this method to see if receptor densities change over time as you subject the cell to various treatments This method was used by Drs Candice Pert and Sol Snyder in order to identify the receptors in the brain that bind and respond to opiates such as heroine Through the use of this method and others we now know that we make internal or endogenous opiates called endorphins which reduce pain and may have other bene cial effects Molecular Models and Computer Graphics One of the most exciting new methods in biology is the ability to build fairly accurate complex 3 dimensional models of proteins based on computer analysis of data obtained by x ray crystallography This method is described on page 247 in your textbook Because it is dif cult to crystallize many important molecules their 3D structure at the atomic level in their native conformation remains illusive However if we learn enough about how amino acid sequence translates into 3D structure we may be able to predict or teach a computer how to predict the 3D structure of a protein from its primary amino acid sequence Because the amino acid sequence of proteins is becoming much easier to obtain through the remarkable progress being made in molecular biology this would be atremendously important breakthrough and would give us new worlds of information about how living things function Focused Reading p236 induced mutations stop at Mutations are p218 quotOnegene onepolypeptide hypothesis stop at DNA RNA 56 p 263 Fig 121 WWW Reading Movie of Microinjection Use of Genetic Mutants Because mutations are changes in the DNA that can alter the activity of one protein they can be used to identify the protein responsible for a speci c function For instance scientists have used genetic mutants to study the process of membrane traf c in the cell Using mutant yeast investigators have identi ed several mutant strains that each have one important protein altered For instance let39s say Mutant strain 1 is missing Protein 1 Investigators nd that this mutant strain cannot transport protein from the ER to the Golgi Mutant 2 is missing Protein 2 This mutant strain cannot transport protein from the Golgi to the secretory vesicle Thus by identifying the protein that is missing and correlating it with the functional de cit in the cell investigators can determine the proteins that are responsible for each step in a biological process We will use genetic mutants to screen compounds to see if they are mutagens We will perform this experiment called the Ames test later in the semester Microinjection There is a very dif cult and labor intensive method to place a molecule of interest inside a particular cell and this method is called microinjection As the name implies you take a very small needle usually made of a glass tube that has been pulled to a very ne point attach the needle to a syringe and inject a cell with a very small volume of solution which contains your favorite molecule Typical molecules that are injected include DNA RNA antibodies and proteins that have been puri ed from other cell types Study Questions 1 Be able to describe each of the techniques outlined above 2 Ifyou had all of these methods available to you in the lab how would you go about answering the following questions Note Just because a method is available does not mean it is the best approach to the problem In each case choose the method or methods that you think provide the most ef cient route to an interesting and substantive answer A Do plant cells use cAMP second messenger systems B Is Ca2 involved as an intracellular messenger in the secretion of saliva from the salivary glands C The micro laments actin and myosin in vertebrate muscle cells are aligned in organized units which produce contraction as described by the sliding lament theory outlined above Are the micro laments of the muscle cells of insects aligned in the same manner D Some forms of breast cancer are stimulated by estrogen a female sex hormone Do these breast cancer cells have a higher concentration of estrogen receptors than normal breast cells tr Plant cells secrete the cell wall Is the secretion of the cell wall constitutive or regulated 57 F Q P What proteins mediate each of the steps that lead from ligand binding to cell division in fat cells Plants and animals both use the inositol triphosphate second messenger system which requires the use of phospholipase C Is the phospholipase C used by plant cells similar in molecular weight and threedimensional structure to the phospholipase C used by animal cells Does the Ca pump inthe SER membrane have the same molecular weight as the Ca2 pump in the plasma membrane qTOP Unit II Genetics Brief Overview Reading Chapters 3 4 912 14 Note you have reviewed much of this already The earth is teeming with living things We can easily see some of the larger organismsitrees grass owers weeds cats sh squirrels dogs insects spiders snails mushrooms lichens Other organisms are everywhere in the air in water soil and on our skin but are too small to see with the naked eyeibacteria viruses protists single celled eukaryotes such as amoebae and tiny plants and animals Life is remarkable in its complexity and diversity and yet it all boils down to a very simple ideaithe instructions for making all this life are written in nucleic acids usually DNA Most organisms have a set of DNA that contains the instructions for making that creature This DNA contains four letters in which these instructions are writteniA T G and C The only difference between the code for a dog and the code for a geranium is in the order of those letters in the code If you took the DNA from a human and rearranged the letters in the right way you could produce an oak treeiarrange them slightly differently and you would have a bumble beeiarrange them again and you would have the instructions for making a bacterium Acting through more than two billion years the process of evolution has taken one basic ideaia molecular code that uses four lettersi and used it over and over in millions of combinations to produce a dazzling array of life forms As far as we know we are the only creatures on the planet that have gured this out The members of our species who get the credit for this discovery are James Watson and Francis Crick although many others helped including Maurice Wilkins and Rosalind Franklin Some believe Franklin was denied the Nobel Prize because of her gender but careful review of the facts will show that she was deceased at the time of the award and the prize is not given posthumously Watson and Crick determined the 3D structure of DNA in 1952 and showed all of the human world and any other species that could understand that all of life is deeply united at the molecular leveliindeed we are all rearranged versions of one another The eld of genetics is the study of how four bases make from aspen trees to zebras Molecular geneticists study how the code is put together how the code is translated into an actual living creature and how the code is passed down from one generation to the next dogs beget dogs oak trees beget oak trees and sh beget sh although the offspring can be slightly different from the parents and from one another In this Genetics Unit we will look at the progress that has been made by researchers in understanding three inherited genetic diseases Cystic Fibrosis Sickle Cell Disease and Huntington s Disease At the end of the Unit we will also discuss some sex linked genetic disorders Many of the diseases that afflict humans have a genetic origin Some diseases are caused exclusively by genetic defects These include cystic brosis Huntington s disease phenylketonuria PKU Down s syndrome Tay Sache s disease sickle cell disease muscular dystrophy and hemophilia A In other cases such as cancer one can inherit a genetic predisposition to a disease but environmental factors also play a major role Most disease conditions are probably in this category which certainly includes diabetes hypertension high blood pressure and most forms of cancer Focused Reading p 3323 hemoglobin stop at receptors and transport p 3334 Receptors and transport proteins stop at Structural proteins WWW reading For J Jquot 39 info when vou have extra time Cystic Fibrosis Web Site Sickle Cell Disease Web Site 50 Huntington s Disease Web Site The three diseases we will investigate in this Unit cystic brosis CF sickle cell disease SC and Huntington s disease HD are caused exclusively by genetic defects CF is the most common genetic disease in Americans of European descent occurring in 1 out of every 2500 births CF occurs with a frequency of l in 17000 African Americans and with less frequency in other races In the US 1000 new cases are diagnosed each year with 30000 CF patients alive in 1996 Victims of cystic fibrosis accumulate thick mucus in the lungs and pancreas produce elevated levels of very salty sweat and frequently develop cirrhosis of the liver Digestion is disrupted in CF patients since pancreatic enzymes cannot reach the intestines The mucus in the lungs makes breathing difficult and exhausting This mucus is also attractive to microorganisms and therefore pneumonia is a constant threat in this disease respiratory infections are the actual cause of death not the thick mucus Untreated children usually die by the age of 4 or 5 and the average life expectancy with medical care is 40 years SC is the most common genetic disease among African Americans af icting l in 400 while 1 in 10 are carriers of the genetic trait Most carriers are unaffected but some suffer from a mild form of the disorder more about this later Red blood cells are biconcave in shape shaped like tiny doughnuts with a membrane across the hole in nonaffected individuals but in this disease they take on the shape ofa crescent moon or sickle which causes several problems see fig 1217 p 235 The sickleshaped cells tend to circulate more sluggishly in the body and clot as they pass through the tiny blood vessels of the tissues thus leading to tissue death andor strokes They are also destroyed more rapidly than normal red blood cells which causes the symptoms of anemiaiextreme fatigue especially upon exertion HD is a fatal neurological disorder that causes severe mental and physical deterioration uncontrollable muscle spasms personality changes and ultimately insanity Perhaps the most troubling feature of this disorder is that the symptoms usually do not begin to appear until after the age of 40 after an individual has already had his or her children Thus until recently people with this disease in their families have had to reproduce without knowing whether they have the disease and run the risk of transmitting it to their offspring The search for the causes and cures of these and other genetic disorders has been the goal of researchers for over 30 years The recent revolution in genetics and molecular biology has dramatically improved our understanding of genetic diseases and greatly enhanced our ability to manipulate genetic systems to produce diagnostic tools and therapies In order to understand how these traits are pass on from one generation to the next we need to understand the process of cell division in somatic cells nonsex cells and gametes sex cells Focused Reading p 199 DNA stop at The genetic material p 157 Eukaryotic cells divide stop at Interphase and the control p 1605 Mitosis stop at Reproduction by MEIOSIS Fig 96 amp 98 WWW Reading Purves6e Ch9 Tutorial 91 Mitosis Cartoon of Mitosis Movie of Mitosis There is one rule that must not be broken for any cell to survive and function properly a cell must maintain the right number of chromosomes at all times This presents a problem for the average cell that is ready to divide Lets say the cell has 23 m it is diploid of chromosomes and it wants to make two new cells The first problem is how can a cell go from 1 X 46 to 2 X 46 chromosomes 51 The obvious answer is that the cell must make 46 more chromosomes before it can divide In its simplest form that is all there is to mitosis duplicate the DNA then divide Of course any process as important and complicated as mitosis must progress in an orderly and stepwise fashion The individual steps of mitosis are outlined in gure 98 You should be familiar with the major steps of mitosis which should not include the cell cycle phase called interphase 1 prophase 2 metaphase 3 anaphase and 4 telophase all 4 phases are reviewed in text p 160165 Two points to note 1 the text includes a 5Lh phase called prometaphase and 2 mitosis does not include cytokinesis but the two are closely associated Now that you have a handle on mitosis we need to see what gametes do when they are formed You know that to form a new individual by sexual reproduction two gametes fuse to form a zygote Since each gamete brings a set of chromosomes to syngamy or fusing of gametes we are faced with a mathematical dilemma How can two cells contribute complete sets of chromosomes to a zygote without violating the cardinal rule of maintaining the proper chromosomal number The answer is in me10s1s Focused Reading p 735 Fig 424 review p 204211 Meiosis stop at Meiotic errors p 1657 Reproduction by meiosis stop at Meiosis Figs 914 amp 912 WWW Reading Purves6e Ch9 Tutorial 91 Meiosis Movie of Meiosis As you read meiosis started off like mitosis with a diploid cell that replicates its chromosomes but instead of a single round of nuclear division there were two rounds of nuclear division This results in haploid cells that have only one copy of each chromosome e g human egg and sperm have 23 chromosomes each Therefore when the two gametes combine their share of chromosomes the zygote is back up to the proper 46 in humans diploid or 2n 2 copies of each chromosome number of chromosomes The important steps of meiosis are again well defined in the focused reading and you should become familiar with them But notice one other very important difference between mitosis and meiosis chromosomes are not solid structures that cannot be modified but they can in fact switch parts with one another in a process referred to as crossing over figure 916 This adds to the variation derived from independent assortment and provides a new source of individuality of each gamete and ultimately the zygote and us Study Questions 1 Be able to outline the major steps in mitosis and meiosis Describe the major steps as if your your younger sibling has asked you why your eyes are the same color as your father s and the inquisitive teen wants a really detailed answer 2 Describe the significance of meiosis in relation to creating variation in the next generation Dr AlfredD Hershey as in the Hershey and Chase experiment died at age 88 on May 22 1997 He shared the 1969 Nobel Prize in physiology or medicine with two other researchers Max Delbruck and Salvador Luria Now we know how cells inherit their DNA from the mother cell and how haploid gametes are formed In the last Unit we saw how a sperm cell tells an egg it has been fertilized Now we need to move on to the genetics the pattern of inheritance Genetics is a very logical discipline but the power 52 to genetics is numbers The more progeny available for study the easier it is to discern the pattern of inheritance Unfortunately genetic experiments with humans are not ethical or practical since the generation times are so long Given this inherent difficulty it is amazing what has been learned about the genetics of human diseases Let s start by putting ourselves in the position of the first scientists who were interested in these diseases Certainly one of the first thing people noticed about CF SC and HD was that they run in families Now because families usually live together and share a common environment you cannot always conclude that something is genetic simply because it runs in families Rather you have to look closely at the inheritance pattern of the disease to see if it ts a classic genetic model of inheritance For instance coronary heart disease runs in families but it does not fit a classic genetic model of inheritance Therefore we hypothesize that environmental factors also play a role in the development of this disease In looking for a classic genetic inheritance pattern in humans the first thing you do is to research the disease occurrence in the family and draw a family pedigree In drawing a pedigree certain rules are followed 1 Squares are used for males 2 Circles represent females 3 Nonaffected individuals are blank while affected individuals are colored or patterned in some way 4 Lines between a circle and square indicate a mating union e g marriage and all offspring of a mating union are drawn on the same level 5 Individuals are numbered from top to bottom and from left to right Here is an exampleia pedigree for a family with cystic fibrosis In this family the woman 2 in the first generation grandma had cystic fibrosis and yet survived long enough to have two children Neither of her children a girl and a boy numbers 3 amp 5 had CF Child 5 and individual 6 produced offspring 10 a normal or wild type child Individual 3 and individual 4 had three children two of whom 7 and 8 have CF 53 Study Questions 1 Given information about a family be able to draw a family pedigree that complies with standard rules 1 Be able to interpret a pedigree drawn by standard rules 2 Draw apedigree for the cross that is outlined in figure 103 p179 qTOP What can we tell about the genetic inheritance of CF by looking at this pedigree Well in order to make sense of this pedigree you have to understand a bit about the alternative ways by which genes can be inherited To understand this we have to go back 130 years to the AustroHungarian Empire and a Catholic monastery Here a monk named Gregor Mendel was conducting breeding experiments with garden vegetables in an attempt to explain how genetic traits are inherited His conclusions stand today as the foundation upon which modern genetics is built Gregor Mendel defined the laws that govern the simple inheritance of traits and traits that are inherited in this straightforward manner are said to be Mendelian traits that obey the laws of Mendelian genetics Focused Reading p 17788 Mendel s work stop at Gene interactions Study Questions 1 Understand all the terms presented in bold face type in your reading assignment and be able to use them correctly in a description 2 Go back to the CF pedigree on the previous page In light of the concepts of Mendelian genetics and the information in this pedigree do you think that CF is a dominant recessive or incompletely dominant trait Explain 3 Label the generations in this CF pedigree using Mendelian terminology eg P F1 F2 4 What are the genotypes and phenotypes of each of the 10 people in the CF pedigree above Use proper Mendelian notation in assigning the genotypes In some cases you will know a person s genotype and in other cases you will have incomplete information Indicate this and be able to explain the rationale you used to assign the various genotypes 5 The mating of person 3 and 4 above represents the F1 of a monohybrid cross refer to figure 104 Draw a Punnett square for this cross Use proper Mendelian notation here Does the actual mating outcome two out of three children with CF match the predicted outcome from the Punnett square Why or why not Ifthey do not match explain why this is the case 6 Be able to solve genetics problems such as the following From Biology by Villee et 611 A In peas yellow seed color is dominant to green State the colors of the offspring of the following crosses l homozygous yellow x green 2 heterozygous yellow x green 3 heterozygous yellow x homozygous yellow 4 heterozygous yellow x heterozygous yellow 54 B Iftwo animals heterozygous at a single locus are mated and have 200 offspring about how many would be expected to have the phenotype of the dominant allele Two longwinged ies were mated The offspring included 77 ies with long wings and 24 with short wings Is the shortwinged condition dominant or recessive What are the genotypes of the parents A blueeyed man both of whose parents were browneyed married a browneyed woman whose father was blueeyed and whose mother was browneyed If eye color is inherited as a simple Mendelian trait it actually is not what are the genotypes of the individuals involved Outline a breeding procedure whereby a truebreeding strain of red cattle could be established from a m a blend of the incompletely dominant alleles for red and white bull and a white cow 0 U P1 Ifyou would like more practice try the questions at the end of the chapter p 1978 NEWS ITEM A collaboration between researchers at the Oregon State University and the University of Bristol in the UK have cloned the gene that encodes for the dwarf trait studied by Mendel The gene is the last enzyme in a pathway that produces the plant hormone gibberellin Without this hormone the plant does not grow as tall This is of more than historical interest Plants that do not grow as tall often produce more seeds or fruit and are less likely to break and fall over since their stems are shorter This is of considerable interest for genetic engineers that want to produce food crops that resist wind damage and produce more Prac Nat Acad Sci Vol 94 8907 1997 Focused Reading p 185188 quotAllelesquot stop at quotGene interactionsquot P 189 quotThe environmentquot stop at end of page When considering CF an individual either expresses the phenotype has two copies of the CF allele or does not express the phenotype has one copy of the mutant allele OR is homozygous wild type This is the phenotypic expression pattern expected when the wild type allele is dominant over the CF allele But now consider the pedigree for a family with members who have sickle cell disease next page Here we see individuals that have 39mild39 cases of the disease How can this be Doesn39t one allele win over the other Well no Some alleles show incomplete dominance In these cases a heterozygous individual shows traits that are 39half way between the homozygous possibilities In sickle cells disease both 39 r39 J 39 and I come into play Penetrance refers to the proportion of individuals that have a particular genotype that show the expected phenotype The predicted phenotype of the mild form of anemia is not always seen in heterozygotes so the mild form of the disease is said to be not fully penetrant Environmental factors can affect 39seeing the sickle cell phenotype Heterozygous individuals may appear unaffected by SC except when faced with conditions of low oxygen such as if they were to run a marathon or go to a vacation resort at a high altitude Here is a pedigree for a family with sickle cell disease l mildform ofSC 13 I severe form ofSC Study Questions All refer to pedigree on this page 1 Looking at the SC pedigree Explain how you can tell that SC is an incompletely dominant trait 2 Label the generations in this SC pedigree using Mendelian terminology 3 What are the genotypes and phenotypes for individuals 112 in the SC pedigree above Use proper Mendelian notation here In some cases you will know a person s genotype and in other cases you will have incomplete information Indicate this and be able to explain the rationale you used to assign the various genotypes 4 In Mendelian terms what type of cross does the union of 6 and 7 above represent eg Monohybrid cross test cross Draw a Punnett square for this cross 5 Individual 13 is still in the womb For each of the following outcomes of this pregnancy 1 indicate the genotypes of the parents 9 and 10 2 What are the odds of these three outcomes a 13 is homozygous wildtype b 13 is heterozygous c 13 is homozygous disease Here is a pedigree for a family with Huntington s disease 3 8 9 1 O 1 1 1 2 1 3 14 15 1 6 1 7 1 8 Study Questions 1 N Looking at this pedigree do you think that Huntington s disease HD is a dominant recessive 0r incompletely dominant trait Explain Label the generations in this HD pedigree using Mendelian terminology What are the genotypes and phenotypes of each of the 18 people in the HD pedigree above Individuals l4 and 15 are not yet old enough to determine whether or not they will get HD What is your prediction about their disease status What are their genotypes Explain Many times people with genetic diseases in their family seek the advice of genetic counselors in trying to determine the probability that they will produce an offspring with the disease Focused Reading p 339 Detecting human genetic variation stop at paragraph beginning Of the numerous p 167 Fig 913 karyotyping Study Questions 1 Individuals 3 and 4 from the CF pedigree are considering having another baby and come to you as a genetic counselor They want to know the chances that this baby would have CF What will you tell them CF pedigree is on p 53 What would you tell individuals 5 and 6 from the CF pedigree under the same circumstances Assume that a person picked from the population at random has a l in 50 chance of being a carrier of a mutant CF allele 3 What would you tell individuals 3 and 4 from the SC pedigree Individuals 9 and 10 SC pedigree is on p 55 Assume that a person picked from the population at random has a l in 100 chance of being a carrier of a mutant SC allele 4 What would you tell individuals 5 and 6 from the HD pedigree Individuals 8 and 9 V39 A couple is planning to have children and comes to you to help them determine the chances that their children will have SC Both parents have a very mild form of the disease What is the probability that their first child will have SC homozygous recessive What is the probability that their first child will be a carrier of SC g not have any SC alleles Iftheir first child has SC what are the chances that their second child will have SC Ifthis couple has three children what is the probability of all three children having full blown SC What is the probability that the first two children will have fullblown SC and the third one will be a carrier What is the probability that one will have SC one will be a heterozygote and one will be homozygous wild type What is the probability that all three of the three children will be homozygous wild type What is the probability that all 3 will be heterozygotes wgt F1190 F9 0 If couples from families with genetic disease decide to conceive and then want to know the genetic status of their fetus what diagnostic tests are now available to them Describe each test Answers to Questions 1 5 1 14 2 150 x14 1200 3 1100 x14 400 1100 x 12 1200 4 0 12 5 A 14 E 34 c 14 D 164 E 132 So far through pedigree analysis of the af icted families we know that cystic fibrosis is a recessive trait Huntington s disease is a dominant trait and sickle cell disease usually behaves as a recessive trait heterozygotes are asmptomatic have no symptoms but sometimes SC behaves as an incompletely dominant trait when the heterozygotes have a mild form of the disease What does all this actually mean at the molecular level What does it mean to have a dominant trait or a dominant allele How do alleles dominate one another In order to examine this question we have to know what genes actually do what they actually are As you know from the previous Unit your life is embodied in your structure mostly proteins and fat and your chemical reactions each one catalyzed by an enzyme which is a protein Your proteins control your life and your genes control your proteins The simplest de nition of a gene one that is outmoded but a good place to start is that a gene is a segment of DNA which encodes one protein This statement is called the one gene one polypeptide theory and it is still basically sound although we now know that the story is much more complicated than this statement suggests Genes encode proteins that is they contain the instructions that the cell can read in order to be able to make all the proteins it needs to live We know from Mendelian genetics that we inherit two alleles for each gene If we use the three genetic diseases we have introduced above as examples we can and investigators do begin speculating about the genes that might be involved In cystic brosis you have too much thick mucus in the lungs and pancreas There must be genes that encode proteins that prevent it from thickening These genes could be involved in the production of mucus the secretion of mucus the control of mucus production and secretion the movement of water into and out of the lungs and pancreas since mucus become thicker when water is removed etc In the rst part of this discussion I will refer to this gene and the mucus gene and its protein as the mucus protein even though this description doesn t explain the high salt concentration in sweat or the liver cirrhosis Nevertheless it gives us a common language with which to refer to the normal gene that when mutated causes cystic brosis Because CF is a recessive disease it is a good bet that the disease allele fails to encode a functional protein In the case of a recessive disease heterozygotes ca1riers do not have the disease because their one wildtype allele is enough to allow them to make all the functional protein they need The second allele is redundant But homozygotes for the disease have no wildtype alleles no wildtype proteins and they get the disease So in the case of a recessive disease we are usually looking for a gene that does not encode for a functional protein In the case of sickle cell disease the phenotype is sometimes incompletely dominantly expressed and sometimes expressed as a recessive trait However at the molecular level SC is always codominantly expressed This usually means as in the case of recessive genetic disease that the disease allele does not encode a functional protein However in the case of incompletely dominant expression the normal allele in a heterozygote cannot fully compensate for the loss of protein caused by the disease allele SC heterozygotes have some wildtype and some SC form hemoglobin in their red blood cells and thus experience some mild sickling in those cells While these cells are usually able to function properly and are destroyed at a normal rate sometimes under extreme conditions heavy aerobic exercise high altitudes they function poorly and produce mild symptoms of SC Thus in this case the trait is incompletely dominant In a heterozygote both wildtype and SC hemoglobin are made but the severity of symptoms in the heterozygote varies widely depending on environmental conditions Because the symptoms of Huntington s disease involve many brain centers a gene that has wide ranging effects on the function of the nervous system must cause the disease Because Huntington s is a dominant trait we would look for a gene that makes too much of its protein or makes a form of the protein that is hyperactive When the disease gene is present it causes its protein to be too active or in too high a concentration but remember that onset of the disease comes around age 40 Regardless of the presence of the normal allele the person has too much of an enzyme or structural protein In the delicately balanced living system having too much of something is frequently just as bad as not having enough Study Question 1 Explain how traits wind up being recessive incompletely dominant or dominant based on the type of defect produced at the level of the protein Give examples for each Do not use CF SC or HD as examples here Your examples need not be diseases They can be normal traits qTOP We need to stop and look at how wildtype genes produce wildtype proteins Genes don t exist as individual strands of DNA but rather they sit one after another in long complexes called chromosomes Chromosomes contain the DNA encoding enzymes as well as the proteins that are involved in packaging the chromosome so it ts into the nucleus and in the control of gene expression Gene expression is the term for the process where the genetic blueprint of DNA is actually converted into a functional protein Bacteria have one circular chromosome and eukaryotes have multiple linear chromosomes Each species has a certain number of chromosomes and humans have 46 However as you know each trait is encoded at a particular locus at which we inherit two alleles one from our mothers and one from our fathers Organisms that have two alleles for each locus or trait are said to be diploid Humans are diploid and therefore their 46 chromosomes actually come in 23 pairs 23 pairs of homologous chromosomes The diagram on the next page is an illustration of the organization of genes on three homologous pairs of chromosomes Note These loci are probably not on the correct chromosomesithis is simply an illustration You can see from the diagram that loci are always identical on 39 Loci are like file folders You have two file folders for a voltagegated K channel one on your maternal chromosome 1 and one on your paternal chromosome 1 The actual file instructions you store in this folder however can be quite different The maternal voltagegated K channel locus contains the instructions for producing a wildtype channel while the paternal voltagegated K channel locus contains the instructions for producing a nonfunctional channel Therefore this organism is heterozygous for the voltagegated K channel It has a heterozygous genotype at that locus The phenotype that results from the expression of these alleles will depend on whether the alleles are dominant recessive or codominant to one another I I I Maternal from the mother I Paternal from the father wildtype voltagegated mutant allele K Channel allele wildtype locus for hemoglobin SiCkle Ge allele allele wildtype locus for adenylate cyclase wildtype allele allele Chromosome wildtype wildtype allele locus for actIn allele Wildtype locus fors ntaXin Wildtype allele y allele Chromosome wildtype wildtype allele locus forthe NaK pump allele Chromosome A number of loci have been added to this genetic map for the sake of illustration and hopefully to alter some misconceptions We concentrate a lot in genetics on the loci that produce individual differences in height eye skin and hair color disease states etc However the vast majority of loci have only one allelic alternative in the species they are monomorphic For instance in humans there is only one allele for the IP3 receptor insulin a hormone collagen the brous component of bones tendons and ligaments keratin hair and nails acetylcholinesterase the enzyme that destroys acetylcholine in the synapse or neuromuscular junction etc etc etc The vast majority of human proteins are encoded by an allele that all humans share there is no variation from person to person Therefore we are almost totally homozygous Loci at which there are a number of alternatives are the exception and are called polymorphic loci and the traits encoded at these loci are called polymorphic traits Most traits are not polymorphic but many interesting ones are including all the features that make us different from one another 61 Study Questions 1 Describe the organization of genes along chromosomes and the concept of homology 2 What is a genetic locus An allele 3 What does it mean when a trait is polymorphic Give an example not given above of a polymorphic trait Give an example not given above of a monomorphic trait So to return to our tale a person with cystic brosis would have two defective genes at the locus that controls mucus production in the lungs and pancreas One defective mucus gene would be on the maternal chromosome the person inherited this chromosome from hisher mother and the other defective gene is on the paternal chromosome the person inherited this chromosome from hisher father A person with sickle cell disease would have two defective alleles at the locus controlling some aspects of the red blood cell s shape A person with Huntington s disease would have w defective allele at the locus controlling an important brain protein This allele could be on the maternal or paternal chromosome Note A person with HD could have two defective alleles but because the disease is so rare it is highly unlikely that two people with HD would mate a J 39 for J J 39 a1 yg 4 HD offspring What is defective about these genes What can a normal gene do that these disease genes can t do In order to address this important question we have to understand what genes do normally Somehow the instructions for making a protein have to be encoded in the DNA molecule in such a way that they can be translated into protein by the cell Overview reading p 205 Fig 117 Focused Reading p 2029 The structure of DNA stop at The mechanism of Figs 118 through 1110 p 209214 The mechanism of DNA replication stop at Practical applications p 2201 RNA differs from DNA stop at RNA viruses p 2235 The genetic code stop at Preparation for WWWeb Reading httpwww mass edn micr 39 39 chimednainde htm look at 3 and 5 DNA polymerase I and 2 Purves6e Chll Tutorial lll DNA Replication The DNA molecule is written in a code that has four letters The four nucleotides letters in DNA are guanine G adenine A thymine T and uracil U In general terms the nucleotides are also called bases In the DNA code three bases in a row equal a word known as a codon and each codon encodes a single amino acid Following this through it is the base sequence of DNA that determines the amino acid sequence of the protein Because amino acid sequence determines native conformation and native conformation determines function the 39 quot1 sequence controls all living processes and structures Study Questions 1 In a basic outline form describe andor draw the structure of DNA What chemical groups does DNA contain and how are they a1ranged in the molecule 62 2 Many times DNA and RNA are described as having a 3 and 5 end Explain what this means in terms of the structure of the molecules 3 How is DNA transcribed into RNA Where in the cell does this process occur 4 Be sure you understand how to interpret the genetic code in Figure 125 p 224 Given the base sequence of DNA or mRNA be able to give the amino acid sequence of the resulting protein 5 What proteins are involved in DNA synthesis and what are their roles during this process At this point you should be able to come up with one hypothesis about what is wrong with the CF SC and HD genes Their nucleotide sequences may be incorrect ie contain some typos Changes in the nucleotide sequence of DNA are called mutations A number of different mutations could be interfering with the function of these genes Focused Reading p 2337 Point mutations stop at Some chemicals You can see by studying the genetic code on page 224 that mutations in the third base of the codon frequently produce no change at all in the amino acid encoded by that codon For instance if the mRNA codon CCU were changed to CCC or CCA or CCG it would still encode the amino acid proline Thus some point mutations have no impact at all on protein structure and function However some point mutations can make a very big difference in the function of proteins By substituting one base for another in the DNA you can change the amino acid at that position in the resulting protein Look at the genetic code on page 224 and see which mutations would make such a difference For instance the code for serine Ser is UCG there are actually 6 codons for Ser while the code for tryptophan Trp is UGG By changing C to G you can change the amino acid at that position in the protein Now look on page 37 at the R groups of the amino acids Serine s R group contains an OH group which means it is m Tryptophan has a large hydrophobic and po R group These two amino acids would behave differently in water and thus this mutation would cause a slight alteration in the 3dimensional shape of the protein Depending on the exact location of this mutation the protein may or may not be significantly altered in its shape Go back to p 224 The code for aspartate Asp is GAU while the code for glutamate Glu is GAA IfU were changed to A glutamate would be put into a protein where aspartate should have been Now go back to page 37 and look at the R groups of these molecules Both R groups are organic acids both are negatively charged Therefore this mutation probably would not have as great an effect on protein structure since glutamate and aspartate would behave very similarly in an aqueous environment Mutations that cause a change in the amino acid sequence of proteins are called missense mutations The ultimate effect of such a mutation on the function of the affected protein as you can see depends on the type of amino acid substitution the mutation produces and the position of the amino acid substitution As you know enzymes receptors transporters and most other functional proteins have active sites ie areas on the protein molecule that actually come into contact with important ligands e g substrates hormones neurotransmitters transported nutrients etc In addition proteins frequently have allosteric sites at which they are regulated ATP 01 GTP binding sites andor phosphorylation sites at which energy is transferred and the protein is regulated 63 Amino acid substitutions at these important sites have a far greater impact on the protein molecule than do mutations that are in the framework or scaffolding areas For instance the change from glutamate to aspa1tate would probably cause no change in function if it occurred in a framework region of the protein However if it occurred at an active or regulatory site it may dramatically alter the protein s function since aspartate is a smaller molecule than glutamate and would alter the topology of the surface of the active site that is so critical to specific binding A slightly bigger or smaller bump at one spot in the binding site may make specific binding to the normal ligand inadequate or impossible A missense mutation is very likely to be the cause of a disease if the protein product is still present but functioning poorly However if the protein is simply not present we may be dealing with a nonsense mutation or an insertion or deletion mutation that has caused a frameshift In either case no protein is made at all HD is dominant therefore we suspect that the protein encoded by the mutant gene is hyperactive We might hypothesize at this point that a missense mutation in the active site increased the affinity of this molecule for its ligand Or possibly and more likely a missense mutation might have destroyed an allosteric site making it impossible for an allosteric modulator to turn the protein off Thus the protein continues to function at a high rate at all times producing too much of something that causes the disease Conversely it does not seem likely that a nonsense mutation is responsible for HD In addition to environmental agents causing mutations irradiation some chemicals and viruses the genetic material itself is constantly changing in ways that may cause mutations For instance genes or pa1ts of genes can be duplicated gene ampli cation methylated this permanently turns the gene off making it unable to be expressed rearranged or transposed moved to another chromosome fig 145 Then of course our cells can make mistakes in DNA replication which can lead to mutations too page 2124 DNA proofreading Any of these natural changes may induce a mutation that destroys or ampli es a protein s function Study Questions Describe the effect of a single point mutation on protein structure and function What types of point mutations are the most harmful The least harmful Explain What two factors play a major role in determining the impact of a mutation on protein function Explain 2 Given the genetic code and the R groups of the amino acids be able to develop a reasonable hypothesis about the effect of a given mutation on protein function 3 Nonsense and frameshift mutations virtually always destroy the gene s ability to produce a product Explain why this is so 4 Explain how a missense mutation may increase the activity of a protein product V39 Describe changes that occur in the DNA without external mutagens that may lead to the development of a nonfunctional or hyperfunctional gene 6 Explain why the five base pair inse1tion mutation described in the NEWS ITEM back on page 29 would cause 300 amino acids to be deleted NEWS ITEM Avery new classification of mutations has been discovered recently This mutation does not happen at the DNA level but at the mRNA level It appears that the RNA polymerase makes certain mistakes frequently such as reading the DNA sequence GAGAG and producing an mRNA that is only GAG a two base deletion This new form of mutation has been discovered in the brains people with Down s syndrome and Alzheimer s disease Science Vol 279 174 January 1998 gt1 Would the form of mutation described in the News Item be passed on from one generation to the next This is a trick question so think about two possible answers To which category of DNA mutations is this mRNA mutation most similar qTOP When we talk about mutations it is a common misconception that we are always talking about changes in the DNA that occur in the individual bearing the trait This is not the case and it is important that you understand this point Mutations can occur in this manner in which case they are called new mutations Some diseases especially some forms of cancer eg skin cancer are thought to be enhanced by new within 39 quot 39 39 39 However the classic genetic diseases are caused by mutations that occurred hundreds or even thousands of years ago in an ancestor and are 39 39 through inheritance to the 39 quot 39 39 39 with the disease Thus even though the disease was originally caused by anew mutation it occurs in individuals as an inherited trait For this reason classic genetic diseases are sometimes referred to as inherited diseases to distinguish them from those that are caused by new mutations in the af icted individuals u WWW Reading SRY paper Human SRY binding to DNA Focused Reading p 215 Fig 1120 Sequencing DNA At this WWW site you will nd a virtual reprint of an article that illustrates how important each and every nucleotide is A Japanese couple has had problems conceiving a child and both of them go to a fertility specialist for some advice This woman a mutation with dramatic system wide phenotypic consequences The paper discusses a person who has a mutation in the SRY gene which is a gene located on the Y chromosome A functional copy of SRY is required for embryos to develop as males rather than females Scott Gilbert s Developmental Biology text gives an overview of how the SRY gene was identi ed You can check it out at httpwwwdevhin 39 17 link1702shtml Study Questions 1 What were the clinical symptoms of the woman described in this paper Which sex chromosomes did she have 2 What kind of mutations did she have in her SRY gene 3 Do you think she inherited this mutation or do you think it is a new mutation in her 4 Be able to explain to your nonscience friends why this woman was infertile 5 What would happen to her if she wanted to compete in the Olympics and was subjected to a 65 karyotype analysis To be precise all of our physical traits originated as new mutations that were passed down to succeeding generations This is one of the major tenets of the theory of evolutioninew mutations arise spontaneously all the time These mutations are either advantageous to the organism the mutant organism lives and successfully transmits these genes to their offspring disadvantageous to the organism the mutant individual is less successful or unsuccessful in passing on these traits or neutral the mutation is of no consequence to survival in the current environmentiit just gets passed along to the next generation Thus as mutations occur and provide advantage to the organisms bearing the mutations they are selected by the environment a process called natural selection and they eventually become a standard trait of the species as more and more individuals who bear this trait outcompete individuals who lack the trait A theory from the tale of human evolution should illustrate this point Humans Homo sapiens first arose in Africa from lower primates that were covered with thick body hair Humans began to lose their thick body hair due to an advantageous mutation What is so advantageous about this remains atopic of debate Upon the loss of thick hair the skin became more exposed to the harmful ultraviolet radiation in sunlight These highenergy rays can mutate thymidine bases and break DNA causing a mutation and skin cancer By the way this is still the caseiexposure of human skin especially the lighter skin colors can break DNA and cause skin cancer These early thinhaired humans had to rely on the expression of genes that control the enzymes that make melanin the dark pigment of skin Having higher production of skin melanin and turned the skin a dark color These individuals didn t get skin cancer as often because their dark skin pigment blocked the penetration of UV light They were healthier and more able to reproduce and raise offspring to maturity These dark skinned individuals therefore became the wildtype phenotype in the population Their pale counterparts represented spontaneous mutations in genes that caused less melanin production Since the pale skinned individual was more susceptible to UV light damage slowly over generations dark skin came to be the dominant trait of the human species which is what we see today in Africans It should be noted that mutations occur all the time on average one mutation per 10 quot12 bases of DNA For instance while some early humans had mutations that increased melanin production in the skin others had mutations that decreased melanin production eliminated vital blood proteins incapacitated vital liver enzymes destroyed the pigments in the retina that produce color vision etc None of these mutations survived in humans because they are not advantageous to the individual and thus do not enhance survival and reproduction Your body contains some new mutations which developed in the egg andor sperm that joined to produce you or in the cells of your body during development in utero or after you were born As you know from the discussion above these mutations can cause a variety of protein changes ranging from no change to complete destruction You may think that your presence on the planet means that none of these mutations is harmful in any significant way However it is quite possible that you Q harbor at least one lethal new mutation destroying an absolutely essential protein but you are protected from its effects by being diploid One of the tremendous benefits of being diploid is that you can have lethal or harmful mutations in a gene and frequently it won t kill you or harm you because the other allele is wild type and compensates for the deficient allele You have built in genetic redundancy that safeguards you against mutations Big multicellular creatures such as ourselves that take a lot of energy to produce are virtually always diploid which provides such an enormous adaptive advantage The presence of a potentially lethal or harmful new mutation makes you a carrier of a defective gene Ifyou mate with someone who is a carrier of a mutation in the same gene you stand a 25 chance of 66 producing an offspring with two mutant alleles at that locusithat child would have a diseased phenotype Because mutations occur spontaneously ie randomly in the DNA it is extremely unlikely that you would pick a mating partner with exactly the same genetic mutation that arose spontaneously in you However because mutations are passed down to offspring they run in families This is why it is a very bad idea for close relatives to form mating unions For instance if a spontaneous mutation occurred in grandma she would pass this down to half of her children who would in turn pass it down to half of their children If these first cousins married they would have a dramatically increased probability of producing an offspring with two bad genes a homozygous individual with serious or lethal genetic problems Most countries have laws or traditions against such incestuous relationships One can only speculate about the origin of such traditions Ifmutations have to confer an adaptive advantage in order to be selected how then do disease alleles manage to stay in the human population and get passed down from generation to generation Recessive disease genes get passed down because individuals can be carriers without actually having the disease Thus heterozygous individuals are just as healthy and able to reproduce as homozygous normal or wild type individuals and the defective genes get passed down The situation is different with incompletely dominant or dominant traits Ifthe disease trait interferes with health and reproduction it should be slowly weeded from the population since anyone with a single diseased allele is not as fit to compete for survival and reproduction Most classic genetic diseases therefore are recessiveinot dominant Exceptions are those diseases that af ict individuals after they have reproduced such as most cancers and Huntington s disease Study Questions 1 Explain the role of new mutations in evolution 2 Explain the difference between a new mutation and an inherited mutation Give examples 3 In animal and plant breeding the concept of hybrid vigor is used to explain why hybrid heterozygous organisms are heartier than inbred homozygous individuals Explain why this is so 4 Most genetic diseases are recessive Explain why this is the case Ifmaladaptive mutations are selected against how do dominant and recessive inherited diseases remain in the population despite their detrimental effect on health Let s return to our study of the cause of these genetic diseases Mutations in actual structural genes may be responsible for producing CF SC and HD However in order to develop a more complete understanding of potential genetic aws we have to look a bit more closely at the process of gene expression Gene expression is the process through which the genetic code is used to produce a functional protein going from DNA to RNA to protein In the following discussion we will explore the possible sources of the genetic defects that cause the classic genetic diseases Focused Reading Fig 124 p 222 p 2223 Transcription stop at bottom of page p 2656 The structures of stop at bottom ofpage p 26870 RNA processing stop at Contrasting eukaryotes p 376 Posttranscriptional control stop at Translational 67 A number of steps comprise the process of transcription A defect at any one step would interfere with the production of an accurate mRNA Without accurate mRNA accurate proteins cannot be produced and genetic disease may occur So in revisiting the three diseases in question what might be causing the problem with the disease alleles other than a direct mutation in a structural gene Well you could have a mutation in a gene that encoded any of the proteins that are required for transcription e g RNA polymerase or transcription factors RNA processing splicing adding a cap or polyA tail or transporting the mRNA from the nucleus to the cytoplasm However if this were the case the cell could make no proteins since all proteins use the same polymerases transcription factors spliceosomes processing enzymes and transport proteins The cell wouldn t exist this would be a lethal mutation so this is an unlikely hypothesis Alternatively the faulty gene might contain a mutation in its promoter This region normally controls the expression of the gene so that it is expressed in the appropriate cells lungs pancreas liver and sweat glands and not expressed in incorrect cells brain bones and kidneys The promoter is a sequence of DNA immediately upstream from the structural gene that is recognized by RNA polymerase and by molecules that specifically control the expression of this gene Thus a mutation in the promoter that changed this recognition area might cause the gene to be expressed too much the promoter is on too often allowing too much transcription too little the promoter is not on enough allowing too little transcription or not at all promoter is nonfunctional and RNA polymerase cannot bind to it Alternatively the mutated and defective gene might be in a region called an enhancer As its name implies an enhancer is a segment of DNA that enhances the expression of the gene The unexpected thing about enhancers is that they can occur several thousand bases kilobases away from the actual gene and can also be found in introns A defect in an enhancer may cause a gene to be 1 J too 39 f 1 quotJ or too f 1 quotJ Athird alternative involves a defect in the introns of the gene In order to be successfully spliced out of the primary transcript to form mRNA introns must contain base sequences that are recognized by the spliceosome and used to determine where the mRNA should spliced If a mutation occurred in these recognition areas of the intron correct splicing may not occur in which case accurate mRNA would not be formed and an accurate protein could be made Finally we could hypothesize that a mutation occurred which made the mRNA more or less susceptible to enzymatic degradation in the cytoplasm Ifthe mRNA remains intact longer than normal more protein than normal could be made Likewise if the mRNA is degraded too quickly less protein than normal could be made Thus the amount of protein may be altered producing a disease state This is a viable hypothesis since the signals for degradation of each mRNA are probably at least partially inherent in the mRNA molecule itself and thus specific to this one gene Thus a mutation need not be in the coding portion of the gene the exons in order to cause a genetic defect It can also be in any of the genetic elements that control the transcription of the gene the splicing of the primary transcript into mRNA or the transport of the mRNA out of the nucleus into the cytoplasm Study Questions 1 What types of mutations may affect protein function besides those within the structural gene Explain how these mutations produce these changes 2 Many proteins are involved in gene transcription Some of them are likely candidates in the quest for the causes of genetic disease and others are not Which of these proteins are unlikely to be the cause of any of the classic genetic diseases and why 3 Describe the role of each of these components in transcription and mRNA processing A RNA polymerase II B The promoter C The spliceosome D snRNPs E The mRNA transport proteins in the nuclear pore F Introns and Exons G Enhancer H Transcription factors 4 What is SRY what is its function In addition to genetic defects in the proteins that control transcription RNA processing and mRNA transport genetic diseases may be caused by defects in the proteins that control translation Focused Reading p 22530 Preparation for stop at Regulation of translation p 231 Posttranslational events p 2767 Translational and post translational control to end p 645 The endomembrane system stop at The Golgi WWW Reading Immuno uorescence labeling of the ER Hemoglobin RasMol Image A defect in 39 quot and p t 39 quot 39 r 39 may be responsible for causing CF SC or HD although it is much more difficult to develop a viable hypothesis about these processes We could hypothesize for instance that a disease was caused by a defect in any of the genes that control the proteins of translation ribosomal proteins initiation factors elongation factors enzymes such as peptidyl transferase etc However as in the case of transcription all proteins are made using the same set of translational proteins and if a defect existed in any of these important molecules the mutation would be lethal and the cell would not exist Another hypothesis could be a defect in the genes that encode tRNA or rRNA If for instance the tRNA that binds to the amino acid alanine were defective alanine could not be activated and could not be incorporated into proteins thus leading to defects Again however this would affect all proteins of the cell and would be a lethal mutation The defect could be in the enzymes that perform posttranslational modifications such as glycosylation sulfhydryl bond formation chain cleavage etc Again these are global or house keeping enzymes that modify all proteins and one would expect to see widespread protein abnormalities if such a mutation existed Study Questions 1 Gene expression is a highly energetic process requiring the expenditure of significant amounts of ATP and GTP Describe the expenditure of energy ATP and GTP during transcription and translation How is the energy expended Which parts of the process 69 require the expenditure of energy 2 Describe the steps of translation 3 How are proteins altered during posttranslational modification 4 Some genes encode processing proteins that control the translation and posttranslational processing steps of gene expression Explain why it is unlikely that CF SC and HD are caused by a defect in a processing protein gene The defect in some genetic diseases may cause the protein to get lost in the cell after it is made Focused Reading p 656 The Golgi stop at Lysosomes p 2303 Polysome formation Stop at Mutations Secreted and membranebound proteins require the presence of a signal sequence for transport into the ER The signal sequence is a stretch of amino acids in the protein that act like a zip code telling the cell where the protein belongs Ifa protein is supposed to be membranebound or secreted a defective form that causes a disease may contain a mutation in its signal sequence In this case the protein could be made but it would never get to the appropriate area of the cell to be used Proteins going to the ER are not the only ones that use signal sequences other proteins contain different zip codes which instructs the cell to send the protein to the mitochondrion the nucleus or the chloroplast Study Questions 1 Describe the process by which secreted and membranebound proteins get from cytoplasmic ribosomes into the ER What role does the signal sequence play in this process 2 Explain how a mutation in a gene s signal sequence could produce a genetic disease STOP While we do not understand the cell or chromosomes well enough to speculate about all the possible mutations that may cause genetic diseases the preceding discussion certainly gives you an idea about the complexity of genetic systems and the incredible number of steps involved in producing a normal protein It is nothing short of a miracle that we exist given all the reactions that have to work exactly right in order for us to produce one gene product not to mention the products of all 100000 genes We have explored many of the possible proteins that may be mutated to cause genetic disease Now let s see what we know about the causes of CF SC and HD and how investigators acquired this knowledge Certainly if you want to know how to cure a genetic disease it would be very helpful if you could find out which protein is defective and how it is defective In the case of sickle cell disease this was a relatively easy process Because the disease symptoms produce disease and because you can actually E the sickled red blood cells RBCs under a microscope it seemed very likely that the 70 defect is in a molecule in the RBC possibly a protein that controls the shape of the RBCs RBCs are normally shaped like this This shape is called a biconcave disk Because of the thermodynamic properties of phospholipid bilayers the most thermodynamically stable shape for a cell is a sphere Like soap bubbles if you don t do something special a cell will always assume a spherical shape So in order to maintain the RBC in this odd biconcave shape the cell has to distort and support the membrane with proteins One such protein is called spectrin and it lies immediately under the cell membrane and holds it in its unusual shape So the sickle cell disease mutation could be in the gene that controls the production of spectrin However investigators noticed that the red blood cells were not always sickle shaped They only became sickle shaped when oxygen levels were low as in the veins as opposed to the arteries The molecule that carries oxygen in the RBC and which changes shape when it binds to oxygen is called hemoglobin see page 41 gure 37 Hemoglobin is a molecule much like chlorophyll we will talk about this more in Unit IH with a porphyrin ring structure containing an atom of iron in hemoglobin instead of magnesium in chlorophyllisee l4l gure 89 to see what a metalbearing porphyrin ring looks like It is the iron atom that actually binds the oxygen RBCs are really bags of hemoglobiniover 90 of their protein content is hemoglobin Investigators were quick to suspect that the genetic defect may be in the hemoglobin molecule Hemoglobin can be isolated from RBC very easily The red cells are burst open usually by osmotic pressure Are they burst by tiny molecular cherry bombs No RBCs are put into pure water which has a very high osmotic pressure Because of all the proteins nutrients and ions dissolved in its cytoplasm the osmotic pressure inside RBCs is low Water therefore moves into the red blood cell All that water makes the RBC swell until it bursts freeing all of its hemoglobin This process is called hemolysis Heme red blood cells lysis slicing open or cleaving The hemoglobin can then be puri ed by a number of processes including column chromatography described in Unit 1 Hemoglobin will be separated from the other proteins in the red blood cell because it moves through the column at its own speci c rate Other proteins will move through faster or slower and thus separation will occur SC hemoglobin and normal hemoglobin can also be compared using electrophoresis also described in Unit 1 If they move at different rates in the electrical eld they are different sizes In this case column chromatography was not sensitive enough to detect a change in hemoglobin Gel electrophoresis detected a slight change in mobility This change indicated that wildtype and SC hemoglobin had some difference in their size and that scientists should look more closely in this direction Note they had to try two approaches that seem to do the same thing separate by size before they saw the difference in the hemoglobinisensitivity counts The approach that got at the difference was to determine the amino acid sequence of the proteins to see if a mutation has produced a change that could lead to an alteration in function Each hemoglobin molecule is composed of four chains or subunits the complete and functional molecule has a foursubunit quaternary structure two alpha chains each 141 amino acids long and two 71 beta chains each 146 amino acids long These four chains each containing a porphyrin ring and an atom of iron interact with one another forming a very large hemoglobin molecule that can bind to four molecules of oxygen one at each iron atom Determining the complete amino sequence of each chain was a timeconsuming laborintensive and tedious process While you certainly don t have to understand the details of this process the following brief discussion should give you an idea of some of the technical difficulties involved in this process Focused Reading p 901 Table 503 focus on enzymes digesting proteins or peptides Amino acid sequencing relies on the use of analytic chemical methods to identify amino acids after the digestion of the protein with enzymes that cleave peptide bonds peptidases or proteases as specific sites For instance if you subject a protein to carboxypeptidase the enzyme will cleave off the last amino acidithe amino acid at the carboxyl terminus of the protein The first amino acid translated always has a free amino group amino H so that end of the protein is called N terminus At the other end of the chain the last amino acid always has a free garboxyl group so it is called the C terminus See page 38 Figure 34 for an illustration Trypsin will cleave on the carboxyl side of lysine or arginine chymotrypsin will cleave on the carboxyl side of phenylalanine tryptophan or tyrosine etc In addition various chemical processes can be used to tag or label the C or N terminus amino acid or other specific amino acids so they can be identified by analytical procedures For instance you could radioactively tag the Cterminal amino acid and then subject the peptide to carbaminopeptidase The analysis of the liberated amino acids should show only one amino acid bearing the tag This is the amino acid at the Cterminus end of the protein You could then subject the protein to carbaminopeptidase for a short period of time and Ih tag the C terminus The tagged amino acids in this analysis should be the second third andor fourth amino acids the enzyme just keeps cleaving amino acids one after another off the Cterminus end While the procedure is very complicated this brief example may give you an idea of some of the technical manipulation that is involved Needless to say amino acid sequencing is not something a laboratory attempts unless it has a very good reason to do so Focused Reading p 3942 The primary structure stop at Chaperonins p 876 Fig 4911 note how RBCs must fit through capillaries p 234 Paragraph beginning In contrast to silent stop at Nonsense mutations another type of mutation WWW Reading Hemoglobin Mutagenesis Page Wildtype 39 39 39 39 called 39 g39 L39 A was 1 J in the 1950s in Germany and the United States Hemoglobin from a sickle cell disease patient now called hemoglobin S was found to be absolutely identical in amino acid sequence except for a single difference at position 6 on the beta chain 6 amino acids from the N terminus Hemoglobin A has a glutamic acid at position 6 while hemoglobin S has a valine at this position Ifyou look at the genetic code on page 224 you see that the difference between the code for glutamate GAG and valine GUG is a single base in the middle of the codon By changing the sequence of the codon for glutamate from A to U valine is put at position 6 instead of glutamate You can see on page 37 that glutamate glutamic acid has a negatively charged organic acid in its R group while valine has a nonpolar hydrocarbon The switch from a charged to a nonpolar R group changes the threedimensional shape of the molecule enough to alter its shape The shape change is in a critical enough point to change the function of the protein thus causing the sickle cell disease Note again that people with SC inherit this mutation from their parentsiit does not occur spontaneously in SC patients The original mutation occurred thousands of years ago In fact this mutation appears to confer some adaptive advantage to heterozygotes Malaria is a dangerous and widespread disease especially in Africa This disease is caused by a protozoan that spends part of its life cycle in the RBC SC heterozygotes are resistant to this phase of the disease and are therefore somewhat more protected from malaria than are normal individual Thus despite its harmful effect in homozygotes the SC gene may in fact have been an adaptive trait for Africans in Africa and naturally selected in heterozygotes This helps explain why SC is so prevalent in African Americans This also provides an example of why mutations are not inherently good or bad iit depends on the environment that the organism is in News Item Genetic mutations are not the only way to make RBCs less effective Exposure to carbonmonoxide CO inhibits the hemoglobin in RBCs from binding oxygen The cells get through the blood vessels but have nothing to deliver No oxygenicells die CO is why car exhaust is poisonous A group at the European Molecular Biology Laboratory EMBL has usedXray crystallography and molecular modeling to visualize the protein and determine cellular mechanisms that block CO binding Their work on sperm whale myoglobin indicates that CO can only bind after two at helices shift position slightly How hemoglobin can tell the difference between oxygen and CO is not yet known but as cities get bigger and we all must drive SUVs its a good research direction to give airtime to GS Kachalova 1999 Science vol 284 p4636 Study Questions 1 Describe the process by which red blood cells are lysed by osmotic pressure Explain why water moves into the cell under these experimental conditions 2 What approach was taken to determine the cause of sickle cell disease 3 What specific genetic defect causes sickle cell disease 4 Describe the selective pressure that may have actually enhanced the presence of the SC allele in the African and African American populations 5 Why is glutamic acid the 63911 amino acid if it is encoded by the 7Lh codon NEWS ITEM The FDA is about to approve a drug that has been used to treat cancer for over 30 years for the treatment of SC The drug is called hydroxyurea and it has the ability to activate the transcription of a gene that is normally silent The gene being activated encodes for a form of hemoglobin that we produced while we are embryos but is silent forever once we are born These fetal hemoglobins work just as well as adult hemoglobin and so it should work as a good alternative for those with SC see article by Robert Finn in The Scientist February 16 1998 Pg 9 6 Is the hydroxyurea treatment considered a cure Will those being treated still be at risk of having children with SC News Item In December 1999 the Associtaed Press reported the success of a new cure for Sickle Cell Anemia A thirteen year old suffering from SC was treated by introducing stem cells from the umbilical cord of an unrelated infant who did not have SC Stem cells are undifferentiated cells found in bone marrow that develop to produce red blood cells The transplant performed Dec ll 1998 is the first time unrelated cord blood has been used to treat sickle cell anemia and is much less painful than bone marrow 73 transplants that have been used in the past His treatment was to provide him with a selfrenewing source of healthy red blood cells the stem cells After one year the cord cells have taken hold in the boy39s bone marrow and are making healthy blood cells so the doctors have declared the child 39cured Do you consider this a cure If he should have children would they be at risk of having SC The sickle cell disease puzzle was solved relatively early because a cellular defect was visible through the microscope and the probable protein affected by SC was fairly easy to deduce Unfortunately the overwhelming maj ority of inherited genetic diseases are much more difficult to investigate In the case of cystic brosis all investigators knew for many years was that the disease altered the way in which mucus is handled by the lungs and pancreas Patients suffered from pneumonia loss of digestive enzymes liver cirrhosis production of profuse sweat with a high salt content and in some cases sterility This mixture of symptoms doesn t immediately point to a culprit We have been referring to the CF gene as a mucus gene but that doesn t explain the all the symptoms of the disease A real breakthrough in CF research came in 1984 from a lab investigating the differences between respiratory cells from CF patients and wildtype individuals This group tested the ability of respiratory cells to respond to second messengers Wildtype respiratory cells pump Cl39 into extracellular spaces in response to the activation of the cAMP second messenger system To review the cAMP second messenger activates CAMPdependent protein kinase which in this case presumably phosphorylates the Cl39 pump and increases the rate at which it pumps Cl39 from the cytoplasm to the extracellular space Because Cl39 exerts osmotic pressure water follows the Cl39 and moves outside the cell in response to the cAMP signal This lab group Sato and Sato found that respiratory cells from CF patients were not able to pump CI39 in response to cAMP activation They asked whether this might be because cAMP cannot activate CAMPdependent protein kinase and they found that the protein kinase M become activated but it does not activate any Cl39 pumping action While we always have to be wary of jumping to conclusions that are insufficiently supported by the data this was a very exciting finding since it correlates with several of the disease symptoms In the lungs and pancreas if Cl39 cannot be pumped into the breathing tubes bronchi of the lungs or secretory ducts of the pancreas water will not follow and the mucus normally found on these internal surfaces will remain thick and dry This condition will harbor bacteria in the lungs causing pneumonia and will block the passage of digestive enzymes from the pancreas to the intestine It looked at this point as though the CF gene produced a defect in the Cl39 pump in the membranes of respiratory cells and possibly the cells of the pancreas Because of the difficulty in working with membranebound proteins and because of the quot 39 quotquot of new 39 39 J in 39 39 biology the next steps in the solution to this genetic disease came not from cell biology or genetics but from molecular biology The nucleotide sequence of an isolated gene can be determined much more easily than amino acid sequences can be determined This allows investigators to work backward using the genetic code to determine the amino acid sequence of the protein Sometimes this amino acid sequence gives a clue about the protein s function For instance membranebound proteins tend to have alternate stretches of hydrophobic amino acids with stretches of hydrophilic amino acids While this pattern does not necessarily mean that it is a membrane protein it gives investigators a clue about where to look Therefore the hunt was on for the CF gene Once the CF gene cold be found investigators would use the gene to determine the structure of the protein involved and then use the protein to determine the cell biology that is actually causing the disease The human genome the sum of all of the DNA in all 23 pair of human chromosomes contains about 6 x 109 base pairs and about 100000 functional genes Over 98 of the genome is non 74 coding sequences So locating a single gene in this gigantic mass of DNA is like looking for a needle in a haystack of DNA but even the haystack is too small to see Investigators working on genetic diseases are trying to nd these needles by some very ingenious techniques we will describe below As an interesting aside the US government has funded an enormous scienti c enterprise called the Human Genome Project The project was first headed by James Watson of Watson and Crick fame and is an internationally coordinated effort to identify the base sequence of the entire human genome in about 15 years Recently a rough draft of the human genome was completed Estimates place the cost of this project at about 35 billion about one dollar per base pair The ethics of this project are currently of being widely discussed The knowledge of the entire base sequence of the human genome will give scientists tremendous power to manipulate the genetics of the human species We have already seen a small glimpse of this power in the ability to detect genetic abnormalities before birth through amniocentesis Many couples have chosen to abort fetuses when they find that they have Down Syndrome called trisomy 21 because it is caused by the presence of an extra chromosome 21 three instead of the normal two The detection of this abnormality does not require the techniques of molecular biology One can only wonder what parents will do when many many more genetic diseases and traits can be diagnosed in utero What if the fetus has genes that predispose it to cancer to heart disease to homosexuality to baldness to being overweight Further in vitro fertilization now allows the predetermination of genetic traits Egg and sperm can be joined in a petri dish producing embryos whose genetic traits can be screened before they are implanted in the woman As we gain more and more knowledge about the human genome more and more traits will be screenable The correc embryos can then be implanted in the woman s uterus and the defective embryos discarded similar to polar body diagnosis described on p 385 bottom of first column See any ethical issues here Obviously this raises significant questions about what we mean by normal and defective One could hold the view and many in the disabilities movement do that we abort Down Syndrome fetuses because as a society we place far too much emphasis on physical and mental perfection As was the case with nuclear technology in the 1940s the knowledge we gain through the Human Genome Project will test our wisdom as a society in unprecedented ways Study Questions 1 Explain the approach taken by Sato and Sato that identified a defect in the respiratory cells of CF patients Describe this molecular defect 2 What are the goals of the Human Genome Project How does this approach differ from the approach taken by investigators studying individual genetic diseases Brie y discuss some of the ethical and economical issues raised by the Human Genome Project qTOP Investigators working on specific diseases usually begin to identify and isolate the disease gene by trying to determine the rough location on a chromosome of the gene so they can limit their search to part of a chromosome rather than the entire genome As a beginning investigators try to determine which one of the 23 pairs of homologous chromosomes bears the locus for the disease gene and its normal allele In order to understand how investigators determine this we need to look at the phenomenon of linkage Focused Reading p 180186 Mendel s experiments stop at Many genes 75 p 190192 Genes and chromosomes stop at Sex is determined Fig 1023 amp 1024 Genes that are on different chromosomes are passed down to offspring through independent assortment as described by Gregor Mendel Here is an example Let s say that the locus controlling CF is on chromosome 10 and the locus controlling some other polymorphic trait let s say blood group is on chromosome 3 For the CF locus you have two alternatives The allele can be wild type or CF As you learned from this reading assignment we now use a more modern terminology to express these alleles In Mendel s notation the dominant allele had a capital letter and the recessive a lowcase letter The letter was determined by the dominant trait eg green G and yellow g green is dominant to yellow However because the recessive trait ie yellow is usually the one that is under investigation as an interesting mutation this notation isn t very helpful Thomas Hunt Morgan devised a system of notation in which the mutant allele is designated by italicized letters and the wildtype allele is designated by the mutant letters with a superscripted Ifthe mutant allele is recessive it begins with a lowercase letter if dominant with an uppercase letter In the case of CF we could use cf to designate the mutant disease causing recessive allele that causes CF Given this nomenclature you could have the following genotypes at the locus in question cf cf wildtype cfl cf heterozygous carrier cf cf homozygous recessive disease phenotype For blood groups you can be phenotypically A B AB or O A and B are codominantly inherited while 0 is recessive Because all three blood types are caused by naturally occurring wildtype alleles we can designate the wt alleles 14 13 and 0 The possible phenotypes and their corresponding genotypes are listed below A AB 0 00 Now if CF and blood groups are on different chromosomes these traits will be independently assorted when they are passed down to the next generation Here is an example Let s say Maria is blood type AB and is a carrier for CF Maria s genotype is Al 3 cf cf Louis is blood type 0 has CF Louis genotype is 0 0 cfcf According to Mendel s first law the alleles at each locus segregate independently of one another when gametes are formed Therefore in Maria s case each egg receives one blood group allele and one CF allele Ifthe alleles are on different chromosomes then they are not linked and they assort independently into the gametes That means that four types of eggs will be produced Egg type 1 Al cf Egg type 2 Bl cf Egg type 3 A cf Egg type 4 3 cf Louis39s alleles also segregate independently during meiosis but because he is homozygous at both loci all of his sperm would get one 0 and one cf IfMaria and Louis should produce offspring and this is fairly unlikely in this case since CF causes infertility in males but let39s say Louis is an exception to the rule this is what the Punnett square would look like Egg Genotypes V A l cf B cf Acf B of Sperm Genotype Ocf Aocfcf 130 cfcf Aocf cf B0 cf of This is a classic Mendelian test cross in which a dihybrid is mated with a homozygous recessive individual Ifblood group and CF are on different chromosomes there are four possibilities for the children carriers of CF with blood type A or B and af icted indiViduals with blood type A or B All possibilities are equally probable If Maria and Louis were elm trees producing thousands of offspring about 25 of the offspring would be in each category Not that elm trees have blood or get CF but you get the point Now let s say that CF and blood groups E on the same chromosomeithat they are linked Here is a picture of what this chromosome homologous pair might look like in Maria and Louis A B Locus for 0 0 Blood Group Cf cf Locus for CF cf of Maria Louis Because A is linked to cf the two alleles go together assort together into the gametes Likewise because 3 is linked to cf these two alleles assort together Thus if Maria and Louis have children under these circumstances this is what the Punnett square would look like Egg Genotypes Acf 13 cf Sperm Genotype Aocfcf B O cf of o of In this case there are only two alternatives for the offspring They are either 1 blood type A and a carrier g 2 blood type B and af icted with the disease Now here s the deal You gather up all of the information you have on many many families that have members suffering from CF and determine the blood type of each member af icted or not By analyzing this information you can see if the traits follow the pattern on the last page 4 possible combinations or the pattern on this one a child that is blood type B always has the disease Ifblood type B is always inherited with of then the two loci are linked on the same chromosome Ifyou know which chromosome carried the bloodtype gene you now know that that chromosome carries the CF locus the same one Ifinheritance patterns follow the example on the previous page you know that CF and blood type are not on the same chromosome so in your search for a chromosomal location you have eliminated one and only have 21 left to go 21 because you also know that CF is not on the sex chromosome because the disease is not sexlinkedithat is it occurs in males and females in approximately equal numbers More on this later Study Questions 1 Explain the Law of Independent Assortment What exactly does this law tell us about genetic inheritance 2 Understand and be able to use Morgan s genetic notation 3 Be able to predict the genotypic and phenotypic frequencies for dihybrid crosses and dihybrid test crosses in situations where the loci are linked and unlinked 4 Be able to solve genetics problems such as the ones below from Biology by Villee et al A In rabbits spotted coat S is dominant to solid coat Sl and black B is dominant to brown B A brown spotted rabbit is mated to a solid black one and all the offspring are black and spotted What are the genotypes of the parents What would be the appearance of the F2 generation if two of these F1 black spotted rabbits were mated The long hair of Persian cats is recessive to the short hair of Siamese cats but the black coat color of Persians is dominant to the brownandtan coat color of Siamese If a pure black longhaired Persian is mated to a pure brownandtan shorthaired Siamese what will be the appearance of the F1 offspring If two of these F1 cats are mated what are the chances that a longhaired brownandtan cat will be produced in the F2 generation What kinds of diploid matings result in the following phenotypic ratios 31 11 933l llll 5 Given information about the chromosomal location of one trait be able to devise a genetic cross that will allow you to determine if a second trait is also encoded on that same chromosome 6 Given data from a linkage experiment such as the one presented above or the one you devised in question 5 be able to interpret the data to assess whether or not the traits are linked STOP Well as is usually the case CF is not linked to something as obvious and easy to detect as the ABO blood group However it is linked to something almost as goodia RFLP pronounced riflip Overview reading Focused Reading Chapter 17 p 315 Plasmid as vector 3 paragraphs p 31213 Restriction endonucleases stop at Recombinant DNA Fig 172 p 3378 DNA marker stop at Human gene Fig 187 WWW Reading Cartoon of Southern Blot Method Real Southern Blot RFLPs can be thought of as genetically inherited traits like brown eyes and dark skin Polymorphic traits such as eye color skin color and RFLPs allow investigators to follow genes on a chromosome As in the hypothetical case of CF being linked to blood groups you can tell CF is on the same chromosome as blood groups because m loci are inherited together they are linked A followed the cf gene and B followed the cf gene Without different allelic alternatives to follow you can t 79 do genetic analysis The problem is as mentioned earlier most human traits are not polymorphic For most proteins every human has exactly the same alleles as every other human So nding polymorphic traits that can be easily detected has been a tremendous problem and barrier to progress in genetics Our problems have been solved by the discovery of RFLPs thanks to the 98 of the DNA in our chromosomes that is noncoding DNA Although 98 of the DNA in the genome does not encode functional proteins these base sequences are passed on from generation to generation You inherit your noncoding DNA from your parents with the same degree of accuracy as you do your functional genes Mutations can occur in these noncoding sequences just as they can in functional genes and these mutations are then passed on to offspring As far as we know mutations in these noncoding areas do not matter much to the survival of the organism so they are not selected against and tend to stay in the gene pool Because these noncoding areas do not code for a protein we cannot analyze them by looking at the amino acid sequence or the function of the proteins they produce Rather if we want to analyze these noncoding regions we have to look at their nucleotide sequence In order to establish the presence of a RFLP on a chromosome or segment of chromosome you have to have a way of labeling certain DNA or RNA sequences so that they can be seen with the naked eye You do this with a probe that traditionally was radioactive because it contains radioactive phosphorus in its phosphate groups and complementary in nucleotide sequence to some chosen sequence of bases Currently most 39 are quot 39 39 to nonMd tive probes since they are cheaper more sensitive and safer Most probes are pieces of DNA isolated from other species For example if we wanted to clone the human version of the glycogen synthase gene we might use the previously cloned mouse version of the same gene as our probe Since they have a highly conserved structure and function we assume that the nucleotide sequences for the two genes would also be conserved Another type of probe is called an Oligonucleotide oligo means a polymer of unspecified length nucleotides are what get polymerized Oligonucleotides are short stretches of singlestranded DNA which are synthesized by a machine called a nucleic acid synthesizer On this instrument is a 4 letter keyboard so you can type in the sequence you want and the synthesizer makes millions of copies of the short nucleic acid chain with the base sequence you typed The machine is loaded with dATP dGTP dTTP and dCTP and in this case the synthesizer is programmed to create an oligo with the sequence 5 AATTCCGGTGGCATTACT 3 Note by convention DNA sequences are always written with the 5 end on the left but where indicated in this illustration we have written some sequences backwards 3 to 5 This oligo is then made radioactive by using a kinase to add a 32P phosphate to its 5 end The radioactive oligo is now ready to be a probe and will bind by complementary base pair bonding with the DNA sequence 3 TTAAGGCCACCGTAATGA5 which becomes our DNA marker It s a stretch of DNA that we can always label or mark with our radioactive probe and follow in a family pedigree Now let s look for RFLPs In order to do this we have to get DNA from many different individuals since we are looking for a polmorphism or genetic variability between individuals Let s say we get DNA from Jack and Jill for starters To get a complete set of chromosomes from a person you simply have to take any cell from their body that has a nucleus Every nucleated cell of the body all 5070 trillion of them contains a complete set of chromosomes This is called genomic DNAiat the genetic level all of your cells are equivalent even though they have quite different phenotypes The genes found in your DNA are expressed differently in different cells so that you wind up with liver cells that look and act differently than hair follicle cells In humans the white blood cell or leukocyte is a popular source of DNA for analysis since sampling merely requires drawing blood You then incubate the DNA from the chromosomes with a restriction enzyme Let s say you choose the restriction enzyme EcoRI pronounced ecoareone 80 and named after the E coli bacterium from which it was isolated This was the first restriction enzyme ever discovered and was called restriction enzyme 1 or R1 This discovery was worth a Nobel Prize This restriction enzyme recognizes the following base sequence and every time EcoRI sees GAATTC the enzyme makes the following cut EcoRI cut Every time this sequence GAATTC appears in Jack and Jill s DNA this enzyme will make this cut as shown below probe sequence top strand TAAGAGT39IG TTC39AATTCCGGTGGCATTAC39TG TTCGATCCAGTCA ATTCTCAACTT GTTAAGGCCACCGTAATGPCTT GCTAGGTCAGT TAAGAGT39IG AATTCAATTCCGGTGGCATTAC39TG AATTGEATCCAGTCA ATTCTCAMITTAA GI TAAGGCCACCGTAATGPCTTAA GITAGGTCAGT T marker sequence bottom strand Thus a restriction enzyme cuts the large chromosomal DNA into small fragments called restriction fragments because they are created by restriction enzymes that can then be electrophoresed and separated by size Because the restriction enzyme digestion of the entire genomic DNA creates millions of restriction fragments of different sizes the bands of this electrophoretic separation are so numerous that it looks like a continuous smear all the way up and down the gel This doesn t help much so we have to use our radioactive probe The DNA separated by electrophoresis is transferred to a piece of special filter paper usually researchers use nitrocellulose and the DNA binds to the nitrocellulose so the immobilized DNA can incubate with a probe that is oating in a solution that bathes the nitrocellulose This process is called Southern blottingiyou have created a Southern blot by transferring DNA from an 39 r gel onto quot 39 The words are big but the process is veg simple Basically if you can make JelloTM and handle paper towels you can do this The DNA is also denatured during this process Denaturing DNA is a bit different from denaturing protein When you denature DNA you unzip the double helix and convert the molecule into two single strands You then apply your radioactive probe allow the probe to bind or hybridize to its complementary sequence wash the blot to remove unbound probe and see where the radioactivity is In order to see this radioactivity you have to place an Xray film over the DNA and give it time to be exposed by the emissions of the radioactive phosphorus Everywhere the probe has bound the lm will be exposed and turn black This process is called autoradiography This whole process is diagrammed and explained in more detail on in your web reading I u u So here are the Southern blots from Jack and Jill after they have been hybridized with the radioactive probe and the resulting blot is exposed to Xray lm In a real blot you would not see the outlined lanes for each sample of DNA We have outlined each lane for illustration purposes Direction of DNA migration gt I l9 JiIIQI I I I 2 Fragment I llll Length 4 62 43 20 171 19 Markers 1 kilobases When restriction fragments are electrophoresed molecular weight or fragment length markers are electrophoresed at the same time These markers are DNA fragments of known length Their lengths are measured in kilobases 1000 bases to a kilobase or kb By running these markers along with the restriction fragments you can estimate the length of the restriction fragments in your sample The restriction enzyme EcoRI has digested Jack s DNA into many many fragments two of which contained the marker sequence 3 TTAAGGCCACCGTAATGA5 For the sake of clarity let s call these Jack 1 on the left and Jack 2 right In Jill s case EcoRI created many many fragments two of which contained the same marker sequence We ll call these bands Jill I left and Jill 2 right Ifwe focus only on the restriction fragments that bear the marker the only ones we can see in a Southern blot Jack 1 and Jill I are the same length about 124 kb For one of their two chromosomes the DNA carried by Jack and Jill are probably identical at this locus However their other chromosome resulted in different size restriction fragments hybridizing with the radioactive probe Here is an illustration with DNA written backwards 3 to 5 with the numbers in the parentheses being hypothetical distances between the given sequences Jack1 or Jill 1 GAATTC113 kbTTAAGGCCACCGTAATGA11 kbGAATTC These fragments are anked by two restriction target sites for EcoRI and contain the marker sequence While we cannot say that Jack 1 and Jill I are identical they may differ in the bases within parentheses above we do know that they both have the marker nucleotides the probe and they both are anked by the target site for the restriction enzyme EcoRI But Jack has 2 bands indicating that the probe hybridized with 2 different size restriction fragments of DNA For this to have happened Jack s 2 copies of this chromosome must not be identical the copy of the chromosome containing the region we call Jack 2 must contain another EcoRI site The same is true for Jill For Jill2 to exist there must be another EcoRI site in this region that puts the probecontaining piece in a 25kb piece of DNA Comparing Jack 2 and Jill 2 we see that these 2 bands are not the same size Jack2 Is 43kb while Jill2 is 25kb Remember both of these fragments m be anked by EcoRI sites and contain the 82 marker sequence Because they are different lengths in Jack and Jill s blots they represent differences in the DNA we call RFLPs restriction fragment length polymorphisms To understand what this means let s look at one possible scenario that would produce this RFLP I 43kb EcoRI fragment Jack 2 Jack 2 GAATTC 81kb GAA TTC 1 8kb CCATTC 14kbTTAAGGCCACCGTAATGA11kbGAATTC Jill 2 GAATTC 81 kbGTAATC 18kb GAATTC14kbTTAAGGCCACCGTAATGA11kbGAATTC 25kb EcoRI fragment Jill2 I kbength of DNA between regions not shown to save space In this case Jack has a 43 kb fragment bearing the marker sequence and anked by two EcoRI sites underlined About 81 kb downstream from the rst restriction site there is an EcoRI site not found on Jack s other chromosome the one that gave 124kb Jackl EcoRI sees this recognition site and cuts Jack s fragment into a 43 kb length This piece of DNA contains the marker sequence in italics so it hybridizes with the probe and is observed on the autoradiograph However Jill inherited a slightly different sequence in this part of her DNA In this copy of the chromosome she did not inherited the EcoRI site J ack2 has but instead has a sequence 99 kb downstream from the first EcoRI target site in which there exists GAATTC This is the target sequence for EcoRI and the enzyme will cut Jill s DNA at that site The digestion of Jill s DNA will produce a 99 kb fragment that does not have the marker sequence so it will not be observed on the autoradiograph and a 25 kb fragment that does contain the marker sequence You should understand that this explanation is hypothetical We usually cannot deduce this much detail from Southern blot data but something like this happens We do know that the EcoRI sites that produced J ack s blot were slightly altered in J ill s DNA She inherited different DNA sequences than Jack did analogous to different alleles and this constitutes a RFLP Different people will demonstrate this particular RFLP if their DNA is digested with EcoRI and probed with the 5 AATTCCGGTGGAT TACT3 probe This type of RFLP analysis can be used to produce a DNA fmgerprint which can be used as a very accurate form of identification in forensics We will perform a different kind of DNA ngerprinting during the last two weeks of lab for a preview see p 32829 RFLPs are so polymorphic in the human population that the chances are virtually zero that you would produce an identical DNA fingerprint to anyone else on the planet except an identical twin if you use several different RFLPs ie different combinations of restriction enzymes and probes WWW Reading Genotyping with RFLPs Study Questions 1 What is a restriction enzyme Where do they come from and what do they do 2 What are restriction fragments Explain the process of electrophoresis When restriction fragments are electrophoresed they produce a banding pattern Why Be able to interpret the band pattern produced by such a technique 3 Why are fragment length markers run along with sample DNA in electrophoresis experiments 4 What is a kb A Mb What do these terms mean 83 5 Explain how a Southern blot is performed What types of information can you get from a Southern blot that you cannot get from simply electrophoresing a sample 6 Explain the process of autoradiography How is this used in the Southern blot 7 Explain as clearly as you can what a RFLP is What does the acronym stand for What is a probe made of and what does it do The discovery of RFLPs has revolutionized molecular genetics Why are RFLPs an important tool in genetic analysis 9 Explain the two parts or components that are required to define a RFLP In other words if I told you that investigators had identi ed a RFLP called DC28036 what information would you expect to get in the published article about this RFLP 9 What is an oligonucleotide and how is it made How are oligonucleotides used in the characterization of RFLPs 10 In recent years DNA fingerprinting has become the basis for conviction in criminal trials If you were called as an expert witness to explain DNA fingerprinting to a jury what would you tell them 11 How are RFLPs related to the process of DNA fingerprinting Optional WWW Reading RF LPs Summary and test your knowledge qTOP In addition to identifying individuals RFLPs are passed on to children just as alleles are passed on To illustrate the power of this multigenerational analysis of RFLPs let s say that Jack and Jill have a child together We ll name the child Payle Let s say we did the same genetic analysis to Payle that we did to Jack and Jill and this is what we found moi I I JillGl I I Payle Q I I I I 1 Fragment Length 039 I I I I I l G Markers 124 62 43 20 171 9 kilobases 39 In analyzing these gels remember that the marker sequence 3 TTAAGGCCACCGTAATGA5 can t simply disappear except through new mutation and we will assume here that new mutations have not happened Jack has two copies of the marker sequence Jack 1 and Jack 2 Jill has two Jill I and Jill 2 Payle inherited two copies of the marker sequence too It appears that he inherited Jack 2 and Jill I and he did not inherit Jill 2 or Jack 1 How can we say that he inherited Jill I but not Jack 1 Don t parents have to pass their genes on to their offspring And how is it that Payle didn t inherit Jill 2 We said above that this marker sequence couldn t simply disappear Well remember that both Jack and Jill are diploid organisms that produce haploid gametes which means they pass only lilftheir chromosomes to their offspring Because Payle had to fetch something from his mother and he did not inherit Jill 2 he had to inherit Jill 1 Likewise because Payle inherited Jack 2 he could not also inherit Jack 1 since Payle can only get one copy from each parent This is one example of the many things you can determine by analyzing family RFLPs You can tell whether an offspring is actually the child of a couple Let s say that the RFLP analysis went like this Jackel I I JiIIQI I I Payle Q l I I l 9 Fragment Length GI I I I I CD Markers 124 62 43 20 171 99 kilobases 39 It is of little concern that Payle did not inherit Jack 2 Payle could have inherited Jack 1 But how did Payle get Payle 2 which is not present in either parent He didn t inherit it from J illishe doesn t have such a fragment and he didn t inherit it from Jackihe doesn t either So the possible 85 conclusions are l Payle has a new mutation in his DNA 2 Jack is not the father or 3 Jill is not his mother which is unlikely if she gave birth to Payle We have analyzed only one RFLP here but in real paternity cases several RFLPs are analyzed Even if one new band in the offspring is due to new mutation the chances are infinitely small that all new bands are due to new mutations Therefore RFLP paternity testing is extremely sensitive and reliable It should be noted before moving on that the process of finding a RFLP has been greatly over simplified in these examples Investigators have to test thousands of probes and scores of restriction enzymes in order to produce the kind of neat package presented here It is a laborintensive process but once the system is set up it is an extraordinarily powerful and reproducible tool in genetic analysis NEWS ITEM Ever wonder what makes a 39Chablis39 a 39Chablis39 and not a 39Chardonnay39 Did all those grapes start out in France or did invaders of long ago bring along their favorites quotPaternity testingquot has now been used to trace the lineage of certain cultivars varieties of wine grapes By examining the DNA at Q different loci scientists have determined that your parents favorite Chardonnay and Melon may be offspring of the same grape parents Bowers I et al 1999 Science vol 285 p 15623 Study Questions 1 Be able to interpret a multigenerational RFLP analysis Be able to explain how the analysis does or does not support the assertion that the child is in fact the offspring of these parents Be able to interpret such an analysis to determine which RFLPs represent a heterozygous trait in the parents Read p3289 DNA Fingerprinting where VNTR s are discussed What is a VNTR How is it similar to a RFLP How is it different NOTE you are determining your own VNTR pattern for the D1880 locus in lab N LapChee Tsui John R Riordan and Francis Collins determined that the CF gene was on Chromosome 7 by finding that it was linked to a RFLP that was located on that chromosome To do this they gathered DNA from hundreds of familiesifamilies without any CF history as well as families af icted with the disease They isolated DNA from carriers the parents of af icted individuals as well as CF patients They looked for linkage between the presence of CF and all the RFLPs they could generate and they found a linkage between CF and two markers on Chromosome 7 Below is a simplified version of their Southern blot data It should be noted here that many different restriction enzymes and probes are used to do RFLP analysis The important element in this approach is that once you have identified a RFLP using a certain restriction enzyme and a probe you must use the same restriction enzyme and the same probe to look for that particular RFLP in everyone RFLP Analysis for CF 86 WTWT WT ltCARRERSgt CF CF CF CF Individual 1 2 3 4 5 6 7 8 9 10 11 Individuals 13 are from families without CF individuals 47 are carriers parents of a CF patient but without the disease themselves and individuals 811 are CF patients Notice that the top band is present in homozygous wt individuals and in carriers but never in CF patients The bottom band is present in CF patients and carriers but never in homozygous dominant individuals The top band contains the marker sequence linked to inherited with the wildtype allele This allele is the only one present in wildtype individual homozygotes The bottom band is linked to inherited with the disease allele the CF causing allele and is the only allele present in CF patients homozygotes Carriers heterozygotes have both bands The RFLP represented by the top band is known to be located on Chromosome 7 That is if you digested each chromosome 1 through 22 plus X and Y individually with the restriction enzyme used in this analysis and applied the probe used in this analysis only chromosome 7 would give you bands at the positions shown above If the wildtype gene is on chromosome 7 the disease gene must be there as well It is important to note that while the restriction fragment with the marker sequence m also contain the CF gene this need not be the case All this analysis shows is that CF and this RFLP assort together and are inherited togetherithey are linked are neighbors on the same chromosome Linkage will be inhereitted within a family so if members of a family have CF linkage of a consistently identifiable RFLP with the CF gene makes it possible to determine whether someone were a carrier wildtype or an afflicted individual using a Southern blot In other words the Southern blot can be used to diagnose the disease state For example if the Southern blot above were performed on a person of unknown disease status and the blot looked like that of Individual 1 one higher band the person would be homozygous wildtype Ifthe blot looked like that of Individual 4 two bands the person would be a carrier Ifthe blot looked like that of Individual 8 one lower band the person would have the disease This kind of diagnosis can be used to determine whether individuals are carriers or even in utero to determine the genetic status of a fetus This has been a real boon to genetic counselors Before this test was available they could only estimate from pedigrees whether or not an individual was a carrier Now they can be more certain and offer the family more realistic information on probability of inheritance The CF gene was initially found to be linked to two RFLPs on chromosome 7 The next step in the isolation of this gene was to try to pinpoint the location of the gene on the chromosome so that its base sequence could be determined There are on average 130 million base pairs on each human chromosome This many base pairs cannot be sequenced easily One has to work with a more manageable unit a much smaller segment of DNA It is much faster to try to pinpoint the general location of the gene on the chromosome and then sequence the DNA in that specific area Once the location has been determined the gene s sequence can be determined So how do you locate a gene on a chromosome In order to understand how this is done you have to know something about a process that occurs naturally during inheritance called recombination Focused Reading p 1802 Mendel s first law stop at Punnett squares Meiosis Review p 16772 Meiosis stop at Meiotic errors Fig on 1689 Fig 1019 p 1902 Genes and chromosomes stop at Geneticists make maps p 341 There are several ways stop at bottom of page Fig 1811 and 1812 p 192 Geneticists make maps 2 paragraphs and Fig 1022 87 Fig 1023 amp 1024 WWW Reading Karyotyping The homologous chromosomes segregate during meiosis and are independently assorted into the gametes Thus at your own fertilization you received chromosome 123 from your mother and chromosome 123 from your father Thus you have two of each chromosomeihomologous pairs You inherit your genes in these chromosome packages Each chromosome is a long line of genes Here s an illustration in which brown eyes are dominant to blue and tall is dominant to short This illustrates the inheritance of only one chromosome This happens to all 23 pairs during inheritance Maternal Chromosome El Paternal Chromosome LOCI Blue Blue Eye Color Eyes Eyes centromere Ear Size Short Short Father Blue Eyes Short Sperm 2 1 Brown Blue Eyes Eyes 7 long Short Egg 2 Sperm 1 J Offspring Type 2 Brown Eyes and Long Ears Offspring Type1 blue eyes and short ears In this example long stays with brown eyes and short stays with blue eyes Therefore you cannot get an offspring from this union that has long ears and has blue eves or that has short ears and has brown eyes So if you wanted that combination in your offspring you would be out of luck Brown is linked to long and blue is linked to short forever and ever and ever We ll modify that statement later Well as you know from your reading genes and chromosomes are not nearly that rigid and immutable they tend to exchange pieces when eggs and sperm are produced In the case above then when the mother created her eggs she produced two types Egg Type 1 and Egg Type 2 However in actuality such a woman could produce four types of eggs because this homologous pair might undergo recombination during meiosis During the S phase of interphase an identical copy of the DNA is made Thus each chromosome goes from being a single linear molecule to a double molecule as follows Maternal Chromosome El Paternal Chromosome oci Ialleles I V V Blue Blue Eye Color Eyes Eyes Ear Size Short Short Father The S Phase of lnterphase Blue Brown Blue Brue Blue Blue Eyes Eyes Eyes Eyes Eye Eyes Short long Short Short Short Short Each chromosome makes an exact copy of itself The copies are attached to one another by the centromere Each half of this double chromosome is now called a chromatid remember F ig96 pl60 The homologous pairs which have been ignoring one another in the cell up to this point nd each other and join together or synapse through a protein complex called the synaptonemal complex as follows Blue Blue 7 Blue BUe Blue Blue Eyes Eyes Eyes Eyes Eyes Eyes Eyes Short Short long Short Short Short Short Mother Father Prophase I Blue Blue Eyes Eyes Short Short Syhaptonemal Complex Syhaptonemal Complex This process where the homologues nd each other and bind is called synapsis and it produces a bundle of four chromatids called a tetrad Enzymes called recombinases reside in the synaptonemal complex and these enzymes can cut chromosomes and swap pieces in the process of recombination The inner two chromatids in the tetrad the ones bound by the synaptonemal complex might swap segments through this process Thus after this process the mother s chromatids would look like this Synaptonemal Complex Recombination during Prophase I The two outer chromatids are the original ones or the parental chromatids However because of the recombination event the two inner chromatids are now different from anything that existed in the mother They are called recombinant chromatids As the mother puts each of these chromatids in different eggs some eggs will get chromatids in which blue eyes are linked to tallness and brown eyes to shortness Four different types of offspring would result from this union blue eyes and short brown eyes and tall blue eyes and tall and brown eyes and short Note here that recombination happens in the father as well when he produces sperm but because he is homozygous at both of these loci recombination does not produce any new combinations He still can produce only one kind of chromatidiblue eyes and short Also note that recombination can happen on all 4 chromatids not just the inner two chromatids as shown in this simplified diagram This feature of meiosis and inheritance was discovered by Thomas Hunt Morgan and is used by nature as a way to increase the diversity in a population thus giving natural selection a greater variety of organisms to work on Nature was recombining its chromatids long long before humans ever populated the earth Determining the location of genes on chromosomes is called chromosome mapping and it relies on a discovery that TH Morgan made about recombination That the freguency of 39 39 quot between two loci is proportional to the distance between the two loci on the chromosome That is if two loci are very far apart on a chromosome say at opposite ends then recombination is veg likely to occur at a point between these two loci thus moving their alleles to 92 homologous chromatids Conversely if two loci are very close together on a chromosome it is veg unlikely that recombination will occur in the tiny stretch of chromosome between them and thus they are likely not to have their alleles separated on different but homologous chromatids Understand Good But how does this allow you to map genes or RFLPs on a chromosome Well if you had a way to measure the frequency of recombination between two loci you could determine how far apart they are on a chromosome In order to do this geneticists have de ned the distance on a chromosome called a map unit A map unit is the distance that corresponds to a recombination frequency of 1 Thus if recombination occurs between two loci 12 of the time these two loci are 12 map units apart on the chromosome This doesn t tell you how may kilobases apart they are but it does give you an approximate distance to use as a starting point You can tell how far apart 3 loci are if you use three loci at a time in your analysis For example let s say you know that Statesville Davidson and Charlotte are all located on the same perfectly straight highway Statesville is 20 miles from Davidson and Charlotte is 50 miles from Statesville If I asked you to draw a map of these cities you would have two alternatives Charlotte 50 miles Statesville 20 miles Davidson or Statesville 20 miles Davidson 30 miles Charlotte In order to choose between these two alternatives you have to know the distance between Davidson and Charlotte If it s 70 miles then the first map is correct Ifit s 30 miles then the second one is correct This is exactly how you map genes on a chromosome You take three points three loci and you find out how far apart each of the pairs of loci is by determining the recombination frequency between each pair and then you map them Such a map is called a genetic linkage map because it relies on the properties of linkage to determine map distances Study Questions 1 Describe the methods used to isolate individual chromosomes Why is this an important component in the process of mapping genes 2 What is a tetrad How do the chromatids in a tetrad assort That is how many and which ones go into each egg or sperm cell 3 What is recombination When does it normally occur What are the genetic consequences of recombination 4 Linkage analysis is based on the idea that recombination frequency is proportional to the distance between loci Explain what this means 5 Given genetic data be able to construct a genetic linkage map STOP Focused reading p192 Geneticists make maps Fig 1022 1023 amp 1024 93 WWWeb reading For further help mapping genes httpwwwwhfreemanpurves6e Click on the Math for Life link and read Topic 71 Mapping Genes Contains how to as well as practice problems and solutions But how do you determine recombination frequency You have to be able to detect the alleles and follow them as they are inherited In the example above this was fairly easyiyou can see eye color and height so you can follow the alleles Even though its a bit more technical following RFLPs and disease states allows you to determine recombination frequencies and thereby determine map distances Ifwe were trying to use RFLPs to develop a linkage map of a chromosome in a organism that produces many many offspringisay Drosophila it would be relatively easy to do so In diploid organisms the simplest way to map chromosomes is to do a dillybrid test cross a heterozygote by a homozygous recessive Here is an example of how this would go Southern Blot for RFLPs Mi y DI I I I63 1 2 Femab FmHFw DI I I I I 3963 1 3 2 4 Let s say we are looking at Chromosome 1 of the fruit y You obtain chromosome 1 digest it with a known restriction enzyme and probe it with two different radioactive probes and you get the above Southern blot The male fruit y has two RFLPs on chromosome 1 whereas the female has fourishe shares two with the male 1 and 2 and has two that she does not share with the male 3 and 4 Thus the male is homozygous for these two RFLPs and the female is a heterozygote While we don t know exactly which RFLPs correspond to which loci chromosome 1 in these ies might look something like this Chromosome 1 RFLP 2 RFLP 4 RFLP 2 RFLP 2 fema ruit y male fruit y As it is drawn band 1 and 3 on the female y s Southern blot are alleles of the same locus and bands 2 and 4 are alleles of a second locus Thus this female y is a heterozygote at both loci The male has identical alleles at the first and second loci thus he is a homozygous at both loci Now when this female y creates her eggs she will make four different kinds of chromatids eggs 1 from chromosome 1 Alleles RFLP l Recombinant RFLP l and 4 RFLP 3 2 Genetic linkage mapping is based on the idea that the freguency with which the recombinant chromatids occur is proportional to the distance between the two loci Let s say you mate this female and male y You do a Southern blot on the offspring and obtain the following data Southern Blot for RFLPs on Chromosome 1 P Male 9 Fruit Fly I I 1 Female Fruit Fly 9 I A l N A G 00 F l 35 of I offspring 35 of I offspring 6 15 of offspring G I 15 of offspring I I I I Because the male y is a homozygote he always passes on RFLP l and 2 Thus all of the F 1 offspring have RFLP l and 2 35 of the offspring also inherited RFLP 3 and 4 Thus they received a chromatid bearing 1 and 2 from their father and a chromatid bearing 3 and 4 from their mother This chromatid from their mother is a parental chromatid and thus these ies are not the products of recombination Likewise 35 of the offspring inherited two copies of RFLP l and 2 Thus they received 1 and 2 from their father and l and 2 from their mother Again they inherited a parental chromatid from their mother and are not the products of recombination 15 of the offspring inherited RFLP l and 2 from their father and RFLP 3 and 2 from their mother A chromatid bearing RFLP 3 and 2 is a recombinant chromatid and thus these offspring E the products of recombination Likewise 15 of the offspring inherited RFLP l and 2 from their father and l and 4 from their mother A chromatid bearing RFLP l and 4 is a recombinant chromatid and thus these ies are the products of recombination Thus in the above example 30 of the offspring are the products of recombination Thus the recombination frequency between these two loci on Chromosome 1 is 30 which represents 30 map units If 10 of the offspring had been recombinant forms then these two loci would be 10 map units apart 3 times closer together than if they were 30 map units apa1t It is a relatively simple task to produce a linkage map of an organism that has many many offspring However this is much harder in humans You can t do recombination frequencies in a single family Rather you have to look at an entire population and determine recombination frequencies there Thus you have to gather many many samples and run many many Southern blotsIf you remember in the case of CF the disease was shown to be linked to two markers on chromosome 7 These markers have names everything in biology has a name they are called MET andD7S8 The lab that characterized the marker chooses the names and they can mean almost anything so don t try to look for a scheme to these namesithere is none You can think of them as human names You name your kid Met or D788 and that s the name that identifies that individual Ifyou do the kind of RFLP analysis outlined above w you look for linkage to the disease at the same time you can determine the order of MET D7S8 and the CF gene on chromosome 7 Consider the following simpli ed and hypothetical data MET and D7S8 MET Just as in the example of the three Cities above you now can determine the order of these alleles on chromosome 7 The only map that works for all the data is MET 4 map units CF6 map unitsD7S8 Thus investigators were able to determine that MET andD7S8 anked the cystic brosis gene This is important information because it de nes the location of the CF gene on chromosome 7 We now know that the gene is somewhere between MET and D7S8 and both of these markers are identi able by the presence of restriction target sites and marker sequences Investigators continued to look for RFLPs in this region of chromosome 7 using many different restriction enzymes and many different markers They found 2 more RFLPs that mapped between MET and D7S8 Recombination frequency analysis and Southern blots using pulse eld electrophoresis determined the order of and distance between these RFLPs to be MET 500 kb quotF 980 kb D7S8 The total distance between MET andD7S8 was determined to be 1480 kb or about 15 Mb million base pairs Study Questions 1 Understand how RFLPs can be used to locate genes Be able to interpret a Southern blot to determine which RFLPs are linked to a disease gene 2 Increasingly RFLP analysis is being used to diagnose the presence of carrier status or the genetic status of fetuses by amniocentesis Ifyou were a genetic counselor how would you explain this process to someone who wanted to understand how her disease status would be determined 3 How can investigators determine which RFLPs are on which chromosomes How are individual chromosomes obtained 4 Ifyou were the technician performing the diagnostic test to determine if someone were a carrier of CF what controls would you run Whose DNA would you sample 5 What is a map unit 6 Explain how linkage maps are created 7 Be able to map a DNA segment given the outcome of dihybrid testcrosses 8 Be able to map a DNA segment given the outcome of a Southern blot analysis of RFLPs resulting from a dihybrid cross The search for the CF gene had been dramatically narrowed by linkage analysis of the RFLPs on chromosome 7 Investigators knew that the CF gene was somewhere within a de ned 15 Mb segment So what now Linkage analysis won t help you any more because the distances between loci in this region are so small that recombinant doesn t occur often enough to be detected So investigators had to turn to a different technique called positional cloning or chromosome walking Focused Reading p 3378 DNA markers stop at Human gene mutations p 3945 paragraph beginning In physical mapping of the chromosome stop at The genomes of several organisms note HGP stands for Human Genome Projectip393 P 34850 Sequencing ofthe stop at The human genome Let s pause for a moment and look at the theory behind DNA marker sequences a bit more closely Ideally a DNA marker sequence would appear only once in the entire genome Ideally a given probe should be able to identify one and only one inherited marker sequenceithis inherited sequence would then be unique in the genomeilike the gene for insulin or the gene for cytochrome c How long does a probe have to be to meet this criterion Well if there are 6 X 109 base pairs in diploid human genome a base sequence should be long enough to have a probability of existing at the frequency of l in 6 billion How long is that Well what are the chances that a given base sequence starts with A The answer 1 chance in 4 since there are four bases we ll assume each is equally probable although that does vary a bit in different species If A is the first letter of our sequence what are the chances that the neXt letter is C Again 1 in 4 But the chances of having a base sequence A followed by C is the product of the probabilities of each letter 14 X l4 or ll6 Well with 6 billion base pairs ifthe chances of AC occurring are l in 16 you are going to have millions of AC combinations in the genome 6 billion l6But let s keep going What are the chances ofhaving the base sequence ACC l43 or l4 X l4 X l4 l in 64 The real question we want to ask is coming into focus To what power do you have to raise l4 to get a chance of around 1 in 6 billion The answer is 14 that is l414 about 3 X 109 So ifyou had a marker sequence 14 bases long the chances are that it is one of a kind in the genome However due to practical considerations like the effects of temperature and salt concentrations on hybridization of complementary sequences probes are usually in the 20 to 40 base range What we want to do is clone the CF gene that is hidden somewhere in a 15 Mb piece of DNA which is anked by two RFLPs MET and D7S8 We have probes for the RFLPs which are located at the two ends of the DNA but we do not have any probes for CF so we will have to walk from one end to the other and look for CF as we walk This process is called chromosomal walking and it allows us to clone an uncloned gene that is hiding near an RFLP The term walking is a nice metaphor since we must use two feet in order to walk Likewise we must use two separate genomic DNA libraries each made from the same genomic DNA but using different restriction enzymes In our eXample we will use the enzymes EcoRI and Sal I Genomic DNA Genomic DNA Genomic DNA 1 l digest DNA digest DNA with EcoRI with Sal I make EcoRI make Sal I genomic DNA genomic DNA library library While the procedures of chromosome walking and jumping are a bit complicated they are based on a fairly simple idea Let s say you have the segment of DNA that contains the CF genei the 15 Mb segment of chromosome 7 First you pull a restriction enzyme out of the freezer Let s say EcoRI cuts your 15 Mb DNA segment into 6 restriction fragments that look like this after gel electrophoresis DNA fragments on a gel Above you see the DNA bands in the gel not a Southern blot From this experiment you learn that EcoRl can cut the 15 Mb portion of the chromosome into six fragments Remember that DNA 99 separates on the basis of size and the arrangement of band on the gel does not have anything to do with the linear order of the fragments in a chromosome To walk between two chromosomal markers you need to determine the correct linear order of these fragments Fortunately you have probesithose RFLPs MET and D788quot and you decide to begin looking for the MET RFLP Because MET is a speci c piece of DNA one and only one of these fragments should contain the marker for MET You perform a Southern blot by transferring these restriction fragments onto nitrocellulose and probing with the MET probe sequence for the sake of simplicity this probe is CCCCCCCCCCCCCC thus it would recognize the marker sequence GGGGGGGGGGGGGG in the DNA Your original gel with all of the restriction fragments looked like this Ollll II I And your Southern blot using the MET probe looks like this 9 CD This result indicates that the third band from the top of the gel contains the MET marker the top of the gel is the end you initially add the DNA to and is always labeled because it is closest to the anode in the electrophoresis unit Now you know that one end the MET end of the 15 Mb fragment is in EcoRI fragment 3 You will need to clone this EcoRl restriction fragment from your EcoRI genomic DNA library see p 35960 and Fig 169 Once you have isolated fragment 3 from the library you are ready to take your first step in chromosomal walking The goal is to walk down the chromosome from MET to D788 In order to walk you need to know which direction to take your next step you want to walk towards D788 and not away from it Therefore you need to produce a restriction map of fragment 3 so you can isolate a piece of DNA from fragment 3 that is on the opposite end of fragment 3 from MET We want to isolate the portion of fragment 3 that is labeled with a in the diagram below the location of fragment 3 has been put onto the 15 Mb DNA so you can see that we have identified only 1 of the 6 EcoRI fragments 100 a Closer look at fragment 3 EcoRI EcoRI MET I I 39 frag I 3 mss entire 15 Mb piece Further characterization of fragment 3 will require you to use several restriction enzymes to digest just fragment 3 You digest fragment 3 with each of 3 enzymes alone and in combinations and run them all on one gel The enzymes you choose are EcoRI SalI the enzyme you made your other genomic library from and BamHI another enzyme to help with ordering This experiment will help in two ways it lets you to place the EcoRI fragments in order and it allows you to identify the SalI fragment that is furthest away from the MET marker so that you can continue walking The DNA banding pattern from your restriction digests might look like this with molecular weights indicated to facilitate mapping Agarose gel of DNA fragments that result from digesting Fragment 3 with the enzymes indicated lOl molecular I I I I I I I weights EcoRI 100 I BamHI 60 I EcoRI BamHI I I Sal I I BamHI Sal I 55 I I 25 I EcoRI Sal I I restriction enzymes If this gel were blotted and probed with MET the resulting autoradiograph might look like this 0 molecular weights EcoRI 100 I BamHI EcoRI BamHI I Sal I I BamHI Sal I 55 I EcoRI Sal I I 102 103 quotDNA FRAGMENTSquot TO USE WHEN PRACTICING RESTRICTION MAPPING The boxes below were drawn to scale using the information found in the agarose gel seen on page 93 The shading of each box indicates the restriction enzyme used to obtain the fragment and only the single restriction enzyme digest information is included Cut out the strips and use the information from the agarose gel single and double restriction enzyme digests and Southern blot to determine the order of the restriction sites in Fragment 3 Fragment 3 100kb BamHI 60kb BamHI 40kb ls Sall 55kb I Sall 1425ka Sal I 25kb 104 105 Again each of the bands seen on the autoradiograph indicates the DNA fragment that contains the 14 base pair long probe sequence and ONLY band that contains the probe sequence When you look at these data you can start to gure out the restriction map for fragment 3 A restriction map is like a road map with several cities ie restriction sites in a row connected by different lengths of highway DNA This type of map is called a restriction map because it locates the restriction sites relative to each other similar to a linkage map The restriction map for fragment 3 might look like this note this is not drawn to scale EcoRI BamHI 3 I Sal I I 40 I 15 I 425 I 25CORI I I I l I MET See if you can reconstruct the restriction map using the information found on the agarose gel of digested DNA and the Southern blot The next page contains a set of quotDNA fragmentsquot boxes drawn on the page Cut out the fragments and line them up on the template for Fragment 3 Remember that the gel lanes containing DNA digested with two enzymes will give clues to help you determine how the fragments overlap This is actually study question 2 below Can you determine the fragment order definitively The next task is to isolate the 25 kb Sal I EcoRI fragment and use this 25 kb fragments as your second probe the Sal I library to isolate the piece of DNA along your walk The probe 25 kb piece will be your second probe because it is the DNA fragment furthest from the MET marker and therefore must be closer to CF When you isolate a piece of genomic DNA from the Sal I library it will overlap some with the EcoRI fragment 3 but then extend further to the right because probe 2 is bounded on the left by a Sal I site You know any new fragment that has Sal I sites on both ends and binds to the second probe will extend towards the right in the direction of CF and D788 First step in a chromosomal walk fragment from EcoR I genomic library RI 8 S RI IMET I I I l Second Probe s RI P B RI s CF is in this fragment from Sal I I I I I I I I I 39 genomic library direction 106 When you nd which Sal I fragment binds to probe 2 you gure out its restriction map the same way we did for EcoRI fragment 3 This process continues until you reach the other end of the 15 Mb fragment which is defined by the D788 marker The final product is a series of overlapping fragments covering the entire 15 Mb piece of DNA and each of these fragments has been restriction mapped You may begin to realize that this is a huge project taking lots of time people and money The final restriction map and the overlapping fragments might look like this 107 IR 4 15Mb gt R SSalI Fragment 3 R s s R I H R S MET I I I S S SR R EcoR I R 2nd probe S R 3rd probe I 4th probe D7 S8 5th probe Using this technique you can walk down the chromosome identifying unique marker sites and restriction target sites as you go and establishing distances between the restriction sites Ifyou keep at this until you reach the D788 marker at the far end of the DNA segment you will have produced a complete restriction map of the 15 Mb DNA segment that contains the CF gene A good restriction map will identify a marker sequence or arestriction site every 5 to 20 kb all along the entire 15 Mb segment Study Questions 1 Explain in general terms how chromosome walking is done 2 Given the kind of data presented on page 9394 above be able to construct a simple restriction map Cut out the paper DNA fragments on page 95 and use them fragments to help you Focused Reading p 355 Genes can be cloned to bottom of page p 360 A DNA copy omeNA can be made 2 paragraphs p 361 Fig 1610 We now have a restriction map of the segment of DNA containing the CF gene How does this allow us to isolate the CF gene We know that all cells contain all genes how has the restriction map helped us narrow our search Well all cells contain all genes but each cell type liver retina and muscle uses only speci c parts of the genome If we look at cells that use our gene we can narrow our search In this case we go to the cells that actually make the wildtype version of the CF protein and isolate mRNA from these cells Because we know that CF patients have problems in their lungs pancreas and sweat glands these cells are a good place to start Investigators took these cells from wildtype individuals and isolated the mRNA from these cells Ifthese wildtype cells make the wildtype version of the CF protein they must contain mRNA for this protein they must 108 use the gene After isolating the mRNA from these cells investigators made radioactive probes that were complementary to this mRNA by incubating the mRNA with radioactive nucleotides and an enzyme called reverse transcriptase Reverse transcriptase as the name implies does transcription in reverse It uses RNA as a template to create a complementag strand of M cDNA so reverse transcriptase is a kind of DNA polymerase too We will talk more about this later when we discuss HIV Because mRNA has already had its introns spliced out it contains only exons of the functional gene When cDNA is made from lung tissue every mRNA made in lung tissue e g cAMP dependent kinase actin elastin spectrin NaK pumps ion channels calcium pumps and the Cl39 channel is converted into cDNA Conversely mRNAs that are speci c to other tissues like opsin in the eye will not be included The cDNA present will be a mixed bag of probes that should contain some CF cDNA It is unlikely that our 15 Mb fragment contains more than one gene since 98 of our chromosomes do not contain any genes Therefore since we know that the 15 Mb chromosomal fragment has the CF gene in it by RFLP mapping a cDNA probe from lung that hybridizes with part of our 15 Mb it will identify the piece of DNA that contains the CF gene Since it is technically possible that there are two genes in this 15 Mb area of the DNA from chromosome 7 one is the CF gene and the other some other lung expressed gene we will still have to prove that we have the right gene We ll get to that in a paragraph or two First let s go back to the mix of lung cDNA probes The mixture of all the cDNAs from the tissue e g lungs is made radioactive and this hot cDNA is used as a probe to see if any of the cDNA binds to the 15 Mb area of DNA After performing a lot of Southern blots you might deduce that the cDNA binds to the DNA in the area of the checkered box as indicated below R S SR H H SR I l l l CF cDNA probe D788 This is a simplified and hypothetical example The real cDNA probe used to find the CF gene was synthesized from sweat glands since those are easier to obtain than lungs and was 6129 bases long The restriction map of the 15 Mb segment on chromosome 7 was much more complicated than this diagram Nevertheless the underlying approach is exactly the same Study Questions 1 What is cDNA and how is it made 2 How was cDNA used to actually pinpoint the location of a gene on a restriction map 3 Given the kind of data presented above be able to pinpoint the location of a gene on a restriction map 109 4 Sometimes investigators probe DNA segments with cDNA and identify the wrong gene Why does this mistake occur Why isn t this system of gene identi cation foolproof Using this cDNA probe method investigators determined that the gene encoding the normal allele at the CF locus is 250 kb long huge and contains 27 exons After mRNA processing the 250000 bases in the gene are reduced to 6129 bases in the nal mRNA This means that 243871 bases in the gene are in introns This mRNA is translated into a protein that is 1480 amino acids long with a molecular weight of 168138 daltons 168 kilodaltons or kD Having pinpointed the gene and knowing which restriction target sites ank the gene this very speci c piece of DNA can be isolated cloned or copied and sequenced Review and Focused Reading p 26216 DNA replication to end of Chapter p 215 Fig 1120 Study Questions 1 Describe the natural process of DNA replication What proteins are involved in the process What role does the primer play in this process What is the primer made of 2 Why is DNA replication called semiconservative What is conservative about it What is semi about it 3 Explain the process of DNA sequencing Why are dideoxynucleotides used in this process 4 Be able to interpret a Sanger sequencing gel to give the correct base sequence of a DNA segment with the correct 5 to 3 orientation negative DNA migration PO399 A T G C long fragments E short fragments positive pole 110 The sequence of bases in the 27 exons of the gene at the CF locus was determined by DNA sequencing Once the base sequence of these exons was identi ed the amino acid sequence of the wildtype protein was deduced using the genetic code on page 224 Investigators noted that in this protein long stretches of hydrophobic amino acids alternated with long stretches of hydrophilic amino acids This pattern of amino acid distribution is consistent with an integral membrane protein Also the amino acid sequence of this protein had a pattern that was similar to several ion channels whose encoding DNA had been sequenced already ie there was some homology Now investigators performed the crucial testithey needed to establish that some of the DNA bases in this gene are different in CF patients than they are in wildtype individuals Remember there is still a slim possibility that this gene could actually encode some other protein made by sweat gland cells investigators had to establish that th gene is altered in CF patients to support their hypothesis that this gene product is involved in causing cystic fibrosis They used the wildtype cDNA as a probe to isolate cDNAs from CF patients and they sequenced these cDNAs After comparing the DNA sequence from the wildtype gene to the sequences of the same gene in people having CF they found that in 70 of CF patients one codon was deleted from an exon in this gene The missing codon encoded amino acid 508 which is a phenylalanine in the wildtype gene The shorthand abbreviation for phenylalanine is F Thus this mutation is called AF508 a deletion A of phenylalanine F at position 508 So it appears as though investigators have found the gene that causes CF at least in 70 of the cases Unfortunately the remaining 30 of cases are caused by over 600 different mutations in the CF geneia very difficult basis for finding a common cure Approximately 4 of CF alleles contain nonsense mutations at different codons The next step in the process was to try to figure out what this protein does and how the AF508 mutation keeps it from doing its job Computer assisted analysis can produce a likely three dimensional structure or topology of a protein from its amino acid sequence by predicting common protein folding patterns or motifs based upon what is known about homologous proteins For instance given the position of polar and nonpolar R groups we can predict which domains probably form an alpha helix like a corkscrew or a S pleated sheet like corrugated cardboard or if this protein is embedded in the membrane Computer assisted prediction of protein conformation is a rapidly growing field but predictions for large proteins are still fairly crude Nevertheless the analysis of the wildtype version of the CF protein clearly predicted that it was a 12pass integral membrane protein A 12pass protein zigzags back and forth through the membrane 12 times like so 111 1111111111 ATP Binding Sltes 1 and 2 2F508 7 Regulatory Site by Phosphorylation 1 The nucleotide sequence in two cytoplasmic areas are predicted to be ATPbinding sites and sites needed for regulation of the protein by ATP binding and hydrolysis This structure with sites for ATP binding is typical of ion pumps and ion channels and is consistent with the hypothesis that this gene encodes a Cl39 ion channel The regulatory domain can be phosphorylated by a cAMP dependent protein kinase sound familiar When R gets phosphorylated then the gate is opened to allow Cl39 ions to move out of the cells The early evidence that lung cells from CF patients cannot export Cl39 when cAMP levels rise correlated very well with the protein structural information acquired through molecular or DNA methods When mutated this integral membrane protein causes CF therefore it was given the name CFTRiCystic Fibrosis Transmembrane Conductance Regulator The conductance being referred to here is chloride ion conductance This is a fairly vague name but good scientists hate to jump to conclusions with preliminary evidence No one wants to be the person who named this protein the cystic brosis ATPdependent chloride ion pump only to nd out a few years from now that it isn t a chloride ion pump at all When something appears in print for all eternity better cautious than wrong At this point we need to figure out why a chloride ion channel would make the mucus in lungs more viscous and all the other problems associated with CF In order to understand this we need to understand osmosis Focused Reading p 8687 Osmosis is passive stop at Diffusion may be p 87 Fig 58 Unlike sodium or calcium water is not a leader but a follower a lamb in a world of Marys Think of ions as Mary as in Mary had a little lamb Wherever the ions go the water is sure to follow All cells have to control the amount of water in their cytoplasm in order to survive This is 112 most obvious in plants that do not get enough water and begin to wilt Cells have to move water to maintain their cell volume and internal pressure but they cannot actually bind water and move it Likewise animal cells and their secretions need to have a balance of water and salt So they rely on the process of osmosis to move water If chloride ions cannot leave the cell and enter the mucus the mucus does not have enough ionic strength to pull more water out of the cells and the mucus is left as a sticky paste NEWS ITEM Having too much water in mucus causes as much trouble as having too little A rare genetic disorder called pseudohypoaldosteronism I PHA causes uid buildup in the airways of the lungs The uid causes wheezing and infection but fortunately the condition is usually outgrown with time The cause A defective epithelial sodium channel that can39t pump sodium out of the cell Using what you know about osmosis why would this result in uid in the airways Why might these people be able to 39outgrow39 their problem The first question you should be able to answer the second requires speculation Reviewed in Dorrell S 1999 MalecMed Today vol 5 p 462 Study Questions 1 Explain the process of osmosis What is producing the force that moves water during osmosis In what way is the process of osmosis an example ofthe concept expressed by the 2quotd law of thermodynamics 2 While the movement of water across cell membranes cannot be directly controlled it can be indirectly controlled Explain how the transport of water is controlled Explain how this process may ultimately rely on ATP as a source of energy 3 What is osmotic pressure What makes a solution hypotonic Hypertonic Isotonic Understand the direction of movement of water under different conditions of osmotic pressure See Fig 58 p 87 Now back to our understanding of CF Where does the AF508 mutation appear in the CFTR It is in the first ATPbinding site Ah ha Good place for a mutation that seriously impairs protein function hypothesis would be that maybe this protein can t bind ATP and therefore can t get any energy to move Cl39 Cl39 cannot move from the cells into the airways of the lungs and pancreatic ducts The water which would have normally followed the C1 by osmotic pressure does not enter the mucus so the mucus becomes thick You get cirrhosis because some other product bile requires this dilution effect as well and when it doesn t happen this dry product clogs the liver ducts causing cirrhosis And finally the sweat glands cannot move Cl39 into the sweat water does not follow and therefore the sweat remains highly concentrated with Na ionsSimple right Well a cardinal rule in science is this An explanation can make perfect sense be awless in its logic E be dead wrong So let s not jump to any conclusions prematurelyithis is only one hypothesis We need to see if experimental evidence about the role of the CFTR in cells supports this hypothesis or if another hypothesis is more plausible Study Questions 1 Draw the hypothetical structure of the CFTR protein and explain each of the significant features of the protein From what experimental evidence and methods is this structure derived 113 2 In what portion of the CFTR protein is the AF508 mutation located Given the location of this mutation describe the most straightforward hypothesis explaining the failure of this protein to successfully move Cl39 WWW Reading in situ methodology You could hypothesize that the protein is in the membrane but cannot function properly because it cannot bind ATP or because it cannot cleave ATP to ADP or because it cannot be phosphorylated by cAMPdependent protein kinase Studies on the normal version of CFTR protein show that phosphorylation by protein kinase A is also a requirement for C139 movement Thus the mutation may make phosphorylation event impossible These questions can be approached in several ways For instance you could hypothesize that the mutation in the CFTR gene keeps it from being transcribed into mRNA To approach this question you would perform in situ hybridization on the usual tissues from a CF patient If you did not find mRNA for CFTR you could conclude that the mutation caused a problem in the creation or stability of mRNA Alternatively if you found normal levels of CFTR mRNA in CF patients you could hypothesize that the mutation keeps the protein from being translated or properly targeted within the cell You could use immunohistochemistry to look for the protein on the cells of CF patients The absence of the CFTR protein would mean a defect in translation or posttranslational processing or transport Investigators looked for CFTR mRNA with the procedure called in situ hybridization In situ means in the normal location in this case in the intact cell and as with all DNA probes the probe hybridizes to its complementary sequence In the case of in situ hybridization the target is mRNA within the cell s cytoplasm For these studies they took radioactive CFTR cDNA and used it as a probe directly in lung tissue All cells containing mRNA for CFTR will become radioactive when the cDNA hybridizes to the mRNA Cells not expressing this mRNA will not become radioactive because the probe had nothing to bind with These studies showed high expression of the mRNA in pancreas sweat glands salivary glands intestine and reproductive tract and lower expression in respiratory tissue So this study demonstrated that CFTR mRNA exists everywhere there are clinical symptoms Does this support our hypothesis above that CFTR is the CF protein Well it is certainly accepted by the scientific community However you will note from this discussion that you can never be absolutely sure you are right Proof in science is based on evidenceisometimes solid sometimes shakyibut only evidence No one ever comes along to say You ve solved it You re right The best that happens is that you and other scientists base many many experiments on your theory and it always holds up That s as close as we come to having scientific proof So even though the mutated version of the CFTR protein is pretty much accepted as cause of CF much controversy still remains about what wildtype CFTR actually does and how the mutation keeps it from doing its job the localization studies did not address that part of the hypothesis Several approaches can be taken in order to try to determine the function of a protein once its gene has been identi ed and isolated Ifyou remember wildtype respiratory cells will pump C139 to the outside when intracellular cAMP levels rise Respiratory cells from CF patients cannot do this One standard approach then is to transfect respiratory cells from CF patients with the dominant wildtype CFTR gene isolated from a wildtype individual In this process the functional gene is transferred into the CF cell to see if this gene can restore the wildtype condition 114 There are several ways to do this You first need to connect the cDNA that contains the CFTR to an appropriate promoter This promoter need not be the CFTR promoter rather it could be a promoter for a gene that is turned on by some easily controlled environmental event For instance the protein hormone insulin is produced when blood glucose levels are high insulin lowers the blood glucose levels Therefore the insulin promoter promotes gene expression in response to high glucose concentrations in the uid bathing the cell If you put the insulin promoter upstream from the CFTR gene this gene will be expressed in response to high blood glucose levels Figure 1713 p323 contains a diagram of an expression vectoria plasmid that allows you to express a foreign gene The CFTR cDNA with its arti cial promoter is incubated with CF respiratory cells in tissue culture Under certain conditions the cells will take up DNA and begin to express this foreign gene as if it were their own The transferred gene is called a transgene and the cell containing the transgene is called a transgenic cell The process by which transgenes are put into eukaryotic cells is called transfection When you transfect CF respiratory cells with the CFTR transgene these cells are restored to wild type function ie when intracellular cAMP levels rises they move Cl39 across their plasma membranes at normal rates This is pretty good evidence that this gene encodes a CF protein that moves C139 in response to a cAMP signal Cl39 movement requires ATP because ATP is a ligand and CFTR is a ligandgated ion channel However the inabilitv to bind ATP is NOT whv AF508 causes CF WWW Reading Immuno uorescence Methodology Okay So if our initial hypothesis is not supported as good scientists we have to modify it Another possibility is that the AF508 mutation effects expression of the gene product the CFTR protein in cells that are affected in CFirespiratory pancreatic hepatic liver and sweat gland cells One approach to studying the protein localization is called immunocytochemistry In previous approaches we used nucleotide probes DNA or cDNA to detect nucleotides DNA or mRNA In this approach you need a way to see or detect a protein To do this you must inject your protein of interest ie CTFR into an animal like a mouse rabbit rat goat etc Because it is a human protein parts of its structure will be foreign to this animal Immune systems react to any protein shape that is not self and the animal will react to this foreign shape by producing an antibody Antibodies are proteins with specific binding sites for foreign shapes These foreign shapes are a kind of ligand called an antigen Thus antibodies bind antigens like enzymes bind substrates like receptors bind hormones like transport proteins bind transported substances etc see a pattern here This binding is speci cijust as in the case of all these other proteins an anti CFTR antibody will bind to CFTR and only CFTR To detect CFTR in cells then you bathe the cells in a solution containing antiCFTR antibody The antibody will bind to CFTR wherever it is located in the cell This antibody is called the primary antibody in the immunocytochemistry see the diagram below Now you have tagged the CFTR with the primary antibody and you need to provide a way to see that tag So you then apply a secondary antibodyione that has been produced 1 to recognize the primary antibody and 2 has been covalently bonded to a uorescent tag that can be seen under the microscope or by a machine For instance if the primary antibody was produced in a mouse the secondary antibody would made by injecting mouse antibodies into a goat to make an antiantibody and then chemically binding the goat antimouse antibody to a uorescent dye This secondary antibody is incubated with the cells from above Every place the antigen CFTR exists 115 the primary antibody binds and then the secondary antibody binds to the primary antibody making the area colored or uorescent Here s a picture Fluoresoe nt orV isible Marker Primary Sec ondary Antibody Antibody Cell Cell In this example the cell on the left bearing the CFTR protein will become uorescent during this procedure while the cell on the right will not Thus you can determine the presence of CFTR and in some versions of this technique you can determine the density of the protein in the membrane and precise subcellular localization When investigators used immunohistochemistry to look for CFTR in the wildtype tissues they found the protein expressed in high concentration in the pancreas sweat glands salivary glands intestine and reproductive tract and lower levels of expression in the respiratory tract However in patients with AF508 all of the CFTR was trapped in the ER 39 this mutation causes the CFTR to be inappropriately sorted it never reaches the plasma membrane and this is the cause of 70 of all CF cases Study Questions 1 What is the cause of CF in patients with the AF508 mutation 2 Describe the process of transfection immunocytochemistry and in situ hybridization How have these approaches been used in CF research 3 We know a great deal about the CF protein but much remains to be discovered If the editor of the prestigious scienti c journal Science called you and asked what were the three most compelling questions remaining about this protein what would you tell him Note he would most certainly want you to explain your rationale for these choices 4 If CF causes cells to die and release their contents why would a physician prescribe DNAase to reduce the viscosity of the mucus 116 NEWS ITEM Just because CFTR has its function at the plasma membrane does not mean that it is always located there Some channels like the GLUT4 channel involved in glucose uptake spend most of their lives in vesicles inside the cell and are only placed in the plasma membrane when they are needed why have a hole in the cell if there is no reason for it Since Cl secretion by CFTR is activated by cAMP researchers at Dartmouth Medical School examined whether cAMP changes the localization of CFTR or if it simply turns the channel on To watch CFTR they made a DNA construct that would code for CFTR attached to the green uorescent protein GFP GFP glows so anywhere this CTFRGFP was found researchers could see it glowing under the microscope The conclusion in the cells tested cAMP acted like a switch to open the channel already located in the plasma membrane not like a moving van that got the channel there in the first place BD Moyer et al 1998 JBC vol 273 2175968 Q Focused Reading p 3478 Gene therapy stop at Sequencing p 31517 Vectors carry stop at Genetic markers identify We now know that the binding of ATP at site 1 converts the channel from a locked mode to an unlocked mode but this does not open the channel ATP binding at site 2 open the channel but only if there is ATP already bound to site 1 and the R domain is phosphorylated This may seem complicated but this is a simpli ed version of a process we don t fully understand It will get more complex each year Aren t you glad you didn t put off Biolll until next year The hope in all of this of course is for a cure to cystic brosis Because it is a genetic disease it could theoretically be cured if a good CFTR gene were delivered to the cells of the CF patient in such a way that it could express a normal protein Such an approach is called gene therapy Because the most lifethreatening symptoms of the disease occur in the respiratory system such a gene could possibly be delivered in an inhalant aerosol spray Several DNA delivery systems are being currently investigated including retroviruses adenoviruses liposomes and DNAprotein complexes As we will discuss in Unit IV viruses function by entering living cells and expressing their genes using the cell s protein manufacturing system If the diseasecausing genes from a virus are removed and a functional CFTR gene added these viruses could enter the respiratory cells and begin expressing the CFTR gene Such a carrier of a gene is called a vector Liposomes small spheres of phospholipid are another way to apply gene therapy By loading a functional CFTR gene onto a liposome and then spraying it into the respiratory tract it may be taken up by respiratory cells the cell membrane will fuse with the liposome as in the processes of endocytosis and may be expressed as a normal gene product Now all this sounds really straightforward but it is a long and journey from an idea to the nished product We don t know for instance if any of these genes will actually be expressed once they are inside the respiratory cells In addition Francis Collins has de ned a number of other questions that must be addressed before a viable therapy is available Science Vol 256 p 778779 1 What are the relevant cells to treat The respiratory tract is full of all kinds of different cells Which ones are the best ones to treat in gene therapy 2 What fraction of the responsible cell types must be corrected to achieve clinical bene t Certainly one would not have to correct the CF defect in every single cell in the lungs in order to reach an acceptable level of health How many cells do you have to treat ll7 3 Is over expression of CFTR toxic One problem with transgenes is that they do wind up at the CF locus of the person s chromosome number 7 and therefore are not subject to the normal genetic control systems of the promoter that function at the level of the clu Over 1 39 J expressioniis a constant threat in gene therapy Would such a thing be toxic to the individual 4 How long will expression persist Even if you can get these transgenes to be expressed will they continue to be expressed inde nitely Transgenes vary widely in their level of stability Some function only very brie y some function for the life of the cell How will these respiratory transgenes behave 5 Will the immune system intervene As we discussed earlier the immune system will respond to anything that is not self Ifthe CFTR protein is not expressed in a particular CF patient it may be seen as foreign by the immune system Thus its sudden expression could cause an immune reaction that destroyed the respiratory cells This process is called autoimmune disease 6 Can safety be insured This is always a question with bioengineered organisms such as the viral vectors in this approach Will they get loose especially if it is delivered in an aerosol and infect everybody thus transfecting normal individuals with the CFTR gene And if this happened would this be dangerous Study Questions 1 Explain the approaches that are currently being tested in gene therapy for CF 2 What are some technical barriers which must be solved before an effective gene therapy for CF becomes available Due to the problems associated with gene therapy researchers are still looking for conventional means for treating CF Recent efforts have focused on the salt concentration in the lungs of CF patients As you should remember from lab isocitrate dehydrogenase IDH experiments proteins do not work well in high salt environments When CF and wildtype lung epithelial cells were grown in culture and incubated with the bacteria most commonly found in CF infections the wildtype cells were able to kill the bacteria while CF cells could not When salt was added to the wildtype cells they were no longer able to kill the bacteria and when the salt was reduced for CF cells the bacteria were killed This suggested that lung epithelial cells secrete a bactericide that is saltsensitive Therefore researchers began to look for other ion channels located in the plasma membranes of lung epithelia Their rationale was to increase the secretion of Cl39 ions which would draw water into the mucus dilute the salt concentration and allow the lung s naturally produced bactericide to function This alternative Cl39 ion channel has been found It is a calciumactivated chlorideion channel which can be stimulated to open when ATP or UTP is administered to the outside of cells This breakthrough has lead to the first clinical trials in which CF patients have been given aerosolized UTP UTP had prior FDA approval while ATP did not Patients treated with UTP are able to clear their lungs better and over time it is hoped they will have fewer infections Meanwhile the search is 118 on for the bactericide in hopes that this could be given directly to CF patients in addition to UTP treatment This work is being headed by Dr Michael Welsh at the University of Iowa and was published in April 19 1996 issue of Cell NEWS ITEM Gerald Pier and his colleagues at Harvard and UNCChapel Hill have determined that the bacterium Pseudamanas aemginasa a cause of chronic lung infection in CF patients binds to CFTR in lung cells In wildtype cells the bacteria bind to the CFTR and are internalized by phagocytosis and killed In patients with AF 508 the bacteria are not internalized and killed which can permit the bacteria to live and reproduce in the lungs Therefore CF patients are hypersusceptible to infection by P aeruginasa See Pier et al Science Vol 271 6467 5 January 1996 Some CF patients suffer from thick mucus and also show altered fatty acid levels in their cellular membranes Juan Alvarez and Steven Freemen Harvard Med School and Beth Isreal Deaconess Med Center created transgenic mice that still had mutant CF TR but corrected the lipid biosynthesis problem Amazingly these mice showed none of the pathology symptoms associated with CF This work may point the way to CF treatments through treating patients with high levels of particular fatty acids Reviewed by Greener M 2000 Malec med today v016 p 4749 As the last part of this unit we will look brie y at the quest for the gene that causes Huntington s disease While the fine points vary from the CF story the approach to identifying the gene was essentially the same However HD investigators did not have a protein candidate early on as in the case of CF In fact the protein that causes HD is still a complete mystery Nevertheless in March of 1993 the HD gene was nally identi ed and cloned HD is a dominant trait and less common than CF in the human population Thus the odds of finding a person who is homozygous for the disease are very low As a researcher why would you want to nd a homozygote to include in your genetic analysis Consider this if you have only homozygous wildtypes and heterozygotes to analyze for RFLP linkage you might get the following Southern blot 119 wildtype Huntington s Disease Individuals 13 are wild type and 47 are HD sufferers Unfortunately there are no obvious bands that correlate 100 with the disease It would be much easier to identify if one person with offspring had two affected HD alleles You could then identify the band that is passed on to subsequent generations and cosegregates with the disease A real breakthrough in HD research came when Dr Nancy WeXler a clinical psychologist and daughter of an HD af icted parent found an area in Venezuela where the incidence of the disease was very high maintained through intermarriage within the town WeXler recommended that investigators study the inheritance pattern of the disease in this group Investigators arrived at the town in 1979 to try to find a homozygote for HD Little did they know they were about to encounter an enormous extended family of over 10000 individuals all with HD or related to someone with HD This is certainly the richest source of familial genetic information for HD that has ever been assembled Out of a total population of 12000 people 258 had the disease and all were direct decedents of a woman who lived in the 1800 s It is believed that this woman had the misfortune of having a spontaneous new mutation which was not present in her parents that caused HD in her and her family A large consortium called the Huntington s Disease Collaborative Research Group was begun headed by Dr James Gusella After collecting samples in Venezuela and identifying individuals that were very likely to be homozygous for HD the offspring of two af icted individuals consortium investigators in returned to their labs and began looking for RFLPs that were linked to the HD gene Usually this process takes years but these people got m lucky and almost immediately in 1983 found a RFLP that was closely linked to the disease This RFLP containing a marker called G8 was always present in af icted individuals and never present in wildtype individuals In addition because these investigators had obtained blood from HD homozygotes they were able to determine 120 the RFLP fragment that contained the normal equivalent of the HD gene It appeared that the quest for the HD gene was going to be short and sweet and everyone was very excited As was the case in CF and every other genetic disease as soon as a reliable RFLP is discovered the disease can be diagnosed by looking for the normal and disease RFLP in a Southern blot So very early on a diagnostic test for Huntington s disease became available The availability of this test forces people with an HD parent to make an agonizing decision Should they have the test or not If one parent had HD they stand a 50 chance of having the disease themselves Most of these people have watched the chronic deterioration of body and mind caused by this disease as their parent died or is dying They now must make a choice about whether or not they want to know if that is the way they will die too This example brings into sharp focus the impact of biotechnology on our lives Because the test now exists children of Huntington s victim must decide what they want to do Even if they decide not to have the test to let nature take its course they have been forced to make a decision that before the technology existed was completely out of their hands Increasingly biotechnology forces us to decideito withdraw a respirator to conduct amniocentesis to detect fetal abnormalities to abort those fetuses we might consider undesirable to register as a recipient or a donor of an organ transplant to be tested to determine if we are genetically predisposed to cancer or heart disease or diabetes Having to make decisions might be the most significant byproduct of the biotechnology revolution But back to the quest for HD As it turned out all the euphoria about how quickly the HD gene would be discovered evaporated as it became apparent that the search would be long and arduous One of the problems was that the HD gene was mapped to the very end of the short arm of chromosome 4 4p This area of chromosome 4 has been described as a gene junkyard and is peppered with many short segments of DNA that could encode short peptide sequences interspersed with intron sections In addition to the difficulties posed by the messiness of 4p the HD investigators did not have a clue about the protein or the cells involved in the development of HD The CF investigators developed cDNA probes from respiratory or sweat gland cells But HD investigators did not know which cells of the brain might be making the normal or abnormal version of the HD protein Well investigators slowly narrowed their search on chromosome 4 by finding RFLPs that were linked more and more closely to the presence of HD that means through a study of the recombination rates the RFLPs segregated from the HD locus less and less frequently This narrowed the DNA segment of the search to a 500 kb region 3 times smaller than the CF region They couldn t narrow their search any more by linkage analysis because they had arrived at a point where the anking RFLPs were so closely linked to the gene that a recombination event was never detected between them an effective chromosomal distance of 00 map units So it was time to walk down the chromosome and create a restriction map of this 500 kb region But the HD investigators had to take a slightly different approach to this problem than the CF investigators because they would never be able to probe their mapped segment with cDNA So now what The HD investigators began by creating a set of overlapping fragments called contiguous fragments of DNA 1 which were mapped as was done for CF 121 Undi ge sted DNA fragment EcioRI Hind III Hind III EcoRI Hind III EcoRI EcoRI EcoRI digestion Contig 1 Area of Overlap I Hind III digestion I I I COHtlg 2 Hind III Ec 0R1 Hlnd 111 By digesting this segment of DNA with two different restriction enzymes two contiguous and overlapping segments were created Sixteen such contiguous segments were created from the 500kb region known to contain the wildtype allele of the HD gene Each overlapping segment or contig was put into a vector Each vector was inserted into a strain of yeast that replicated the human DNA along with its own DNA Thus as long as these yeast kept dividing and they do that as long as you feed them they kept making copies of these 16 contiguous segments of human DNA which contain the HD gene By the way this shows how simple DNA cloning is but don t tell anybody because everyone will want to major in biology Then they did something quite interesting called exon ampli cation They transfected a monkey cell line so that 16 different petri plates of cells each received a different contiguous segment of DNA Researchers then looked for an mRNA that resulted from transcription of any genes contained within that segment of DNA If a segment contains a real exon from the HD geneione that contains a coding sequence and is anked by the proper recognition sites for mRNA splicing then a new mRNA product will be created in the monkey cells Ifthe segment does not contain a real exon but only introns or junk DNA that does not encode a peptide no new mRNA would be created By doing this and mapping the exons to their exact location based on which segments they were on they identified a functional gene isolated cloned and sequenced it The wildtype gene is 210 kb long ouch and creates a processed mRNA transcript that is 10366 bases long that s huge and 120000 bases of the gene are in introns This transcript is translated into a protein that is 3450 amino acids in length and has a molecular weight of 348 kD a large protein Because huge computer libraries of amino acid sequences are available and M can access them via WWW you can enter the sequence of their newly found protein and ask the computer to compare the sequence of this protein to the sequence of all known proteins When the HD investigators did this the computer told them that their protein was not similar to any other known protein It would have been nice to have a known homologiit would have provided a clue about what this protein does as was the case of the CFTR protein looking like other ion channels But alas not so lucky As it stands we have no clues about the function of this protein even though we know the sequence of the entire gene 122 The HD investigators did notice something quite unusual about this gene however At the 5 end of the coding area the codon CAG repeats itself many times CAG is the codon for the amino acid glutamine This type of nucleotide pattern is called a trinucleotide repeat In the normal HD gene from nonaf icted individuals CAG is always repeated between 11 and 34 times in this region In itself this is not so unusual Many functional genes contain trinucleotide repeats However the HD gene from afflicted individuals always contains from 38 to over 100 copies of CAG in this region This increase in the number of codons is a kind of mutation called a trinucleotide repeat expansion This mutation accounts for the difference between the HD gene and its wildtype alleleithe number of times CAG is repeated at the 5 end of what appears to be the coding area of the gene We will see something like this during the last two weeks of lab While this doesn t give us much help in understanding the protein defect in the HD gene this type of mutational change is also found in at least three other less wellknown genetic diseases myotonic dystrophy fragile X syndrome and spinal bulbar muscular atrophy see p 339 and fig 189 Therefore this trinucleotide repeat expansion a form of insertion mutation is a type that has been shown to produce at least three other genetic diseases This greatly strengthens the evidence that investigators have actually found the HD gene In November of 1995 researchers made a startling discovery When the protein encoded by the HD gene now this protein is called huntingtin has 38 or more glutamines in a row it has very different binding properties form meets function again Huntingtin normally binds to another recently discovered protein called huntingtin associated protein number 1 HAP 1 When there are 38 or more glutamines in huntingtin it appears to bind more tightly to HAPl The increased binding causes a change in the level of activity of a dimer of huntingtin and HAPl such that only one mutant allele of the huntingtin gene is sufficient to cause a dominant disease In wildtype individuals huntingtin and HAP1 probably have the same function still unknown as of July 2001 as in affected individuals but this function is properly regulated in healthy people Too much of this activity leads to neuronal cell death Researchers have used immuno uorescence to determine that huntingtin is present in every cell of the entire body Why only neurons are affected is unclear but this phenotype may have to do with which cells express HAP1 Locating and characterizing the function of huntingtin HAP1 will occupy investigators for a long time The brain is one of the most complicated chemical systems in the body and the most mysterious But in the search for huntingtin investigators will undoubtedly learn much about the biochemical function of the normal brain as well as coming to a better understanding and possibly cure of the biochemical defect that causes Huntington s Disease NEWS ITEM We know that the mutant form of huntingtin contains a polyglutamine region that is toxic There are fourteen known neurological diseases that have this sort of repeat or a trinucleotide repeat region that causes the protein to not be translated at all Recently a group from Canada has shown that PKR a doublestranded RNAbinding protein PKR preferentially binds mutant huntingtin Previously PKR had been linked to been linked to a form of virallyinduced and stressmediated cell death called apoptosis Could it be that the neurological defects in Huntington s and other trinucleotide repeatbased diseases are due to mutant RNA transcripts interacting with PKR and initiating something like apoptosis PKR is found in the right tissues at the right time so researchers will be looking in this direction in the future A L Peel et al Hum Molec Gen 2001 Vol 1015311538 July 2001 Study Questions 1 In attempting to locate and characterize the genetic defect causing a disease explain why it is helpful to have an individual in your sample who is homozygous for the disease trait 123 7 The cells that express the HD protein are unknown to investigators How did this fact change the approach taken by HD investigators when compared to that taken by CF investigators What is contiguous DNA and how was it used to clone the HD gene What is exon ampli cation and how was it used to pinpoint the location of the HD gene HD investigators determined that the normal version of the HD gene is not similar to any other known protein in structure How do they know this What is the actual genetic defect in HD What is this type of mutation called Why does the presence of this type of mutation in the HD gene strengthen the evidence that investigators have located the gene that actually causes Huntington s disease Look at the gure called HD pedigree showing anticipation on the Biol 11 Home Page What do you think caused the patients to get HD at younger ages with each generation NEWS ITEMS In January 1998 another international team has isolated a disease version of a potassium channel that appears to cause some cases of schizophrenia The interesting cause of the defective ion channel is that there is a trinucleotide repeat which causes too many glutamines to occur in a row in this disease allele It appears that this allele over stimulates some neurons in the CNS and causes other proteins to change their function which leads to the mental state we call schizophrenia see January 1998 issue of Molecular Psychiatry STOP And nally a note about sexlinked genetic disorders such as bluegreen color blindness hemophilia A and Duchenne s muscular dystrophy de ned p 377 structural proteins Focused Reading p 1956 Genes on sex chromosomes to end of chapter p 1956 Fig 1025 amp 1026 Here is a pedigree for a family with hemophilia A 124 1 2 I I0 4 5 6 7 8 9 1o 11 12 13 Males are hemizygous analogous to being haploid for sex chromosomes because they are XY and the Y chromosome is greatly reduced in length compared to the X chromosome Thus the genes on the X chromosome have no corresponding alleles on the Y Sexlinked genetic diseases all map to the X chromosome and most are expressed in a dominant fashion in males and in a recessive fashion in females Because females can have a wildtype allele to counter balance the defective one on the X homologue they frequently escape the effects of sexlinked genetic diseases However because males are haploid at the sex chromosome if they inherit a single diseased copy they have the disease In the pedigree above individual 1 amale has the disease The disease is carried on his X chromosome Therefore he cannot pass the disease on to his sons because they must receive his Y chromosome in order to become male However all of his daughters will inherit his X chromosome that is what makes them girls they must inherit an X from both parents Individual 12 inherited his diseasebearing X chromosome from his mother who inherited it from her father Therefore all mothers of hemophiliacs must live with the knowledge that they are the genetic source of their sons disease This is good news for researchers because a genetic disease that is sexlinked is easier to identify and isolate since the researchers start out knowing to which chromosome the gene maps Study Questions 1 What are the genotypes of all of the individuals in the hemophilia pedigree assuming individual 2 is homozygous normal Assuming individual 2 is a hemophilia carrier 2 How did individual 1 1 get hemophilia E Given the genotypes of individuals bearing sexlinked traits be able to predict the genotypes and phenotypes of the offspring e g Male with no disease crossed to a female carrier etc 4 Test your understanding of the overall concepts in this Unit by thoroughly explaining this newspaper article to a classmate How do you think these investigators approached this problem Upon what classic genetic principles was their work based What aspects of modern biotechnology made this discovery possible Based on information in this article would you classify Alzheimer s disease as a Mendelian genetic disease Why or why not 125 126 From the M 39 quot Star Tribune August 13 1993 Most common form of Alzheimer s linked to cholesterol processing gene From News Services Washin on DC n I have linked the most common form of Alzheimer s to a gene that helps process cholesterol enabling them to identify some patients who are virtually certain to develop the minddestroying disease in their elderly years The discovery could account for half of all patients with the common neurological disorder they said and it points the way toward devising treatments to block or at least delay the ultimately fatal symptoms of the 39 39 39 illness About 4 million Americans suffer from Alzheimer s and the number is expected to increase sharply as the population ages In research on 42 families where lateonset Alzheimer s is common Duke University scientists found a 90 percent risk of the disease by the age of 80 among people with two copies of a gene variant called rquotrr quot E type 4 or APOE4 Copies ofthe APOE4 gene also was linked to people developing Alzheimer s at an early age said Dr Allen D Roses of Duke What this shows is that APOE4 increases the risk and lowers the age at which you get the disease he said It looks like virtually all will develop it the disease by the age of 80 if they have two 11 A report on the study appears in today s issue of the journal Science Dr Zaven Kachaturian director of Alzheimer s Research at the National Institute of Aging one of the National Institutes of Health said the research has caused a great deal of excitement among Alzheimer s researchers because it links the most common form of the disease with a speci c gene factor APOE4 that can be measured It could become a diagnostic tool said Kachaturian We may be able to screen for this and be able to make judgments about whether a person s likelihood of getting the disease is high or low or early or late It has that potential The Duke researchers cautioned that their conclusions now can be applied only to families where members have lateonset Alzheimer s the most common form of the disease Additional studies to verify the nding will be required before the conclusions can be applied to the general population said Kachaturian In the latest finding the researchers studied a gene that allows the body to manufacture apolipoprotein E or ApoE an essential protein that shepherds 127 Scientists have known for years that the gene comes in three varieties called E2 E3 and E4 and they have known that patients with the E4 version of the gene have a small but notably elevated risk of cardiovascular disease The new work demonstrates that possession of the E4 variant is an even greater risk factor for Alzheimer s disease than it is for heart disease Studying 234 people from 42 families af icted with lateonset Alzheimer s the researchers found that those patients with two copies of the E4 gene had eight times the risk of having the neurodegenerative disease that people had when their two copies of the apolipoprotein gene were some combination of either E2 or E3 varieties All genes of the body come in two copies one donated by the mother the other by the father Even inheriting one copy of the E4 gene turns out to be bad news doubling or tripling the risk of Alzheimer s over that of people having no E4 genes at all Researchers do not yet know how the gene predisposes people to the disease but although it has not yet been proven Roses say he believes there is a direct cause and c0pies holesterol loodstream 128 through the effect Unit 111 Bioenergetics Brief Overview Reading Chapters 26 Certainly one of the primary differences between biological creatures and inanimate objects is their structural organization Organisms are built from cells and while cell structure varies dramatically from one organism to another all cells share many common features e g plasma membranes genetic material enzyme systems receptors membrane transport systems etc In trying to de ne what we mean by quotlifequot this structural difference serves us well If you look through the microscope and see cells you are certainly looking at a biological creature But is it alive How do you distinguish living cells from dead cells Living creatures from dead creatures Well okay dead creatures don t move or vocalize or breathe or eat ie they can t Q anything Doing something requires the contraction of muscles the beating of cilia or agella or the secretion of products In addition as far as we can tell dead creatures don t sense anything They don t see hear feel or taste That is they have no quotsensory functionquot And again as far as we living types know dead creatures don t think about anything or have any emotions they don t remember plan enjoy problem solve love hate or do homework If you look for the common denominator in all these activities moving sensing thinking and feeling you nd that they all require ENERGY Only living creatures can use energy to accomplish these things these characteristics of life You can have the structure of a biological creature but without energy you cannot be alive Energy is an odd and sometimes hard thing to study It s much less tangible than matter which you can see weigh and measure directly The effects of energy are manifested in movement e g actin and myosin filaments sliding past one another ions traveling up their concentration gradient or in increases in temperature We also have sense organs that can sense the presence of certain kinds of energy For instance our eyes can detect the presence of electromagnetic radiation with wavelengths between 380 and 750 nm visible light Our ears can detect the vibration of the air at certain frequencies sound waves However we have no sense organs for many forms of energy including radio waves your radio receiver can detect these but you can39t radioactivity a Geiger counter can detect these but you can t or neutrinos they are passing through you right now but you can t sense them So what is energy anyway Focused Reading p 28 Most biological stop at Acids Bases p 256 quot Chemical Reactions quot stop at quotWaterquot p 9599 quotEnergy conversions quot stop at quotChemical reactions quot Energy is the capacity to do work In order for this definition to make sense you must think of work in the very broadest sense of the word work is an hing that changes the position or state of matter Matter at absolute zero no energy is absolutely still and immutable no movement or change of any kind y movement or change in the structure of matter requires the input of energy And that is how energy is defined It39s circular reasoning but reasoning all the same That which moves or changes matter is energy And ENERGY IS MEASURED BY THE AMOUNT OF 114 MOVEMENT OR CHANGE IN MATTER THAT IS PRODUCED Big change or big movement equals big energy Little change or little movement equals little energy In many ways this is just common sense Does it take energy to move a barge up river Yes Does it take more energy to move a large barge than to move a small sailboat Yes Does it take more energy to move a barge up river than down river Yes Energy and matter functioning on the molecular level are NO DIFFERENT Does it take energy to move a molecule across a cell membrane Yes Does it take more energy to move a big molecule than to move a small one Yes Does it take more energy to move a molecule up its concentration gradient than down its concentration gradient Yes Concepts concepts concepts there are only a few but they apply in many many situations Study Questions 1 What is energy Give one of the classic de nitions and then de ne it in your own terms 2 How is energy measured How do you know that a lot of energy is being expended versus a small amount of energy 3 How do kinetic and potential energies differ Give some examples not found in lecture or your textbook of the two forms of energy 4 What are the two laws of thermodynamics Define them in everyday terminology This unit is about how biological creatures harvest energy from their environment and use it to live The sun provides the energy we need to live but in order to convert this energy into a usable form biological creatures have had to develop elaborate systems for energy harvesting storage and use This system is called metabolism and its study is the field of bioenergetics In this Unit we will look at four examples of cells that harvest store and use energy in different ways We will find out why the United States government spayed paraquat on Mexican marijuana why cyanide is used by terrorists to poison consumer goods why vegetarians eat tofu and how a rusty nail can kill you 115 QUESTION 1 THE US GOVERNMENT V MEXICAN MARIJUANA FARMERS Rolling Stone April 6 1978 Whatever Happened to Mary Jane by Michael Roger The case of the poisoned Mexican marijuana started late in 1975 when the United States faced with an abrupt increase in the amount of heroin entering from Mexico began to assist that government with an elaborate program of spraying poppy fields with powerful herbicides From the beginning however that aerial attack was equally aimed at marijuana fields e program has been a success the Mexican heroin supply in this country has declined dramatically But is has also meant that approximately twenty percent of the Mexican marijuana entering this country is contaminated with a dangerous herbicide an estimate based on government analysis of marijuana samples confiscated recently in the Southwest The herbicide in question is paraquat an exceedingly toxic chemical that less than a month ago was placed on the Environmental Protection Agency s restricted list meaning that only licensed applicators may purchase it and which some observers feel may be banned altogether in this country Paraqua remains in the body even longer than DDT and has no kno antidote thus figuring occasionally in fail safe suicides At present the maximum paraquat contamination that the EPA allows in foodstuff is 005 parts per million Confiscated marijuana samples analyzed last November contained an average of 177 parts per million with a high of 655 One recently tested sample reportedly contained 2200 parts per million It is not yet clear what paraquat will do when burned and inhaled although the National Institute on Drug Abuse is doing its best to find out The current and hopeful guess of course is that the compound is rendered harmless during combustion Even assuming that to be the case however what about oral ingestion brownies majoun and the like The Drug Enforcement Administration estimates that 2700 tons of marijuana enter this country each year from Mexico Assuming that only one percent of that produce is eaten and that only twenty percent of that has been contaminated with paraquat that still means that almost fourteen tons of poisoned marijuana have been eaten in this country since the spraying program began I not clear what sublethal doses of paraquat can do because most cases reported have involved lethal doses due to the ingestion of pure material Evidence suggests that damage would occur first in the lungs liver and kidneys t w Rolling Stone May 4 1978 Poison Pot In the weeks since Michael Rogers s Alternating Currents column RS 262 described the possible health hazards of ingesting herbicide contaminated Mexican marijuana the situation has changed for the worse Scientific studies have revealed that the herbicide involved paraquat can survive the burning 6 process and be inhaled directly into the lungs Paraguat is so exceedingly toxic that on March 12th HEW Secretary Joseph Califano issued a warning that heavy use of contaminated marijuana could lead to irreversible lung damage At present the only drug analysis laboratory with an effective paraquat test is in California Consumers may send a one half gram sample one joint of suspected Mexican marijuana wrapped in foil to PharmChem Research Foundation 1844 Bay Road Palo Alto CA 94303 Enclose five dollars for lab costs plus any five digit number After ten days the result of the analysis may be learned by calling 415 322 9941 and giving the identification number PharmChem also requests the following information about the sample city and state where purchased street price paid and what it was sold to you as Colombian Mexican Hawaiian etc Brief Overview Reading Chapters 7 amp 8 Why would our government want to spend tax dollars to spray paraquat on Mexican marijuana To answer this question we need to know that paraquat is an herbicide It kills almost all plants except a few plants that are resistant to it How does paraquat kill plants Why might it be dangerous to humans Before we can answer these questions we need to understand how plants do what they do best harvest energy from the sun and turn that energy into sugars which are then used to support all life A plant is nothing less than a miracle Plants are able to harvest the energy of the sun and use it to convert COZ into food And in the process of doing this plants produce a waste product called oxygen All animal life depends on plants to harvest energy make food from a gas in the air and produce the oxygen we breathe If there were no plants there could be no animals whereas without animals many plants would be just fine We need them more than they need us Remember that the next time you walk on the grass or forget to water your houseplant or lean against a tree or read about the rapid loss of the planet s rain forests So how do plants turn sunlight into sugars How do they harvest energy use that energy to create food from C0 and excrete oxygen Energy is harvested and oxygen is produced in a process called the light reactions of photosynthesis The creation of food sugars proteins nucleic acids and lipids from C0 occurs in a process called the dark reactions of photosynthesis or the Calvin Benson cycle Both processes occur in the leaves of plants A typical plant leaf is illustrated in fig 819 p149 of your text The leaf is covered by a skin or epidermis which secretes a waxy coat called the cuticle The epidermis protects the plant and the cuticle prevents water loss on exposed surfaces Under the epidermis lies the mesophyll a tissue that contains the photosynthetic cells of the plant First let s look at the ligl1t reactions of photosmthesis Remember during this process the marijuana plant will harvest the energy of sunlight and give off oxygen What do we mean by the term quotharvest the energy of sunlightquot How would M harvest sunlight energy if you were asked to do so The word quotharvestquot implies that the energy is gathered and stored in a form that can be used at a later time the harvest contains potential energy Going out and eating a field full of corn would 7 not be considered quotharvestingquot the crop So using sunlight energy to do something e g illuminate a room warm your skin dry your clothes is not harvesting energy because you have already quotusedquot it none of the energy is stored for use at a later date Of course you can t destroy energy and in the process of using it the energy has simply been converted to another form namely to heat energy which is eventually radiated into space Have you thought of a way to harvest sunlight One high tech example of harvesting sunlight energy is the solar cell The cell collects sunlight and uses it to separate charge create voltage This voltage can produce current to run electrical devices A lowtech example would be hanging a blanket out in the sun to warm it and then using the blanket to warm yourself The radiant energy of the sun increases the kinetic energy of the blanket which can be used to warm you as it is released from the blanket Study Questions 1 What major events happen during the process of photosynthesis 2 In general what happens during the light reactions of photosynthesis During the dark reactions 3 We say we use energy to perform tasks However the first law of thermodynamics instructs us that energy cannot be created or destroyed What happens to the energy we quotusequot to live our lives The task of the green plant is to collect the energy of the sun and store it in a form that can be used later to do work In order to understand this we have to know a little more about sunlight a form of radiant energy and chemical energy the kind of energy organisms use to run their lives Focused reading p 1389 quotProperties of Light and Pigmentquot stop at end of page Radiant energy comes in various forms including radio waves microwaves gamma rays Xrays visible light and infrared Each of these waves has a characteristic wavelength The wavelengths of visible light are between 380 and 750 nm Because we are primates and can see color our eyes can distinguish the various wavelengths from one another and we experience these different wavelengths as differences in color For instance when light at 400 nm hits our eyes we experiences this as violet while light at 600 nm will give us a yellow sensation The relationship between colors and wavelengths is illustrated on page 139 Figure 85 Visible light has some of the properties of waves and therefore has a wavelength However light also has some of the properties of particles These particles are called photons They can be thought of as packets of energy Each photon has a certain quantity of energy a quantum plural quanta The energy level of photons is inversely related to the wavelength Thus a photon of red light wavelength 750 nm has about half the energy of a photon of violet light 3 80 nm short wave length high energy So during the day you and the Cannabis plants are being bombarded by these photons of light acting like waves and particles at the same time Zillions of photons per millisecond hit us each 118 one having a particular energy level wavelength and color There is no such thing as a white photon the color white is caused by photons of all the different energy levels or wavelengths colors striking your retina simultaneously White sunlight contains blue photons and red photons and violet photons and yellow photons etc all mixed up together IT IS THE ENERGY OF PHOTONS THAT THE GREEN PLANT HARVESTS But how The first thing the plant has to do is absorb the energy of these photons Most of the world around you absorbs photons In fact anything with any color or pigment is absorbing photons The grand mixture of photons in white light hits an object some of the photons are re ected back to your eye while some are absorbed by the object Ifthe object is colored it contains atype of molecule a pigment molecule that is chemically structured in such a way that it can absorb some photons energy Each type of pigment molecule will absorb photons based on their energy levels Some pigments only absorb blue photons some only absorb red some absorb yellow and blue etc Ifno pigment molecules are present then all the photons are re ected and the object appears white If all the photons are absorbed the object re ects no light and appears black Ifonly red photons are absorbed the rest of the photons are re ected back minus red photons and the color will be a mixture of violet blue green yellow and orange no red If red orange and yellow pigments are absorbed the remaining colors violet blue and green will be re ected back and the object will appear to be some shade of blue If a tree re ects green photons in the forest but no one is there to see it is it really green Because photons are a form of energy when colored objects absorb photons they are absorbing energy and become warmer due to an increase in the kinetic energy of the molecules in the colored object Thus black clothing absorbs all photons and heats up while white clothing re ects all photons and remains cool Study Questions 1 Describe the components of white light Which components have the highest energy The lowest What is the range of wavelengths spanned by visible light 2 Chemically and physically what makes something appear to have color 3 Visible light is an example of electromagnetic radiation What are some other examples of this type of energy Focused Reading p 1402 quotLight absorption and quot stop at quotElectron ows quot WWW Reading Relative Sizes from glucose to cells and larger Chlorophyll a and b are green pigments and carotenoids are shades of yellow and orange as in ca1rots and fall leaves Since chlorophyll a is the dominant pigment in most plant leaves most plants appear green But if you look at plant leaves closely you ll note that each plant and each leaf on each plant is a slightly different shade of green This is due to a shift in the proportions of the various pigment molecules in the cells 119 Let s focus on chlorophyll a It appears green So it re ects green light So it does not absorb gleen photons Because colors are so complex however it s really hard to say what colors absorbed This has to be measured using a spectrophotometer The absorption spectrum for chlorophyll a and b are illustrated in Figure 87 on page 140 You have used a spectrophotometer in lab and should understand how this piece of equipment works You have also constructed an absorption spectrum so the gure should be easy to interpret Ifit doesn t look familiar refer to your laboratory manual IDH labs This absorption spectrum shows that chlorophyll a absorbs maximally at about 450 nm it prefers to absorb highenergy blueviolet photons and also at 670 nm orangered photons It does not absorb bluegreen yellow or true red photons so they are re ected back to the eye of the observer The ultimate color produced by this absorption pattern is green It is the energy of the blueviolet and orangered photons that is harvested by chlorophyll a in the green plant In addition to chlorophyll a most plants have accessory or secondary pigments e g chlorophyll b and the carotenoids which absorb photons at other wavelengths Therefore plants can frequently harvest photons across the entire spectrum of white light Study Questions 1 Explain how a spectrophotometer works 2 What is an quotabsorption spectrumquot How is it obtained 3 What wavelengths and colors of light are absorbed by the chlorophyll a Explain how this results in its green appearance 4 Why does the absorption spectrum of chlorophyll a differ from the spectrum of an entire chloroplast So the leaves of green plants are full of these pigment molecules that absorb photons Where are these pigment molecules Floating free in the cytoplasm Attached to a membrane These are generally the two options in cell biology Well photosynthesis is a COMPLICATED PROCESS It involves dozens of enzymes performing dozens of tasks in a set order This is very much like assembling an automobile you cannot put in the stereo before you have assembled the dashboard you have to do things in order So instead of having the molecules involved in the process oating around haphazardly in the cytoplasm they are attached to membranes in macromolecular complexes These complexes are organized so that the molecules involved in each reaction are kept near the next molecule in the sequence The 39 serves as a scaffolding or frame that holds these molecules in position and carrier molecules travel between those positions The membranes that hold chlorophyll and all the other molecules associated with photosynthesis are found in the chloroplast Focused Reading p 689 quotPlastids photosynthesize or store materials stop at quotOther types quot Look at the picture of chlorophyll a on page 141 Figure 89 You ll remember that cell membranes have hydrophobic lipid cores Therefore the nonpolar hydrocarbon tail of chlorophyll 120 a dissolves with great stability in the lipid membrane of the thylakoid in the thylakoid membrane The highly polar porphyrin ring containing the Mg atom is the portion of chlorophyll that interacts with light Thus part of chlorophyll is designed to anchor it to the membrane maintaining its orderly relationship to the rest of the molecules of photosynthesis and the other part is designed to harvest light energy Study Questions 1 Describe in general terms the chemical structure of chlorophyll a Focus on the structural characteristics of the molecule that are signi cant for its function 2 Explain why it is advantageous to embed macromolecular complexes in cell membranes 3 Describe the structure of the chloroplast including the structure and location of thylakoids grana and stroma Describe the location of chlorophyll a in the chloroplast and explain how the molecule is anchored into the membrane Focused Reading p 13940 quotAbsorption ofa photon quot stop at Light Absorption p 140 Fig 86 Each pigment has a particular photon energy level that quotfitsquot it perfectly such that photons of that energy level can be absorbed while photons of other quotmisfittedquot energy levels cannot be absorbed This absorption results in a transfer of photon energy to an electron in the pigment molecule This electron normally at ground state or in its normal nonexcited position in an orbital around the nucleus is boosted to a higher orbital an excited state by the absorbed energy In regular pigment molecules such as the ones in your clothes when the electron is in its excited state it quickly returns to ground state and gives off the absorbed energy as light or heat However in the chloroplast chlorophyll a is anchored in the thylakoid membrane in a macromolecular complex One of the associated molecules is the primary electron acceptor When the electrons of chlorophyll are boosted to an excited state before they have a chance to fall back to ground state this primary electron acceptor takes them away from the excited chlorophyll Because biologists and chemists have to give names to everything this process where an electron is transferred from one molecule to another or when an electron moves closer or farther away from a molecule without actually being transferred to another atom is called oxidation reduction or a redox reaction Focused Reading p 11506 quotRedox Reactionsquot stop at quotThe coenzyme NAD quot p 116 Fig 72 WWWeb Reading A quick reference for reaction equations can be found at httpwww 39 f com purves6e choose the Math for Life icon and the Biochemical reactions in the reference table section Study Questions 1 What is oxidation Reduction Give examples What is areducing agent An oxidizing agent Give examples 2 What is electronegativity Electropositivity In which way will an electron naturally tend to ow from electropositive to electronegative or vice versa 3 What would you suspect is true of the primary electron acceptor in the thylakoid membrane it is relatively electropositive relatively electronegative or about in the middle Explain your answer STOP So this is how chlorophyll harvests light energy Well it s a long way from this little excited electron to using paraquat to kill marijuana In order to get there we have to talk about the other kind of energy involved in photosynthesis chemical energy Potential chemical energy food and fuels of all kinds is said to be stored in the bonds of molecules Covalent bonds as you know are shared electrons These electrons are being shared because each element in the bond quotneedsquot the stability that sharing electrons brings The elements are more stable or at a lower energy level if they are sharing electrons with one another Each atom is trying to ll an electron shell with the correct number of electrons and covalent bonds help the molecule do this Refresher Read pages 2024 Molecules contain POTENTIAL CHEMICAL ENERGY We say that the potential chemical energy is quotin the molecules bondsquot but this is in many ways misleading Potential chemical energy can be thought of as the capacity to produce molecular change to do chemical work Thus if a molecule is fairly UNSTABLE it is likely to change to a shape or con guration that is more stable The energy that is released when this change towards a stable con guration occurs is the heat of the reaction AH H is referred to in the book as enthalpy and it is a measure of how much potential energy was stored in that unstable molecule Actually it is a measure of the difference in the potential energy stored in the reactant and the potential energy stored in the product since the product could go on to react and become even more stable and release even more energy By convention when a reaction gives off energy this energy is usually given off as heat but it might also be light electrical current or movement the AH of the reaction is designated as negative Thus a reaction that gives off energy eg burning fuel has a AH and is said to be exothermic Conversely reactions that proceed only when energy is added usually in the form of heat but it might also be light electrical current or movement the AH of the reaction is positive Thus a reaction that requires the input of energy draws energy from the environment has a AH and is said to be endothermic Focused Reading p 96100 quotEnergy Conversionsquot stop at quotATP Transferring WWW Reading Animation of Photosynthesis from Virtual Cell 122 When a chemical reaction gives off energy e g when gasoline is burned in a car engine most of the energy given off by the reaction is given off as heat or car movement Heat and movement represent M Car movement propelling the car and Heat increasing the movement kinetic energy of molecules However some of the energy given off by the reaction is not represented in either heat or movement is not represented by work Rather this energy is represented by a change in the entropy of the gasoline molecules The chemical reaction is Oxygen Gasoline gt Carbon Dioxide Water Gasoline a long chain hydrocarbon is more organized than C02 and H20 Therefore gasoline has less entropy or randomness than C02 and H20 Some of the energy given off by this reaction has been used to increase entropy change low entropy more organized molecules into higher entropy less organized molecules This change in entropy level AS is not available to do work in this case provide heat or movement Reactions e g burning gas in which entropy is increased have a positive AS while reactions e g re ning gasoline in which entropy is decreased have a negative AS Thus in all chemical reactions the energy source for virtually all biological function two kinds of changes occur changes in potential energy of the molecules AH and changes in entropy AS Usually reactions that give off energy to do work exothermic or AH reactions also involve an increase in entropy have a AS Such reactions in biology include the burning of food for energy Big complex organized molecules proteins carbohydrates lipids and nucleic acids are broken down to simple small molecules of CO2 and H20 Much energy is given off in the process AH and the entropy of the molecules is dramatically increased AS Conversely reactions that absorb energy endothermic or AH usually involve a decrease in entropy have a AS 1 such reactions in biology include the building up of structures during growth Simple small molecules such as amino acids nucleotides and monosaccharides are linked together into large organized molecules such as proteins nucleic acids and polysaccharides Much energy is required for this process AH and the entropy of the molecules is dramatically decreased AS The production of sugars by plants is an example of an endothermic reaction that decreases the randomness in the world a comforting thought Biologists are very interested in the AH of reactions because this determines when a given reaction will be able to supply energy for life and when a reaction will require the input of energy from the organism However another factor is also very important to biologists and that is the free energy of the reaction AG This factor determines whether a reaction will proceed or not Reactions that proceed on their own without energy input from the cell beyond activation energy are called spontaneous while reactions that will not proceed unless energy is added are called non spontaneous Spontaneous reactions are said to be exergonic and have a AG while non spontaneous reactions are said to be endergonic and have a AG Usually exothermic reactions are exergonic and endothermic reactions are endergonic but not always If an endothermic reaction takes heat from the environment AH involves a large increase in entropy AS then it may be spontaneous have a AG even though it requires the input of energy Melting ice is an example of a reaction that requires the input of energy heat is removed from the environment thus the reaction 123 has a AH but results in a dramatic increase in entropy AS as ice goes from an organized crystal to a disordered liquid form Cheat Sheet AS product has less randomness AS product has more randomness AH rxn gives off energy exothermic AH rxn takes in energy endothermic AG rxn is spontaneous exergonic AG rxn nonspontaneous endergonic The friendly relationship between AH and AS given on page 121 of your text is AG AH TAS T is the temperature in Kelvin units Thus to determine whether a reaction is spontaneous or not and to determine how much of the reaction energy is actually available or free to do work you must subtract any gain in entropy multiplied by the temperature Kelvin degrees from the total change in potential energy of the reaction We do not introduce all this to confuse you although it must seem like it Rather we face a dilemma in teaching you In chemistry you are learning about AH which is widely used by chemists to describe the simple thermodynamics of chemical reactions Biologists however focus less on the chemical reactions themselves and more on what the chemical reactions can do for biological creatures ie we are interested in that portion of the energy that is available to run biological creatures Thus we have to introduce AG and we have to tell you how it relates to AH so you can integrate what you are learning in the two classes Study Questions 1 How is energy stored in molecules 2 Be able to explain these terms exothermic endothermic AH AH endergonic exergonic AG AG spontaneous nonspontaneous entropy AS AS 3 What determines whether or not a reaction will proceed without an input of energy from the cell Given examples of the types of biological reactions that tend to be exergonic and examples of those that tend to be endergonic 4 Explain the second law of thermodynamics as you would to a junior high school student in a science class Give an example of how the second law of thermodynamics is important in the study of biological systems 124 The second law of thermodynamics governs all chemical reactions that means your entire life Now this is just ne if you want to do something exergonic Ifyou provide an enzyme to lower the activation energy barrier the reaction will proceed just ne The problem is most of what you really want to do move pump blood breathe think see hear secrete etc is decidedly ENDERGONIC This is another way of saying that living is an energyabsorbing activity Living is endergonic and endothermic So you have a problem because endergonic reactions don t occur spontaneously you have to add energy to the reaction to get it to proceed While ultimately this energy you live on comes from the food you eat which ultimately comes from plants which synthesize it using the energy harvested from the sun the DIRECT SOURCE OF ENERGY FOR MOST ENDERGONIC REACTIONS 1N LIVING THINGS IS Focused Reading p 100102 quotATP Transferring quot stop at quotEnzymes Biological catalystsquot Study Questions 1 Describe and draw the reactions converting ATP to ADP and vice versa What is the AG of each reaction 2 Be able to describe the process of energy coupling by phosphate transfer outlined in Figure 610 on p 102 For the most part as long as you maintain an adequate supply of ATP you can live your lif ATP will supply the energy required for your endergonic reactions And the same is true of all other creatures on the planet including marijuana As long as we have enough ATP or other highenergy nucleotides that function in the same manner as ATP we can do all the endergonic reactions we must do to stay alive However you can see that ATP is converted to ADP during the process of providing energy for endergonic processes Thus living cells are constantly sapping ATP39s energy so cells must have a way of constantly replacing this lost ATP Marijuana plants can do this directly by using solar energy photosynthesis or indirectly by burning fuel molecules cellular respiration Animals can do this only by burning fuel molecules The Cannabis plant has two biological needs 1 It must provide itself with enough ATP to stay alive and 2 it must provide enough nutrition in its seeds to nourish its offspring which will allow them to sprout and in turn harvest energy on their own In the process of harvesting energy and storing energy marijuana leaves also happen to produce oxygen as a waste product which animals gratefully inhale we re referring to the oxygen OK Harvesting Energy and Generating Oxygen The Light Reactions of Photosynthesis Focused Reading p 1368 quot Identifying photosynthetic reactants quot stop at quotProperties of light quot p 1002 quotATP Transferring energy quot stop at quotEnzymes quot 125 p 1426 quot Excited chlorophyll quot stop at quotMaking sugar quot WWW Reading httpwww 39 f com purves6e Ch8 Tutorial 81 Photophosphorylation Diagram of NADP conversion to NADPH httpwww 39 f com purves6e Math for Life Biochemical reactions With the help of photosynthesis CO2 and H20 are converted to sugars e g glucose C6H1206 lipids amino acids with the addition of nitrogen and nucleotides with the addition of nitrogen and phosphorus This process is HIGHLY ENDERGONIC and the energy to power this non spontaneous process is provided by the sun Your text uses the example of the synthesis of glucose from C02 and H20 and we will use this example too However you must remember that plants can make all the nutrient classes not just glucose via photosynthesis The overall balanced reaction for glucose synthesis then is 6C02 12HZO light energy gt CEHIZO6 602 6HZO This overall reaction is actually a redox reaction The light reaction is as follows 12H20 light energy gt 602 24e39 24H At this point we need to stop and talk a bit more about hydrogen and its propensity to fall apart into an electron and proton Hydrogen is extremely electropositive meaning that the nucleus of hydrogen composed of only one proton and zero neutrons does not have very much affinity for electrons it does not pull very hard or attract very tightly the electron in orbit around it Thus it is relatively easy for an electronegative molecule one that has a high affinity for electrons and attracts them very strongly to take hydrogen39s electron away from it rather than sharing the electron in a covalent bond Thus in the presence of electronegative molecules such as NADP NAD and the cytochromes hydrogen s electrons are more attracted to electronegative molecules than to the hydrogen nucleus they leave orbit and are added to the electronegative molecules eg converting NADP into NADP etc The quotnakedquot hydrogen nucleus having lost its electron becomes a proton or hydrogen ion H These protons simply oat around in the cytoplasm During the light reactions of photosynthesis the 24 hydrogens on the 12 water molecules are removed leaving 6 molecules of 02 which the plant gives off to the environment The 24 hydrogens are split into 24 protons H and 24 electrons e The 24 electrons are added to quotcarrierquot molecules called NADP l2 carrier molecules pick up the 24 electrons 2 electrons per NADPgt NADP 39 and 12 protons l proton each NADP39 gt NADPH To form reduced NADPH NADP the oxidized form picks up 2 electrons and one proton One electron neutralizes the NADP to NADP The second electron plus the proton forms a hydrogen atom and is added to the molecule to form NADPH The other 12 protons simply oat free in the thylakoid space of the chloroplast lowering its pH By causing chlorophyll to lose an electron solar energy converts chlorophyll into a powerful oxidizing agent it will get reduced Because chlorophyll quotwantsquot to replace that electron m y it is able to take the hydrogens away from oxygen in molecular water By taking water s 126 hydrogens chlorophyll gains back the electrons it loses by photooxidation Again hydrogens are split into electrons which enter the chlorophyll molecule and protons which oat freely in the thylakoid of the chloroplast Water is a very stable molecule so removing its hydrogens is not easy oxidized chlorophyll is one of the most electronegative molecules known far more electronegative than oxygen That s how it is able to take oxygen39s hydrogens away in a water molecule Study Questions 1 Explain why the addition of a hydrogen atom to a molecule is reduction How does the electropositive nature of hydrogen allow it to function as a reducing agent Very speci cally how does solar energy cause the splitting of water into hydrogen and oxygen during the light reactions What happens to the oxygen released from the split water What happens to the hydrogen released from the split water Describe how the carrier molecule NADP works Why is it called a carrier What does it carry To what molecule that you have studied in this course is it most closely related besides NAD and FAD Is NADP a protein lipid carbohydrate or nucleic acid Describe the processes of cyclic and noncvclic 39 39 ylatiou What is being phosphorylated in these reactions How do these processes different from one another Which process evolves oxygen Explain the mechanism through which this process evolves oxygen while the other process does not Which of these processes produces NADPH Explain the mechanism through which this process produces NADPH while the other process does not The ultimate products of the light reactions of photosynthesis are NADPH ATP and 02 Be able to describe how each of these products is formed What is a cytochrome What is an electron transport system Upon what basic concepts is this model based ie what attracts the electrons down the system Describe the chemiosmotic theory and explain how it works to produce ATP in the chloroplast during the light reactions of photosynthesis Explain in the simplest possible terms for a younger brother or sister how green plants harvest sunlight energy qTOP Synthesizing Food The CalvinBenson Cycle 127 Focused Reading p 148 Fig 817 p 1378 The two pathways stop at quotProperties of light quot p 1468 quotMaking sugar quot stop at quotPhotorespiration quot The light reactions of photosynthesis generate 02 ATP and NADPH Oxygen is a waste product to the plant but ATP and NADPH are required by the plant to make nutrients from C02 For the synthesis of nutrients the plant requires an energy source provided by the ATP generated during the light reactions a source of carbon C02 from the atmosphere and a source of quotreducing powerquot Look at the second half of the photosynthesis equation the part that synthesizes sugar 24e39 24H 6C02 energy ATP gt glucose CSHIZOS 6HZO In this reaction C02 is reduced to glucose the carbon atoms have 12 hydrogens added and 6 oxygens removed This reduction requires a reducing agent and that reducing agent is NADPH itself reduced in the light reactions with electrons and hydrogens from water Ultimately the hydrogens used to reduce CO2 to glucose come from water Despite the fact that glucose was used in this example of photosynthesis the molecule we should focus on is glyceraldehyde 3 phosphate often abbreviated as G3P The structure of this molecule is shown on p147 G3P is a threecarbon sugar and it is the starting molecule precursor for the synthesis of several sugars not just glucose and lipids Amino acids used for protein synthesis can also be made using PGAL as a precursor in addition to a source of reduced nitrogen The overall reaction of the Citric acid cycle is 3C02 from the air or water l G3P 9 ATP from the light reactions gt 9 ADP 8 Pi 6 NADPH from the light reactions 6 NADP The complete cycle is outlined in gure 817 on page 148 Study Questions 1 What role does quotreducing power play in photosynthesis What molecules provide reducing power directly to the CalvinBenson cycle Where and how do these molecules obtain their reducing power 2 Explain how the photooxidation of chlorophyll a is related to the reduction of C02 in photosynthesis Trace the connection in general but accurate terms ie you need not list every chemical in each pathway but you need to list each pathway and discuss its signi cance 3 What is the product of the CalvinBenson cycle Why is this molecule of pivotal importance in the life of the plant 128 4 Rubisco is one of the most important enzymes in the entire biological world What does rubisco do that is so impressive What is rubisco s full name Explain this name in terms of the enzyme39s function 5 Explain the CalvinBenson cycle in general terms What is important about this cycle What does it do What are its products what happens to them and why are they important 6 Based on what you know about the role of phosphorylation in chemical reactions develop a hypothesis that explains why 3phosphoglycerate is phosphorylated in the second step of the CalvinBenson cycle The phosphates come right off again in the next step Why do you suppose the cycle doesn39t simply convert 3phosphoglycerate to glyceraldehyde phosphate in one step This would save 6 ATP per cycle and would be of great adaptive advantage to the plant Use an energy diagram to explain your hypothesis NEWS ITEM Macromolecular complexes appear to be very common for proteins involved in photosynthesis Darl Heinz 51155 has evidence that suggests that Rubisco is anchored to the thylakoid membrane via the ATPsynthase If this is true it demonstrates that many proteins may have a primary function synthesis of ATP and a secondary function anchor rubisco This is the kind of dual function allows a duplicated gene to give rise to two similar proteins with very different functions the kind of variation that is critical to evolution See Suss Z Naturfarsch Vol 45c 633637 1990 Now the marijuana leaf has harvested sunlight energy and stored it in the nutrient G3P As the first law of thermodynamics tells us energy cannot be destroyed or consumed it can only be conve1ted to another form of energy As a summary of the process of photosynthesis let s brie y describe the harvesting of energy through the light reactions Remember you measure energy by the effect it has on matter so to follow energy we describe what gets quotenergizedquot during this process 1 The electrons in chlorophyll get energized and jump to a higher orbital N These electrons pass across an electron transpott system ETS and transfer their energy to the proton pumps which use the energy to move protons up their concentration gradient The energy of sunlight is now contained in the high concentration of protons in the thylakoid space E The protons fall down their concentration gradient and transfer their energy to the ATP synthase which energizes ADP by phosphorylating it to become ATP 4 The highenergy electrons tumbling down the ETS in Photosystem I don t transfer all their energy to the proton pumps Much of the energy remains in the electrons and is transferred to the NADP as it becomes NADPH V39 NADPH and ATP both contain much of the energy originally reaching the plant in sunlight During the Calvin cycle this energy is transferred to C02 in the form of highenergy electrons and hydrogen ions as it becomes G3P 129 At every step in this process the energy transfer is not 100 efficient a percentage of the energy is not transferred to the next step but is given off as heat to the environment This is the nature of energy transfers They are never 100 efficient However the energy transfers of photosynthesis are among the most efficient Study Question 1 Be able to explain the transfers of energy outlined in steps 15 above Make sure you understand the nature of each energy transfer and the nature of energy transfers in general G3P has been synthesized and the marijuana plant uses G3P in the following ways 1 The leaves send G3P to the mitochondria inside the mesophyll cells G3P is oxidized in the mitochondria to C02 and H20 The energy released by this process is stored in ATP which powers the living processes ofthese leaves 2 Plants synthesize glucose fructose sucrose a fructoseglucose disaccharide and starch polyglucose in the chloroplast The starch is a storage form of sugars that the plant can live on in times of darkness when photosynthesis cannot occur The mono and disaccharides are stored in the mesophyll as well but are also transported to all the cells that do not photosynthesize roots stems and owers These sugars are used A As an energy source the sugars are burned for energy by these cells B As a source of glucose for the production of cellulose the major structural component of cell walls C As a source of glucose for producing starch in nonphotosynthetic plant cells D As a precursor for amino acids and nucleic acids made in the cells of the root See Fig 822 p152 for a diagram of how the CalvinBenson cycle fits in plant metabolism Back to our original question Why did the US government use paraquat on marijuana plants to kill themquot Paraquat is very very electronegative and binds to a protein near photosystem I When light hits chlorophyll at the reaction center the electron is excited and sent to the primary electron acceptor Rather than entering the electron transport pathway it is sucked up by paraquat As a result no NADPH is produced So Later in this unit we will see why it might be so harmful to humans 130 stroma thylakoid membrane thylakoid lumen NADP reductase Q 89 high energy electron Study Questions 1 Why does paraquat kill marijuana What affect would this have on marijuana s ability to make G3P 2 Ifyou wanted to design a weed killer what other steps in photosynthesis could be exploited The New York Times Magazine November 19 1978 an excerpt from P01son0us Fallout From The War On Man uana by Jesse Kornbluth Mexican marijuana growers had learned that parquatdrenched plants might still be sold as commercialgrade marijuana if they could be harvested before the herbicide turned the leaves brittle and the taste harsh Because their illegal crop meant the difference between a subsistence income of 200 a year and a cultivator s income of as much as 5000 the Mexicans unhesitatingly harvested the poisoned marijuana And then they sold it to Americans The dangers of paraquat were no secret to the State Department Swallowing as little as a half ounce is suicidal paraquat gravitates to the lungs where it causes such massive damage that death almost invariably occurs within two weeks There is no known antidote But whether paraquat that has been burned and then inhaled produces those same deadly results was unknown In 1975 when State started funding the Mexican program there had been no inhalation studies There would be none until 1977 when Senate investigators forced the issue This month Secretary of Health Education and Welfare Joseph Califano announced the disturbing results of those tests Heavy users of this tainted marijuana might develop fibrosis an irreversible lung disease and clinically measurable damage might befall less frequent smokers In the furor that followed the Administration explained that there was nothing it could do but warn smokers against Mexican marijuana the Government of Mexico selected this herbicide independently purchased it from a British company with its own funds and sprayed marijuana mostly when opiumpoppy fields the true targets of the Americanfunded program lay fallow Among the many accomplishments of the MexicanAmerican eradication program are these unforseen results Contrary to the original widely publicized White House announcement this poisoned marijuana is generally indistinguishable from the ordinary Mexican product Because of the distribution of patterns of Mexican marijuana paraquatsprayed marijuana is sold mostly on the West Coast to teenagers on the East Coast in ghettos and across the nation to the estimated 200000 Armed Forces enlisted personnel who smoke These are the three groups least likely to have heard Secretary Califano s warning or to believe it if they did Con icting statistics released by various Government agencies have caused widspread confusion Secratery Califano s announcement indicated that onefifth of the marijuana confiscated at the Mexican border had been contaminated by paraquat some of it at concentrations 40000 times greater thatn the Environmental Protection Agency allows for domestic use In August the Center for Disease Control tested paraquat positive marijuana forwarded by PharmChem the California laboratory which had received more contaminated samples than all other private labs combined PharmChem s findings that as much as 39 percent of its 10000 samples were paraquat poisoned had been widely publicized when the CDC discovered that only 2 percent of this laboratory s contaminated samples were paraquatpositive PharmChem rexeamined its testing procedures found them to be inadequate and suspended its operations In the confusion which surrounded these developments the CDC s warning against paraquat test kits devices which might enable consumers to resolve their doubts at home hurt sales of the one kit said to be reliable a simple chemical test developed by University of Mississippi marijuana researcher Dr Carlton Turner for Landis Labs of Horsham PA Last month when the National Institute of Drug Abuse announced that paraquat was as prevalent and as potentally dangerous as Secretary Califano origianally indicated this news went almost unreported STOP 132 Question 2 Why Do Vegetarians Eat Tofu Focused Reading p 64041 quotnitrogen xation quot stop at quotSome plants p 889 Food provides stop at quotAnimals need mineral elements quot p 890 fig 505 WWW Reading What are Tofu Pups Tofu is made from soybeans and soybeans are excellent sources of protein It harvests the sun39s energy gives off oxygen for our consumption AND stores nutrients in its seeds that contain an unusually high concentration of protein As you know protein gives us our structure which allows us to function Without protein we can39t produce any chemical reactions pump any ions phosphorylate any substrates or send any electrical impulses to name a few functions that depend on proteins We animals need a constant source of protein in our diet Ifyou are a carnivore you get much of your protein from meat the muscle cells of other animals However this is an inef cient source of protein It takes at least 10 times more energy to create a gram of animal protein than it does to create a gram of plant protein Therefore with the human population explosion and hunger and starvation a constant threat it makes sense for humans to consume less meat and eat more plants in order to conserve the precious energy resources of the planet Unfortunately many plants are poor in protein but the soybean is an exception A note here about quotcompletequot and quotincompletequot proteins All 20 amino acids must be available to you on a daily basis in order for you to make the proteins you need to be healthy You need to consume eight amino acids the essential amino acids in your diet every day see gure 505 on p 890 From these you can biosynthesize the other 12 the non essential amino acids thus giving you all 20 In case you are interested the essential amino acids are isoleucine leucine lysine methionine phenylalanine threonine tryptophan and valine Because most animals especially vertebrates are composed of the much same proteins you are if you eat animal muscles you will automatically take in the correct amino acids in about the right proportion for your dietary needs We ll ignore fats and vitamins for now However plants are quite different in their amino acid compositions and are present in proportions that vary signi cantly from humans Therefore if you eat only one kind of plant say wheat or corn or rice you usually get too much of some essential amino acids and not nearly enough of others You will be eating quotincomplete proteinquot Therefore if you are vegetarian you should eat legumes peanuts soybeans garbanzo beans navy beans kidney beans pinto beans etc and grains wheat rice oats corn etc These two types of plants provide a quotcompletequot protein mixture by compensating for each other s missing essential amino acids Study Question 1 When do you classify an amino acid as quotessentialquot In order to remain healthy why must vegetarians eat meals containing both legumes and grains Focused Reading p 635 Autotrophs stop at quotHow does quot 133 Sugars and lipids contain only carbon hydrogen and oxygen like G3P Therefore G3P can be used as the precursor for the biosynthesis of carbohydrates and lipids without the addition of other elements As is true for all synthetic processes these processes are ENDERGONIC and require an energy source in the form of ATP However proteins which are composed of amino acids contain carbon hydrogen oxygen and nitrogen Therefore in order to synthesize amino acids and therefore protein the plant must have a source of nitrogen Focused Reading p 46566 quotNitrogen and Sulfur metabolismquot stop at quotProkaryotes and their quot p 641 quotSome plants and bacteria quot stop a quot Biological nitrogen fixation quot The equation for nitrogen xation is as follows N2 8H 8equot 16ATP gt 2NH3 H2 16ADP 16Pi As you can see this is a redox reaction in which nitrogen is reduced hydrogens are added to nitrogen Thus the reaction requires reducing power which it gets from NADH produced during bacterial metabolism see below The reaction is also m endergonic requiring at least 16 ATP per reduced nitrogen molecule Some estimates of the overall energy requirements of nitrogen xation place this figure at 2535 ATP per nitrogen molecule Thus the creation of amino acids the raw materials of protein synthesis is itself a costly endeavor for biological creatures Nitrogen fixing bacteria contribute about 2 x 108 tons of ammonia NH3 to the soil each year for plant growth and produce many times more soil ammonia than is provided by agricultural fertilizers The soybean obtains nitrogen in the form of ammonium from the Rhizobium residing in its root nodules Sucrose is transported to the plant roots where it is converted to alphaketoglutarate Root cells synthesize the amino acid L 39 by 39 39 39 alpha 39 39 and ammonia as follows 039 o o39 0 H20 H20 th3 NADPH H gt 13 H20 H2 CY H2N O O alphaketoglutarate Lglutamate The amino acid Lglutamate can be used as a source of amino groups to make all of the other amino acids The amino acids are transported all over the plant to meet its own protein synthesis needs In the case of the soybean amino acids are also supplied in large numbers to the developing soybeans These soybeans then are a rich source of protein for humans and other animals when harvested 134 And with this came the invention of soyburgers and tofu pups what some vegetarians eat on picnics and at ball games NEWS ITEM The recommended daily allowance of vitamin E 10 134IU can be easily obtained in your daily diet However higher levels of the vitamin 1001000 TU have been found to decrease your risk of heart disease and some cancers and improve the immune system This level was almost impossible to reach without vitamin supplementsuntil now Soybean oil is one of the main sources of dietary vitamin E and researchers in Nevada are working to bioengineer a better bean A precursor of vitamin E is abundant in plant oils but most of it doesn39t get converted into the vitamin By cloning in a gene that overexpresses the necessary biosynthetic enzyme scientists are able to shift the equilibrium of the reaction from precursor to product and increase the overall vitamin E content CD Shintani andD DellaPenna Science 1998 vol 282 p 2098 Study Questions Describe the various ways in which G3P is used by the green plant in general terms 2 In what form must nitrogen be supplied to plants in order for them to incorporate the nitrogen into amino acids How is this form of nitrogen provided to nonlegumes 3 Describe the symbiotic relationship between legumes andha39zobz39um What does the legume gain from this relationship What does the Rhizobium gain 4 Describe the efforts of genetic engineers and selective breeding to increase the protein productivity of crops Why is this work important What is the problem with simply fertilizing crops to provide more ammonia and nitrates 135 QUESTION 3 WHY IS CYANIDE THE TERRORIST S POISON OF CHOICE The Cyanide Scare A Tale of Two Grapes by Bill Grigg and Vern Modeland excerpts from FDA Consumer March 1989 marked the most intensive food safety investigation in Food and Drug Administration history Millions of tons of fruit became suspect when a terrorist 6000 miles away apparently made good on a phone call threatening to poison this nation s fresh fruit supply Fruit in stores was returned or destroyed and shipments coming into the country from Chile were halted In Chile seasonable fruit and vegetable exports are second in importance only to copper to the national economy In the United States the cost of the terrorist s call might reach 50 million the estimated value of 45 million crates of nectarines plums peaches apples pears raspberries strawberries blueberries and table grapes that faced destruction How did it happen it Since it was his turn as duty officer Dick Swanson wasn t surprised when the black box on his belt beeped at 720 pm Friday March 3 Ever since the 1982 Tylenol tampering crisis his wife only half counted on him on Fridays A second beep sounded as he reached his door so he headed straight to the telephone and called the number that had appeared Hi the Ibeeper 2 UJS Customs official came on the line He told Swanson that a cable from the US Embassy in Santiago Chile had informed Customs ON MARCH 2 AT 1550 HOURS AN EMPLOYEE OF THE AGRICULTURE PUBLIC HEALTH INSPECTION SERVICE RECEIVED A CALL FROM A SPANISH SPEAKING MAN WHO SOUNDED MIDDLE AGED AND WHO SPOKE WITH AN UNEDUCATED ACCENT THE MAN STATED THAT FRUIT BEING EXPORTED TO BOTH THE UNITED STATES AND JAPAN WILL BE INJECTED WITH CYANIDE IN ORDER TO FOCUS ATTENTION ON THE LIVING CONDITIONS OF THE LOWER CLASSES IN CHILE HE FURTHER STATED THAT TOO MANY PEOPLE IN THE COUNTRYSIDE WERE STARVING DUE TO INCREASED LIVING COSTS AND WERE UNABLE TO BUY SUFFICIENT FOOD TO SURVIVE 136 The caller said killing policemen and placing bombs had not solved the problem and he wanted to involve other countries Although the Manuel Rodriguez Patriotic Front and the Leftist Revolutionary Front had been attacking policeman and placing bombs to bring about changes in the country and government of Augusto Pinochet the caller did not say if he was involved with either group Saturday FDA Commissioner Frank E Young MD PhD and others met at FDA headquarters in Rockville MD They continued to confer on Sunday But by Monday the State Department had concluded the telephone call was probably a hoax FDA then released news of the call and State s view of it as a likely hoax FDA said fruit had been temporarily held but was moving again Few newspapers reported FDA s announcement The crisis appeared over The terrorist called the embassy in Santiago again on the eighth of March and again on March 17 warning that the March 2 threat was no hoax FDA began to step up inspections mostly at the Port of Philadelphia where 80 percent of all Chilean fruit imported by the United States arrives First to be inspected would be the Almeria Star which had sailed Feb 27 from Santiago with 364000 boxes of fruit in her holds On Sunday March 12 investigators began examining a representative 12000 boxes of fruit To examine the mountain of Chilean fruit the FDA Philadelphia district office needed extra help Among those assigned to the temporary duty was William Fidurski from FDA s North Brunswick NJ resident inspection post He was one of some 40 FDA people assigned to inspect fruit at the Tioga Fruit Terminal in Philadelphia They were right on top of the box Fidurski recalls The red seedless grapes were discolored They had damaged skins That s about all remembered about them out of the 2 million grapes FDA investigators saw that day Being careful not to disturb anything in the box Fidurski turned the crate over to his supervisor It went among others containing damaged or discolored fruit to the FDA Philadelphia laboratory for closer examination There color photos were taken that showed rings of a crystalline substance surrounding what might be puncture sites The grapes then were sliced carefully and placed in small glass flasks In the flasks the slices were squeezed with a glass rod to release the juice and a solution of dilute sulfuric acid was added Sulfuric acid will cause chemical changes to cyanide compounds releasing hydrogen cyanide This cyantesmo test would detect the presence of as little as 10 millionths of a gram of cyanide Within minutes it did The analysts then did a Chloramine T test which produces a pink purple color in a reactive solution The second test confirmed results of the first Those two red grapes contained cyanide in amounts far too small to cause death or even illness to anyone eating them And because crystalline potassium cyanide and sodium cyanide change to hydrogen cyanide gas in acid fruit which can then dissipate FDA scientists couldn t determine how much of the poison might have originally been injected into the grapes But cyanide was present FDA Commissioner Young said Very low levels Very low 003 mg vs 20 mg to hurt an adultquot The newly confirmed Secretary of Health and Human Services Sullivan was briefed The many political and financial ramification of a quarantine were discussed They agreed that HHS and FDA weren t charged with foreign policy considerations and commerce 137 A news release was drafted in case it was needed Copies were passed around the table and quickly approved The FDA said today it has found and confirmed traces of cyanide in a small sample of seedless red grapes from Chile and as a result is detaining all grapes and other fruit from that country The news was made public on the evening newscasts on March 13 Why is cyanide poisonous How does it kill people To answer these questions we need to learn how all organisms generate ATP from sugars like glucose Nonphotosynthetic organisms are called heterotrophs troph to feed on hetero other therefore quotone who feeds on othersquot as opposed to photosynthetic organisms which are called autotrophs quotones who feed themselvesquot Animals many bacteria most protists and non photosynthetic plant cells roots stems owers must get ATP by nonphotosynthetic means These means are called fermentation and cellular respiration Fermentation does not require the presence of oxygen we ll cover this later while as the name implies cellular respiration does While all nutrient molecules be burned to obtain energy by far the molecule most frequently used for this purpose under normal circumstances is the monosaccharide glucose Glucose is the predominant sugar in human blood Homeostatic mechanisms maintain the plasma glucose concentration at about 80 mg per 100 mls ofblood Focused Reading p 108 quotMetabolism and the quot stop at quotEnzyme activity is subject quot p 145 Fig 7 lnote relationship between autotrophs and heterotrophs p 4346 quotCarbohydrates quot stop at quotDerivative carbohydrates quot The energy of sunlight has been harvested and stored in the glucose molecule through the process of photosynthesis Humans eat sugars and complex carbohydrates which are converted to glucose for consumption by the cells The energy stored in glucose by the green plant is thus released to the cell and converted to ATP during the process of cellular respiration Complex carbohydrates come in three varieties starch cellulose made by plants and glycogen made by animals All of these polysaccharides are polyglucose Because they contain alpha glycosidic linkages people can break down glycogen and starch to glucose molecules that serve as fuel for the cells of the body Because it contains beta glycosidic linkages cellulose cannot be broken down to glucose by us You do not have the enzyme required to break the betaglycosidic linkage therefore lettuce celery carrots broccoli etc actually contain thousands of calories but you can t get at these calories because you can t break down the primary bulk of the vegetables cellulose So it simply passes through your body as quotroughagequot The overall equation for cellular respiration is C5H1205 602 6HzO gt 6COZ 12HZO energy ATP Heat You will immediately recognize this as the reverse of photosynthesis Photosynthesis is an endergonic reaction with a AG of 686 kcalmole Conversely cellular respiration is an exergonic reaction with a AG of 686 kcalmole Thus for every mole of glucose oxidized by the cell 686 kcal of energy becomes available to do cellular work However as in the case of photosynthesis each of 138 the many energy transfers in cellular respiration is inefficient Thus about 254 kcal of this total energy is given off as heat while only about 432 kcal is successfully stored in ATP This heat is de nitely used by humans to maintain their body temperature and cannot be considered wasted energy However in warmer environments much of the heat is quotdumpedquot into the air by cooling mechanisms most notably perspiration Nonetheless the transfer of energy from glucose to ATP is about 63 efficient 432 kcal stored out of 686 available By the standards of other biological processes as well as those of humanbuilt machines this is an extraordinarily efficient process In addition to being highly exergonic cellular respiration like photosynthesis is a redox reaction Focused Reading p 11516 quotObtaining energy and electrons quot stop at quotAn overview quot Cellular respiration happens in two basic processes 1 The oxidation of glucose and water glycolysis and the citric acid cycle also called the Krebs cycle after the scientist who first described it 2 The reduction of oxygen oxidative phosphorylation While ATP is synthesized from ADP and Pi Pi HZPO4 throughout both halves of cellular respiration the vast majority of ATP is synthesized during oxidative phosphorylation During glycolysis and the citric acid cycle the hydrogens highenergy electrons plus protons are removed from glucose which becomes C02 in the process This equation is CEHIZO6 6HZO gt 24e39 24H 6C02 The 24 hydrogens that are removed from glucose and water come off as 24 highenergy electrons plus 24 protons As in the case of photosynthesis the 24 electrons and some of the protons are transferred to carrier molecules In cellular respiration this carrier molecule is NAD the same molecule as NADP but minus one phosphate and in one case FAD NAD and FAD pick up the 24 electrons from glucose and water that are released during glycolysis and the citric acid cycle As in the case of photosynthesis the hydrogens picked up by NAD and FAD are highenergy electrons plus protons NAD becomes NADH by picking up two electrons and one proton while FAD becomes F ADHZY by picking up two electrons and two protons These highenergy electrons are carrying the energy that was originally carried to the earth as photons In the second part of cellular respiration called oxidative phosphorylation oxygen is reduced to water The equation is as follows 24e39 24H 602 gt 12H20 The 24 electrons required to reduce oxygen are donated from the carrier molecules NADH and FADHZ which picked up the hydrogens during glycolysis and the citric acid cycle Some of the 24 protons come directly from NADH and FADHZ while others come from the pool of H s in the cytoplasm This should all sound vaguely familiar Same idea as photosynthesis shuttle high energy electrons plus protons hydrogens from one molecule to another using a dinucleotide e g 139 NAD as an intermediate Nature has a few good ideas and they provided a selective advantage over and over again One idea that should not escape you in all this is that electrons are not all equal in energy level When an electron is bound into covalent bond between hydrogen and carbon as in glucose amino acids lipids etc it has a relatively high energy level When it is bound into a covalent bond with oxygen as in water it has a relatively low energy level Thus the transfer of hydrogens from a carbohydrate forming COZ to oxygen forming HZO constitutes an exergonic process in which the energy level of the electrons falls This loss of energy is converted to ATP and heat which is released into the environment Study Questions 1 Explain the process of homeostasis in relation to thermodynamics What is it and why is it important 2 In this unit on bioenergetics you have now encountered the four biological processes that yield the ATP that living creatures use to power their lives What are they 3 Analyze the cellular respiration equation as a redox reaction What is being reduced What is being oxidized During which processes do each of these reactions occur Be able to do the same for the photosynthesis equation 4 Explain how hydrogens are shuttled from one process to the other in cellular respiration What molecules do the shuttling 5 Explain the concept of highenergy electrons storing energy How is this energy released How is it stored in the first place qTOP So how is this all accomplished Cells in our body get glucose from the blood The concentration of glucose is always very low in the cytoplasm for two reasons 1 glucose is constantly being burned for energy and 2 as soon as glucose enters a cell it is immediately converted to glucose6phosphate whether it enters glycolysis or not Glucose6phosphate is not the same as glucose thus glucose is removed from the cytoplasm by phosphorylation and the cytoplasmic glucose concentration remains very low Being a hydrophilic organic molecule and not a simple ion glucose must cross a cell s plasma membrane by being transported by a glucose transport protein and not a glucose channel However because the concentration of glucose is higher outside the membrane in the blood than in the cytoplasm the process can be passive not requiring ATP Focused Reading p 856 Physical nature stop at Osmosis p878 Diffusion stop at quotActive transport quot WWW Reading Relative Sizes 14o The model for the glucose transport protein is very much like the active transport models you studied in Unit 1 However there is only one substrate binding site on the molecule a site highly speci c for glucose The affinity of this site does not change as the protein opens to the inside and then the outside of the membrane Let39s say the concentration of glucose is 100 fold higher outside a cell than inside Thus when the glucose transporter is open to the outside the cell it is 100 times more likely that a glucose will hit its binding site on the transporter and stick before the transporter ips to the inside After the transporter ips because the bonds between glucose a ligand and its transporter are weak glucose wiggles free through its own kinetic energy Now it is 100 times likely that a glucose molecule from the cytoplasm will bind to the site before it ips back to the outside After it ips it is 100 times m likely that glucose will bind Thus for every glucose molecule that is transported outward 100 are transported inward and the net transport is inward without the expenditure of energy in the form of ATP One more thing the glucose transporter does have one additional site on it for allosteric modulation This causes the transporter to ip faster or slower allowing the rate of transport to be increased or decreased Molecular oxygen is hydrophobic because the double bonds between the two oxygens are not polar both oxygens have equal affinity for the electrons Therefore oxygen can enter a cell by passive diffusion across the phospholipid bilayer Since oxygen is constantly being converted to water by cellular respiration the oxygen concentration in the cytoplasm is lower than in the blood outside the cell Therefore oxygen enters down its concentration gradient In fact this is a self regulating system since a rapid consumption of oxygen due to increased cellular respiration increases the concentration gradient across the plasma membrane and causes oxygen to enter a cell at a faster rate by passive diffusion Study Questions 1 The transport of glucose into most mammalian cells is a passive process Explain the conditions that make it possible for glucose to cross the membrane without the expenditure of energy in the form of ATP 2 Explain the passive transport process of glucose In what ways does it differ from active transport How can the rate of passive transport of glucose be changed 3 Explain how the delivery of oxygen to cells is a selfregulating process that adjusts as the rate of cellular respiration changes 4 Along the lining of your intestines there a different glucose transporter a symporter that uses the Na gradient to power glucose uptake See Fig 513 p 90 Why is this necessary Once the glucose is inside the cytoplasm it can be oxidized for energy Focused Reading p 68 quotMitochondria are quot stop at quotPlastids photosynthesize quot p 312 quotFunctional groups quot stop at quotIsomers have quot p 31 Table 220 p 11625 In the presence stop at The respiratory quot In lab we worked with IDHFind the Krebs cycle step catalyzed by the mitochondrial form of IDH WWW Reading Glycolysis Summary Look over all the steps in glycolysis and the Krebs cycle and try to understand each one While you do not have to memorize these steps you will understand the overall concepts a lot better if you have some understanding of the individual steps in the process Here are some chemical rules that will help you understand glycolysis and the Krebs cycle In general alcohols end in 01 e g ethanol butanol and estradiol endin 39 Jegfor quot39 39 ketones end in 0ne eg cortisone acetone acids end in ic acid or ate e g carbonic acid carbonate phosphoric acidphosphate oxidation oxidation RCOH gt RCO gt RCOOH RCOH lt RCO lt RCOOH reduction reduction OH ICI ICI R C H R CH R C OH alcohol aldehyde acid 0 R C C or ketone When alcohol groups are oxidized they become aldehydes or ketones When aldehydes or ketones are oxidized they become acids Conversely when acids are reduced they become aldehydes or ketones which become alcohols when they are reduced For example 1 2 CH3CH20H lt gt CH3CHO lt gt CH3COOH ethanol acetaldehyde acetic acid The forward reaction is oxidation while the reverse is reduction In reaction 1 the oxygen in the hydroxyl group breaks its bond with hydrogen and the carbon breaks its bond with one of its hydrogens and carbon and oxygen form a double bond This carbonoxygen double bond is a carbonyl group If this occurs at the end of a molecule it is an aldehyde group if it occurs anywhere but the end it is a ketone group This is the loss of hydrogen or oxidation 142 In reaction 2 the carbon breaks its attachment to the hydrogen and bonds with a hydroxyl group When a carbonyl and hydroxyl are bonded to the same carbon this is an acid group During this process the ketone or aldehyde gained an oxygen thus this process is oxidation Sugars contain one carbonyl group and several hydroxyl groups Therefore they are not very highly oxidized or in other words they are highly reduced Through the process of glycolysis and the Krebs cycle more and more hydroxyl groups are converted to aldehyde ketone and acid groups by the process of oxidation Finally the most highly oxidized form of carbon is produced carbon dioxide One more rule that may help if a compound ends in ate it is the ionized form of an organic acid For instance because acetic acid is an acid when you put it in water it quotdonatesquot a proton as follows CH3COOH lt gt CH3COO39 Ht acetic acid acetate Thus when you call a molecule glutamate or pyruvate or oxaloacetate you are indicating that the molecules are acids that have ionized In their nonionized forms they are glutamic acid pyruvic acid and oxaloacetic acid Biologists frequently use the ionized and nonionized names interchangeably so don t be thrown off by this Study Questions What is the difference between substrate level phosphorylation and oxidative phosphorylation What is being phosphorylated in each process 2 For what purpose is ATP spent during the first few reactions of glycolysis If glycolysis is supposed to yield energy not cost energy why is the cell doing this 3 While glycolysis is considered to be aredox process really only one step in the pathway is a redox reaction What happens at this step 4 Given just the names of compounds in reactions be able to determine whether the reaction is an oxidation or a reduction For example Dformaldehyde to formate phosphoglyceraldehyde to phosphoglycerate 5 Explain the difference in the chemical structure of a molecule whose name ended in quotatequot as opposed to quotic acid 6 What is the overall reaction of glycolysis What goes in and what comes out What is the fate of all products 7 What is the overall reaction of the Krebs cycle including pyruvate oxidation What goes in and what comes out What is the fate of all products 143 9 What is cyclical about the Krebs cycle Explain in general how carbons cycle through this pathway 9 Ifyou had to summarize the processes of glycolysis and the Krebs cycle in the simplest terms how would you describe it If your life depended on clearly conveying what happens in these processes in two or three sentences what would you say 10 How do our cells obtain glucose from the blood Does this process require the expenditure of ATP Explain 11 While the overall reactions of glycolysis and the Krebs cycle yield energy the process also costs some cellular energy in the form of ATP What steps in the process require energy and why By how much is the total ATP yield reduced by these endergonic steps NEWS ITEM A group of researchers at Duke University have located a second protein that interacts with huntingtin and it also interacts with HAP1 The new protein is glyceraldehyde3phosphate dehydrogenase the first enzyme in the energy harvesting half of glycolysis This is the first protein in the HD story that has a known function The scientists are entertaining the idea that HD and four other less common neurodegenerativeDdiseases have reduced energy production due to a molecular interference with glyceraldehyde3phosphate dehydrogenase See related summary Marcia Barinaga Science Vol 271 pp12331234 1 March 1996 A group from Syracuse University has used antibodies to localize the enzymes involved in glycolysis in Drosophila flight muscles Surprisingly these cytoplasmic proteins were found spaced in regular intervals over the striations in the muscles When mutations were made in these enzymes so that they were still functional but no longer located over the striations the Drosophila was no loner able to y Therefore glycolytic enzymes are necessary for energy production but it appears that this production must be located in specific areas inside some cells in order for the cells to function properly Molecular Biology of the Cell Vol 8 1665 September 1997 qTOP For every one glucose molecule and six molecules of water that enter glycolysis and the Krebs cycle a cell makes six molecules of CO This C02 is hydrophobic and it leaves the cell by passive diffusion across the lipid bilayer As in the case of oxygen increased levels of C02 in the cytoplasm which would occur if cellular respiration rates increased would increase the concentration gradient This would in turn increase the rate at which C02 diffuses out of the cell Thus C02 elimination is a selfregulating process too To make this C02 cells transfer 24 hydrogens 24 highenergy electrons plus 24 protons to carrier molecules two at a time You need 12 carriers to transfer 24 electrons 10 NADH and two FADHz While these electrons have lost some of the energy they had when they were in glucose they haven39t lost very much and they continue to be quothighenergy In addition to the 24 hydrogens we have a net synthesis of four ATP two from glycolysis and two from the Krebs cycle produced by substrate level phosphorylation These four ATP are a net gain and can be used by the cell for anything it wishes ATP made in glycolysis is in the cytoplasm ready to be used The ATP generated in the Krebs cycle is in the mitochondria and can be used there or can be transported across the mitochondrial membrane into the cytoplasm for use there Because ATP is in such high concentration inside the mitochondria ATP can go down its concentration gradient on a transport protein into the cytoplasm by the process of passive transport 144 The majority of ATP is synthesized by the cell from the energy stored in the highenergy electrons found in NADH and FADHZ The process of oxidative phosphorylation harvests this energy Focused Reading p 125 The respiratory chain quot stop at quotThe respiratory chain transports quot p 12628 Active proton stop at Two experiments WWW Reading Animation of Photosynthesis The process of ATP synthesis in photosynthesis and oxidative phosphorylation is essentially the same This process must be very ancient having evolved before plants and animals separated during evolution In fact because bacteria synthesize ATP this way too it must be one of the most ancient quotgood ideasquot in the biological world Bacteria pump protons toward the outside across their plasma membranes Protons then reenter the cell via an ATP synthase and ATP is synthesized Thus in bacteria the plasma membrane has a function that is equivalent to the inner mitochondrial and thylakoid membranes in eukaryotes NEWS ITEM The study of apoptosis programmed cell death has become providedpotential links to bioenergetics and to neurodegenerative disease Some of the main players in apoptosis are proteases called caspases that help to kill the cell by degrading it from the inside out Dr Shim izu and colleagues have shown that caspases are activated when cytochrome C is released from the mitochondrion When apoptosis is initiated cytochrome C which normally acts to help harvest energy acts as a messenger of doom to activate the caspases so that they can destroy the cell Caspases also appear to be activated in Huntington39s disease In this case it seems that the presence of the CAG repeats activates the apoptosis cascade leading to neuronal death in both mice and humans This finding leads to hope for treatment of HD since caspase 39 ibitors injected into the cerebrospinal uid slowed progression of the disease See Nature 1999 v01299 p411 and 263 NADH FADH 2 inner mitochondrial membrane Study Questions 145 1 Explain the process by which ATP is synthesized from ADP and Pi using the energy of the highenergy electrons from NADH and FADHZ You need not memorize the name of the cytochromes but you should understand the process and be able to explain it accurately 2 What role does oxygen play in oxidative phosphorylation Why is oxygen a good molecule to play this role why not carbon or neon or hydrogen 3 Approximately how many ATP are synthesized in oxidative phosphorylation per glucose molecule Most cells in our body do not absolutely have to use glucose as a source of energy They can oxidize lipids or amino acids to make ATP While most cells are like this the cells of the brain M burn glucose no other fuel will do Thus if you suffer from low blood sugar hypoglycemia hypo low glyc sugar emia in the blood you may experience loss of ability to concentrate to speak coherently and even to stay conscious all signs of compromised brain function If a source of lipids is available a cell will burn lipids along with glucose for fuel Fats are digested into glycerol and fatty acidsboth of which can 39feed39 into metabolism Glycerol is converted into glyceraldehyde phosphate and used in glycolysis This releases a little energy but most of the energy from fat is stored in the fatty acids A process called beta oxidation breaks fatty acids into 2 carbon units that can enter cellular respiration as acetyl coA If glucose and lipid levels are low the cell will begin to burn amino acids for fuel This can be detrimental to your health because the amino acids must be deaminated in order to be burned and the brain and kidneys have a hard time dealing with the extra ammonia that is produced Focused Reading p 13032 quotMetabolic pathways quot stop at quotRegulating energy quot p 4951 quotLipidsquot stop at quotPhospholipids quot Study Questions 1 Fat stores more energy per gram than carbohydrates What part of a fat molecule stores the most energy How is that part broken apart so that components can enter metabolism 2 How is glycerol burned for fuel Where does it enter the cellular respiration pathway 3 What must happen to amino acids before they can be burned as fuel One common point of entry for amino acids into the Krebs cycle is at alphaketoglutarate Refer back to the section on nitrogen fixation and the production of amino acids in this unit and explain how an amino acid such as L glutamate might enter the Krebs cycle at alphaketoglutarate 146 Intestinal cells work very hard all the time and have a fairly constant metabolic rate In contrast cardiac myocytes have a fairly low metabolic rate when you are sleeping and a very high metabolic rate when you are exercising Therefore the at which glucose is burned must be regulated so that you don39t waste energy burning a lot of fuel when little energy is needed or starve for energy burn very little fuel when a lot of energy is required All cells must be able to regulate the rate at which glucose is burned and ATP is created Focused Reading p 1324 quotRegulating the energy pathwaysquot to the end Study Questions 1 Explain in chemical terms how the rate of glucose oxidation is controlled by environmental conditions Why is this evolutionarily adaptive 2 Describe the structure of phosphofructokinase How many sites binding does it need to perform its function What molecules bind at each Explain the name of the enzyme What makes it a good enzyme to function as a rate regulator for cellular respiration Now that we understand how cells get energy from sugar we can understand why cyanide is so lethal and so popular with extortionists Cyanide its chemical formula is ON has a negative charge as the name indicates since it ends with the suffix ide As you know from your basic chemistry negative ions are attracted to positive ions cations Unfortunately some of our vital enzymes use cations as a part of their structure One class of enzymes that use iron ions is cytochrome oxidases As the name tells you they oxidize cytochromes by taking away an electron and these oxidases are located in the inner mitochondrial membrane The highenergy electron temporarily binds to the iron in the cytochrome oxidase before the electron is passed onto the next cytochrome in the electron transport pathway as seen in this diagram cytochrome cytochrome high energy electron cytochrome oxidase 147 As a result our enzymes and our very lives depend upon cytochrome oxidases being able to carry highenergy electrons temporarily Cyanide has the unfortunate tendency to bind irreversibly to the iron ions in cytochrome oxidases If the iron is occupied by CN39 then it cannot accept another electron from a cytochrome that is carrying a highenergy electron N N N N But CN39 does not bind to all of the cytochrome oxidases only the next to last one So what s the problem As you know most of the H ions are transported into the mitochondrial intermembrane space before this next to the last step of the electron transport pathway How could missing out on the last two steps kill you Think of yourself in a bucket brigade where each person passes one bucket of water onto the next and receives another bucket of water from the person upstream You are the next to last person passing on buckets in a long line of bucket passers All of a sudden the person you normally give your bucket to has stopped he has been given an ice cold glass of sweet tea and cannot be bothered to accept your bucket What are the repercussions for every one else in this long line of bucket passers Since you cannot get rid of your bucket the person who normally passes a bucket to you cannot unload her bucket and a domino effect rushes backwards until every person in the long line is left holding a bucket of water with no one to accept it This is what kills you with cyanide poisoning The next to last cytochrome oxidase is gummed up with CN39 therefore it cannot relieve a cytochrome of its highenergy electron and this clogs up the entire electron transport pathway NADH FADHZ l inner mitochondrial membrane 148 Therefore no H ions are transported into the intermembrane space no H gradient is created which results in an absence of the chemiosmotic generation of ATP You die by a deprivation of ATP you run out of energy even though you have already generated lots of NADH and FADH2 Study Questions 1 Given what you know about electron transport why might paraquat be harmful to humans 2 How does cyanide kill 3 What do photosynthesis and cellular respiration have in common How do they differ NEWS ITEMS A single base pair substitution has been identified in patients who suffer from severe infantile lactate acidosis and encephalomyopathy These symptoms were due to a genetic disease but surprisingly the gene is not located in the nucleus The base pair substitution occurred in a mitochondrial gene which encodes one subunit of the mitochondrial ATPsynthase The mothers of each patient contained 11 mixtures of wildtype and mutant mitochondrial DNA suggesting that each mother had inherited mutant DNA from their mothers too See Haustek et al Biochemical etBiaphysica Acta Vol 1271 349357 1995 In the 29 April 1997 issue of Prac Nat Acad Sci a team from UVa has identified a gene that is mutated in many patients with Alzheimer s Disease This gene is located in mitochondrial DNA and encodes for one of the 13 proteins that make up cytochrome oxidase These data are consistent with two points 1 It has been suspected for many years that AD patients may suffer from poor energy metabolism This would suggest that oxygen free radicals 0 cause neuronal damage 2 Children of mothers with AD are more likely to get AD than children whose fathers have AD We inherit almost all of our mitochondrial DNA from our mothers Science Vol 276 682 May 1997 4 Mitochondrial genes encode several components of the ATP synthase complex One family has been identified that has a missense mutation in subunit a of the synthase and this gene is mitochondrial gene patients suffer from neurogenic muscle weakness Draw a pedigree for this family 5 Hypothesize why paraquat is probably toxic to animals Sour Grapes Land US in the Dock by Dan Charles excerpted from New Scientist 16 March 1991 The US Food and Drug Administration may have botched tests that appeared to detect cyanide in grapes from Chile two years ago ON the basis of the tests the US banned imports of all fruit from Chile for five days Last month Chilean fruit growers filed a legal claim against the US government arguing that the FDA s mistakes in analytical chemistry cost them more than 400 million Manuel Lagunas Solar a radiochemist at the University of California Davis has spent the last two years injecting grapes with cyanide and trying to duplicate the FDA s results From his research which was paid for by Chile s fruit growers one thing seems clear the grapes were not contaminated with cyanide when they left Chile Lagunas Solar suspects that the grapes were never contaminated at all 149 The central problem with the FDA s results is that they found too much cyanide says Lagunas Solar His tests show that the chemistry of grapes breaks down and detoxifies cyanide with remarkable speed The FDA detected 62 micrograms of cyanide in the pulp of the two grapes According to Lagunas Solar this would mean large amounts must have been injected into the grapes just a few hours before the tests But the grapes were on the docks in Philadelphia or in the custody of the FDA for longer than that before the tests were carried out Working backwards Lagunas Solar estimates that a terrorist in Chile would have had to inject a minimum of 4000 micrograms of cyanide into the grapes in order to produce this result It is more likely that ten times this much would be necessary he says But the larger of these quantities cannot physically be injected into grapes and even the smaller amounts would have damaged the grapes and contaminated other grapes in the package The grapes the FDA analyzed were in good physical shape and they did not find any other contaminated grapes even in the same bunch We were able to rule out with confidence the hypothesis that cyanide tampering could have occurred in Chile says Lagunas Solar Bill Grigg a spokesman for the FDA rejects Lagunas Solar s conclusions The FDA s own studies confirm that cyanide does disappear rapidly from grapes and other kinds of fruit But in one FDA study two grapes did retain large amounts of cyanide for between 3 and 6 days without having much effect on the look of the grapes No one has been able to explain this result A further puzzle in the saga is that the FDA was also unable to find any traces of cyanide on the other grapes from the same bunch even using their most sensitive techniques Lagunas Solar s experiments show that traces of cyanide from contaminated grapes will show up throughout an entire crate of grapes STOP 115 QUESTION 4 Why would authorities ask you to update vaccinations after a ood Bacteria are stunningly diverse and comprise an entire domain Eubacteria It is impossible to cover the bioenergetics of this entire domain in any meaningful detail However brief introduction to the metabolic diversity of bacteria will help broaden your understanding of the variety of ways organisms can acquire energy Focused Reading p 4667 quotA small minority quot stop at quotProkaryote quot p 4645 Prokaryotes have stop at Nitrogen and sulfur p 469 Fig 2612 WWWeb reading httpwwwcdcgovncehemergency oodhtml Choose Immunizations We will focus on one bacterium Clostrz39dz39um tetam39z39 the organism that causes tetanus During the summers of 1993 and 1995 the Mississippi River and its tributaries ooded and caused billions of dollars in property damage and catastrophic losses for thousands of people in the Midwest The Red Cross responded to this natural disaster by providing shelter food clothing and tetanus vaccine Why in the midst of chaos and misery did the Red Cross spend time and money delivering this vaccine and what does this have to do with studying bioenergetics Well herein lies the tale Tetanus causes all of the skeletal muscles of the body to contract into rigid paralysis If untreated the disease is fatal the diaphragm the skeletal muscle that facilitates breathing contracts into a rigid paralysis along with all the other skeletal muscles Because it cannot relax the victim cannot exhale and subsequently suffocates Tetanus is caused by a protein toxin poison released by the bacteria Clostridium tetam39z39 Because this toxin is released by the bacteria as a soluble molecule it is called an exotoxin Other bacteria called gram negative because they do not stain with a gram stain such as Salmonella contain a toxic molecule in their outer membrane called lipopolysaccharide or LPS Because LPS remains bound to the bacterial membrane and is not released as a soluble product it is called an endotoxin Exotoxins are very dangerous and often lethal eg tetanus botulism diphtheria cholera whooping cough while endotoxins have lower levels of toxicity and are rarely fatal The exotoxin produced by Clostridium tetam39z39 is called a neurotoxin because it attacks the nervous system If the tetanus bacteria is growing somewhere in the body and is releasing this toxin the toxin is carried throughout the body by the blood When the toxin reaches the brain it binds to and inactivates components within the membranes of neurons The inactivation of these components inhibits the nerve impulse and blocks contraction of the muscles on the other side of synapse While it is apparent that your brain causes muscles to contract we often forget that your brain must also inhibit contraction or cause relaxation For instance in order for you to ex your arm the muscles that extend your arm must relax Otherwise both sets of muscles would contract into a tug of war and your arm would be rigidly paralyzed Tetanus toxin prevents the victim39s muscles from relaxing and all movement is halted in rigid paralysis NOTE Rigid paralysis can be contrasted with accid paralysis a condition in which muscles cannot contract at all the body cannot move because it is limp or accid Study Questions 1 What is the difference between an exotoxin and an endotoxin 147 2 What are the symptoms of tetanus What happens at the cellular level to cause these symptoms The genus Closm39dz39um also contains other pathogens ie diseasecausing agents such as the organism that causes botulism Clostridium botulinum a form of severe and often fatal food poisoning as well as the organism that causes gas gangrene C lostridz39um perfringens Other Clostrz39dz39a are nonpathogenic and are used to produce valuable fermentation products such as various alcohols and organic acids or to fix atmospheric nitrogen All bacteria in the genus Closm39dz39um are soil bacteria and all are obligate anaerobes Anaerobes harvest energy in the absence of oxygen The metabolic pathways we have discussed so far need oxygen that39s why they are called cellular respiration So how do these bacteria generate ATP They rely on a bioenergetic pathway that looks very familiar but has a different ending They rely on fermentation a metabolic pathway that oxidizes glucose to pyruvate using the reactions of glycolysis producing NADH and ATP in the process no oxygen required Then instead of having further energy harvested from pyruvate via respiration these organisms use NADH to reduce pyruvate to lactic acid or to ethanol and C02 The energy yield is less than aerobic cellular respiration but some ATP is harvested 2 ATP per glucose and NAD is regenerated so it doesn t build up in the cell And a large industry has been built around the production of ethyl alcohol Focused Reading p 129130 quotFermentationquot stop at quotConnections with other pathwaysquot Study Questions 1 What is the difference between a facultative anaerobe and an obligate anaerobe If you were to do a protein analysis of a facultative anaerobe and an obligate anaerobe what differences would you nd In other words what enzymes would you expect to find in the facultative anaerobe that would be missing from the obligate anaerobe and vice versa 2 Explain the process of fermentation The absence of oxygen is a requirement for the fermentation process Explain why this is the case 3 Compare and contrast the production of ATP through aerobic and anaerobic metabolic processes How is ATP made in each process Which process yields more usable energy for the cell By how many fold Explain What are the end products of each process Explain how these end products are produced 4 Facultative anaerobes need a control mechanism that responds to presence or absence of oxygen Based on what you know about molecular control systems develop a reasonable hypothesis that describes such a functional control system for facultative anaerobes Because the C lostrz39dz39a are obligate anaerobes they are killed by oxygen Thus they must live in an environment in which oxygen levels are extremely low While it is not clear how oxygen kills these microbes the dominate hypothesis is that they are unable to detoxify eliminate the toxic by products of oxygen reduction hydrogen peroxide H202 superoxide 0239 and hydroxide radicals OH39 These byproducts are toxic to all cells but facultative anaerobes and aerobes contain enzymes that immediately destroy these substances as soon as they are formed while obligate anaerobes lack these enzymes 148 Getting away from oxygen on this planet is no small task Thanks to the phototrophs air is 20 oxygen a lethal level for anaerobes They must therefore live in places that are deprived of oxygen such as deep soil sediments of rivers and lakes bogs and marshes canned foods intestinal tracts of animals sewagetreatment systems or injured tissue which has had its blood supply interrupted Because Closm39dz39um tetam39z39 lives in soil and the intestinal tract of animals wounds that come in contact with dirt or animal feces are particularly susceptible to the development of tetanus This is why people who work with animals professionally or as a hobby should be sure they have their tetanus vaccinations up to date The common practice of bleeding a wound especially a deep puncture wound is a good one since the bacterium enters the body through a wound and blood carries oxygen to the area which can kill the tetanus bacterium So why give tetanus vaccinations to ood victims Because the oodwaters would wash the tetanus bacteria from soils water treatment plants and animal feces If an open wound came into contact with this oodwater it might become infected with the tetanus Losing your possessions to a ood is one thinglosing your life is something else When C lostridz39um tetam39z39 enters a wound if the oxygen level is very low it will begin to divide and produce a colony This bacterial colony does not invade the body but excretes the toxin that is carried from the wound into the body and eventually into the central nervous system The exotoxin enters neurons by endocytosis review on p 111 and travels retroaxonally in reverse direction to nerve impulses to reach the spinal inhibitory intemeurons Tetanus toxin is a protease it degrades proteins but it is a very selective one Its substrate is VAMP the integral membrane protein that facilitates neurotransmitter release Remember way back when we talked about neurotransmitter release Why don t you go back and take a quick peak at Unit I For a good summary see Montecucco and Schiavo TIBS 18 324329 1993 By blocking the release of inhibitory neurotransmitters no muscles get the message to relax and get stuck in a contracted state Tetanus vaccinations are aimed at this toxin rather than the bacteria itself The tetanus vaccine contains purified tetanus toxin which has been denatured with formaldehyde Because protein function is dependent on its 3D structure denaturation makes the toxin inactive In this form the toxin is called a toxoid The immune system however will react to the toxoid in the same manner that it would a toxin Thus the body produces an immune response antibodies against tetanus toxoid which neutralizes the real toxin should it ever be encountered before it reaches the brain NEWS ITEM During the summer of 1997 millions of waterbirds died to a mysterious illness The common symptom prior to death was accid paralysis and the disease was initially called limbemeck The cause was eventually identified an outbreak of botulism caused by Clasm39dium botulinum Science Vol 278 1019 November 1997 Study Questions 1 What kind of paralysis is caused by tetanus toxin and how does it cause paralysis 2 Are we capable of anaerobic metabolism If so when and where 3 What kind of toxin would cause limbemeck and why would this be fatal As you know we are capable of burning glucose anaerobically for short periods of time It has been determined that our muscles contain about 5 millimoles of ATP per kilogram This ATP supply is depleted in a few seconds when we begin to exercise After 10 seconds we use ATP that has been 149 generated by an enzyme called phosphocreatin kinase that rips a phosphate from phosphocreatin and adds it to ADP After one or two minutes of hard breathing you will be using anaerobic metabolism glycolysis to generate ATP and lactic acid which is why your muscles burn with extensive exercise Eventually this oxygen dept must be repaid so your muscles can return to aerobic metabolism which is why we are obligate aerobes TWO RESEARCH QUESTIONS AND APPROACHES IN BIOENERGETICS Focused Reading p 3312 stop at quotHemoglobinquot p 3401 quotScreening for abnormal quot stop at quotThere are several Genetic Defects in Metabolism Sometimes a mutation will occur in an organism that interferes with its ability to metabolize fuels properly Because mutations alter genes and each enzyme in the metabolic pathways is produced by one gene each mutation in a single gene should alter or destroy only one metabolic enzyme By studying the results of these mutations some of the complexities of the metabolic pathways can be unraveled For instance 1 in 15000 human infants is born with the inability to metabolize oxidize the amino acid phenylalanine This condition is called phenylketonuria or PKU Normally excess phenylalanine is converted to either fumarate or acetyl CoA and is oxidized in the Krebs cycle for energy However this normal oxidation pathway cannot occur in people with PKU because ability to make the rst enzyme in the pathway phenylalanine 4 monooxygenase is destroyed by a mutation in both alleles of this locus Thus excess phenylalanine must be eliminated by an alternative pathway in which it is converted to phenylpyruvate Phenylpyruvate accumulates in the blood is excreted in the urine and causes irreversible brain damage and mental retardation People with PKU can escape the effects of this disease by eliminating phenylalanine from their diet including Nutrasweet Focused Reading p 21820 quotOne gene quot stop at quotDNA RNA p 219 Figure 121 Metabolic mutations naturally occurring or induced by agents such as xrays in lower organisms eg yeast and bacteria can be especially useful in helping us characterize metabolic pathways For instance in 1941 GW Beadle and EL Tatum induced a series of mutations in the mold Neurospora See Figure 121 on page 219 for a diagram ofthis study Normal or wild type Neurospora can synthesize all 20 amino acids Therefore it will grow on a simple medium containing only glucose and a source of nitrogen in the form of ammonia Mutants however cannot grow on this simple medium but rather each mutant needed one nutritional supplement in order to survive For instance one mutant could only live if the amino acid arginine was added to the medium This meant that this particular mutant was defective in one of the enzymes that synthesize arginine Without arginine the mold could make no proteins and died In looking further at the arginine mutants Beadle and Tatum discovered that there were several different types of mutants Class III mutants required arginine in the medium or it would die Class II mutants could live on arginine or citrulline Class Imutants could live on arginine citrulline or omithine Thus Beadle and Tatum were able to arrange the three terminal enzymes in the proper sequence of arginine synthesis Since these experiments were performed investigators have used metabolic mutations in other pathways to characterize many of the enzymatic steps in metabolism Spectroscopic Analysis of Cytochromes The oxidation and reduction rates of the cytochromes can be measured using a spectrophotometer because the cytochromes change color as they become oxidized and reduced This is very much like hemoglobin the molecule that carries oxygen in 150 mammalian blood When oxidized by the gain of oxygen hemoglobin is bright red When reduced by the loss of oxygen the color shifts to a more bluishred or reddish purple Because spectrophotometers can analyze the wavelengths of light being absorbed by a substance and since the wavelength of absorbance changes as the color changes the oxidized and reduced forms of the cytochromes produce different absorption spectra When the cytochromes are spectroscopically analyzed across time their rate of oxidation and reduction can be measured The affect of various factors on the rate of electron transport can then be tested Study Questions 1 If you are given data similar to that in figure 121 p219 be able to determine the order of genesproteins in a metabolic pathway STOP 151 Unit IV Other Interesting Topics In this Unit we will cover Cancer 11 AIDS III Genetic Engineering 1 Cancer WWW Reading Cancer Statistics 20year trends One person in ve in the developed world will die of some form of cancer For 25 years cancer research has been among the top priorities of the biomedical research community in the United States We have learned much about this disease and in the process about the function of normal cells Focused Reading p 3423 quotCancer Cellsquot stop at Some cancers Cancer is de ned as the presence of a malignant tumor in the body A neoplasm or tumor is a relentlessly growing mass of abnormal cells that are dividing in de ance of normal restraints on growth However most tumors are benign that is all of the cells of the tumor remain in the tumor mass and do not invade other tissues Benign tumors are not cancerous but they can be life threatening if they occur in places in the body from which they cannot be removed without causing serious damage e g some places in the brain Such tumors are said to be quotinoperablequot However most benign tumors are not life threatening and can be easily treated by surgical removal A tumor becomes malignant or cancerous when its cells invade the other tissues Invasiveness usually implies that the cells of the tumor can break loose travel to a new site in the body through the blood or lymph and establish secondary tumors Such a tumor has metastasized The process of spreading is called metastasis and it is the hallmark of cancerous tumors While benign and pre metastatic cancerous tumors are relatively easy to cure by surgery or localized radiation or chemotherapy metastasized tumors are very dif cult to treat Because cancer arises from a single cell that is growing out of control in order to cure the metastasized cancer eve single cancerous cell in the body must be destroyed This is virtually impossible in widely disseminated cancers The type of cell that becomes cancerous de nes the name of the cancer Focused Reading p 695 quotTissues quot stop at quotNervous tissue quot p 695 Fig 402 Animal tissues come in four varieties l Epithelia lines the inside and outside surfaces of the body e g skin lungs blood vessels etc and provides the bulk of functional cells in internal organs eg endocrine glands liver pancreas kidney Cancers of epithelial cells are called carcinomas 2 Connective tissue is a very broad category of tissue which includes blood bone cartilage fat tendons ligaments and the strong protein bers that hold all the organs together Cancers of the connective tissue cells are called sarcomas Cancer of the white blood cells the leukocytes is called leukemia 3 Muscle forms the mass of the skeletal muscles creates the walls of blood vessels and internal organs smooth muscle and forms the wall of the heart cardiac muscle 152 Cancers of muscle cells are also called sarcomas 4 Nervous tissue forms the brain spinal cord and nerves in the body Cancers of the nervous system are called neuromas if they involve actual neurons and gliomas if the involve the supporting cells of the nervous system While these terms de ne broad categories of cancer each type of cancer has its own distinguishing name For instance basal cell carcinoma is a kind of skin cancer caused by the cancerous growth of a basal cell in the skin an epithelial cell Melanoma is a different form of skin cancer caused by the cancerous growth of a melanocyte the pigment producing cells of the skin Both types of cancers are carcinomas but they have very different characteristics the former being very easily treated and almost never fatal while the latter is much more life threatening Most cancers 85 are carcinomas and in fact an agent that causes any type of cancer is said to be a carcinogen or to be carcinogenic Below you will nd a list of some of the most prevalent cancers in the United States Lung cancer has the distinction of having the highest incidence of any single cancer at over 150000 new cases per year d the highest death rate at 87 Actually this method of calculating the death rate understates the threat of this disease Over 90 of lung cancer victims will die within one year of diagnosis The other feature that distinguishes lung cancer is that of all the cancers listed below it is by far the most preventable The vast majority of lung cancer victims smoked cigarettes or lived with a heavy smoker As in the case of AIDS this sadly preventable disease continues to claim lives needlessly Eve year lung cancer kills three times as many people as died in the Vietnam War Cancer US Site of New Cases Deaths in Female Cancer in 2000 2000 2000 Adapted from Greenlee RT Murray T Bolden S Wingo PA CA Cancer J Clin 2000507 33 To begin looking at what might cause cancer here are a few things we know from simple observation 1 Cancers tend to run in families However very few cancers exhibit Mendelian inheritance ratios indicating that most are heavily in uenced by nongenetic factors 2 Exposure to certain agents in the environment certain chemicals and irradiation is associated with the development of cancer We call these agents carcinogens 3 Ifwe perform the Ames test for mutagenicity we find that all carcinogens are mutagens 153 However not all mutagens contribute to the development of cancer 4 Malignant cancer cells have at least two things wrong with them 1 They can t stop dividing and 2 They leave their normal tissue beds and take up residence in areas of the body that are completely foreign to them So at a minimum based on this information we should be able to hypothesize that 1 Cancer is caused or enhanced by changes in the DNA which may be a inherited mutations since predisposition for cancer runs in families andor b new mutations since carcinogens cause mutations in the DNA 2 Cancerous cells have a defect in the molecules that control communication about a when to stop dividing and b in which tissue the cell should exist The genetic changes could be in the molecules that control communication The mutations could be in genes that encode transcription factors needed to transcribe the genes that encode communication molecules Keeping this in mind let39s look at what we know about the normal signals that control cell division in normal cells Focused Reading p 1579 Interphase quot stop at quotEukaryotic quot p 1589 fig 94 and 95 Cell division is almost always studied by placing cells in tissue culture an experimental approach that grows mammalian cells in a petri dish Many types of animal and plant cells can be removed from an organism and if provided with the right combination of nutrients the right gas mixture and the right kind of substrate to sit on they will not only live they will also continue to divide Lots of picky details insetting up the system but a great way to get at how a cell really works without dealing with an entire pesky organism The tissue culture cells can then be treated in such a way that their cell cycles are synchronized Normally cells divide on their own inherent timetables regardless of what their neighbors are doing Having a culture of cells that are all at different stages in the cell cycle is not very helpful In order to study the changes that occur in the cell as it moves from stage to stage it is easier to look at a large population of cells in one stage in one dish and compare them to a large population of cells in a different stage in a different dish Certain drugs are used which arrest cell division at a given stage As each cell enters this stage it gets stuck there Since the cell cycle is just thata circle39 no matter what stage a cell was in when you added the drug sooner or later it will come around to the drugblocked step and get stuck Given enough time every cell in the petri dish will be ready and waiting stuck at the drug block By removing the drug all the cells resume dividing but now they are all sta1ting at the same point and will be in synchrony Why both getting synchronized cells Here is an example of a kind of experiment you can do with them Scientists hypothesized that a soluble factor in its cytoplasm stimulated cell to go past the G1S boundary This boundary is a step that commits the cell irreversibly to DNA synthesis and mitosis This point is also known as the restriction point or Gunget it a cell must pass 39go To test 154 the hypothesis they synchronized one dish of cells in G1 and another in S They then mixed the cells together and caused them to fuse so that they ended up with giant double cells This quotdouble cellquot has two nuclei with DNA in different stages of the cell cycle but all of the cytoplasmic molecules have mixed together Thus after fusion these two sets of chromosomes receive the same cytoplasmic signals When cells in the S phase were fused with cells in G1 the S39 DNA stayed the same but the G1 DNA began to replicate Thus there was some soluble signal molecule in the S phase cells that caused the G1 cells to enter the S phase Investigators wanted to know if this factor was made in S phase and then stayed as soluble factor in the cytosol for the rest of the cycle g was the factor destroyed after the S phase So they fused G2 cells with G1 cells This fusion did not result in the replication of G1 phase chromosomes Thus they hypothesized that this soluble factor was no longer present in the cell after the S phase was complete This soluble factor was called the S phase activator A rise in the concentration of this molecule in the cell triggers or facilitates the transition of the cell from G1 to S Normally a cell that enters the S phase has passed the restriction point and will undergo mitosis However another control molecule must signal that the S phase is complete before the cell will enter mitosis M If S phase has begun but DNA synthesis is artificially blocked so that it cannot be completed the cell will not enter mitosis until the block has been removed Also if a G2 phase cell is fused with an S phase cell the G2 phase chromosomes will wait for the S phase chromosomes to complete their duplication before they enter mitosis Therefore investigators hypothesized that there is a quotdelayquot molecule which prevents mitosis from beginning until the Sphase is complete After this quotdelayquot molecule has been inactivated the cell needs yet another signal to progress into mitosis the M phase promoting factor MPF IfMphase cells are fused with cells in any other phase the double cell will immediately enter mitosis even though the division will be unsuccessful for any chromosomes that have not replicated their DNA Thus MPF can override the quotdelayquot factor and therefore must not be present in the cell during S phase Otherwise the quotdelayquot signal would be overridden and the cell would enter mitosis prematurely The MPF is described further in your text The investigation of the cell cycle in wildtype and mutant yeast has proven to be one of the most powerful tools used to investigate the cell cycle In fact many cancer researchers actually study yeast This is because a number of mutant yeast strains exist which are deficient in different proteins required at different stages of the cell cycle These yeast strains are called cell division cycle mutants cdc mutants Therefore by determining which protein a given strain is missing and correlating it with the stage of cell division that is eliminated or dysfunctional in that strain the role of various proteins in the process of cell division can be determined So far over 50 genes have been identified that act to control some phase of the cell cycle In some cases these genes are wellknown biochemical entities in the cell For instance one cdc mutant strain that cannot go through the S phase has a defective gene for DNA ligase while another such mutant cannot Jutl i 39 from 39 391 However other genes encode true control molecules such as MPF Sphase initiation factor mitosis inhibition factor etc J In addition to identifying the intracellular proteins that control entry into the various stages of cell division investigators have also recently identified a number of growth factors which exist outside the cell and whose stimulation is necessary for cell division in many cases The following is a list of some of the major growth factors and the types of cells that responds to each 155 Factor Effect Plateletderived growth factor PDGF Stimulates connective tissue cells and supporting cells of the brain Epidermal growth factor EGF Stimulates many cell types Insulinlike growth factor I and II Collaborates with PDGF sand EGF simulates roliferation of fat cells and connective tissue cells Transforming growth factor B TGFB creases the sensitivity of most cells to other growth factors regulates differentiation in come cells Fibroblast growth factor F GF Stimulates cell division in many cell types including cells of connective tissue blood vessels and muscles Interleukin2 Stimulates cell division in T lymphoc es Nerve growth factor NGF Promotes growth in size of neurons Many blood cell growth factors Promote growth and development of all the cell types in the blood Study Questions 1 Understand the meaning of the terms that are used to describe tumors and cancers 2 List the phases of the cell cycle including the phases of mitosis and explain the significant events that happen in each phase 3 Understand the mechanisms used by the cell to produce two genetically identical daughter cells during cell division While these cells are genetically identical they may not be identical in other ways Explain 4 Describe the factors that have been shown to play a role in controlling triggering or inhibiting cell division eg nutrients cell size growth factors etc 5 What is the restriction point When does it occur and what is its significance 6 Discuss the structure and function of MPF What is structure of this molecule Through what mechanism does this molecule39s concentration rise and fall in the cell What is the role of this molecule in cell division What specific functions does this molecule perform 7 Discuss the following methods and their application to the study of cell division Give one example for each method illustrating the type of information that can be obtained using this approach A Cell synchronization in culture B Cell fusion C Yeast cdc mutants 8 Be able to interpret results from a cell fusion experiment in which cells of different phases of cell division are fused For instance if you learned that when G1 cells and S cells are fused the G1 phase chromosomes replicated their DNA what would you conclude 156 STOP What do we know now about cell division that will help us gure out what causes cancer V Cell division is carefully synchronized and controlled by proteins which must be encoded by genes N V Cells respond to signals from their environment to quotdecidequot whether or not to divide Each of these signals must be quotreceivedquot by the cell and responded to through a receptor system and second messenger system mediated by proteins that are ultimately controlled by genes External signals include the presence of adequate nutrients in some cases the presence of speci c growth factors the degree of contact with neighboring cells the degree of attachment to a substrate Note In this case substrate or substratum means a layer of protein bers that underlie cells and anchor them in position bP N A defect in any of these processes may cause a cancerous transformation And because cancers arising in different tissues or organs have very different characteristics different cancers may have very different causes What approaches can we use to gure out what exactly is wrong in a cancerous cell Well one approach that has been extraordinary helpful in cancer research has been the experimental use of viruses that are known to cause cancer in animal cells Such viruses are called tumor Viruses The first tumor virus to be discovered the Rous sarcoma virus discovered by Dr Rous causes connective tissue tumors in chickens Since this discovery several other tumor viruses have been identi ed and characterized Virus Species Tumor Rous Sarcoma Virus Chicken Connective Tissue FBJ osteosarcoma virus Mouse Bone Simian sarcoma virus Monkey Connective Tissue Abelson murine leukemia virus Mouse Leukemia Avian erythroblastosis virus Chicken Bone Marrow Harvey murine sarcoma virus Mouse Connective Tissue Avian MC29 Chicken Bone Marrow myelocytomatosis virus I Humans I Tumor I I Papillomavirus HV I Uterine Cervical Carcinoma I 157 HepatitisB Liver Carcinoma EpsteinBarr virus EBV Burkitt s lymphoma B cell cancer Nasopharyngeal Carcinoma Human TCell Leukemia VirusI Adult Tcell Leukemia HTLVI A 39 Herpes Simplex virus variant Kaposi s Sarcoma AIDsrelated opportunistic infection Note that the only way you can be m that a virus causes cancer is to inject the virus into an organism and watch for the development of the tumor with the aid of proper control injections Because you cannot do this with human subjects and because viruses are species speci c you have no way of de nitively determining if the human viruses listed above actually cause or contribute to the development of cancer However we do know that people who are infected with these viruses have an increased likelihood of developing the tumors listed in the table On the other hand infection with these viruses does not guarantee the development of a tumor it only increases the likelihood It is probably the case that human tumor viruses contribute only minimally to the overall incidence of cancer in humans However tumor viruses have been of exceptional importance in the study of cancer When a known tumor virus is placed in culture with its target cell the cell will become cancerous a process called cellular transformation If a biologist tells you a cell population has been quottransformedquot you need not ask quotinto whatquot D By definition it is a tumor cell By studying the differences between a cell population before and after transformation scientists can gain an understanding of the changes that occur during the development of cancer What happens to these cells in the process of transformation Well it depends on the cell and the virus but here is a summary of some changes that occur when cells are transformed I Plasma membrane related changes A Enhanced transport of nutrients B Excessive blebbing of plasma membrane small areas where the membrane balloons out like a weak spot in a garden hose C Increased mobility of the plasma membrane proteins 11 Adherence abnormalities A Diminished adhesion to surfaces B Disorganization of the cytoskeleton C High production of protease causing increased extracellular protein degradation 111 Growth and division abnormalities A Growth to an unusually high cell density B Lowered requirement for growth factors C Less quotanchorage dependencequot Can divide even without attachment to a solid surface This is highly unusual in normal cells Can continue to divide indefinitely immortality in tissue culture Can cause tumors when injected into animals P19 The actual growth of tumor cells in culture is amazing to see Depending on the cell type they can be large misshapen cells with little interest in attachment to the culture dish They divide while 158 they oat in the medium draining the culture medium of nutrients in a very short time Ifthey are quotfedquot that is given fresh growth media they will continue to divide inde nitely We would have absolutely no trouble lling Dana Science Building with the offspring of one wellfed tumor cell in a surprisingly short period of time This week s puzzlah a la Car Talk for fun only Average cell volume 125 pL l picoliter 0001 nanoliter and l nanoliter 0001 microliter Dana Science Building 50000 m3 Average cell cycle l division every 12 hours How long would it take to ll Dana Science Building Focused Reading p 2401 Viruses reproduce quot stop at Bacteriophages p 342 quotSome cancers are caused quot stop at most cancers p 244 g 135 as example ofa retrovirus The key to understanding cellular transformation is to look at the genetic changes that occur when the tumor virus infects the cell To study this investigators have focused on tumor retroviruses since as you know these viruses actually insert genes into the cell s genome that are then passed to the next generation of cell Thus these genes become a genetic characteristic of the tumor The rst such tumor RNA retrovirus studied was the Rous sarcoma virus RSV RSV inserts its entire genome into the host cell during the transformation event so it would be dif cult to determine which of these viral genes is responsible for the cancerous transformation However as is the case with all viruses RSV mutates at a rapid rate and investigators were able to nd a viral strain that seemed like a perfectly healthy virus it was able to infect cells insert its DNA and make new virus but it did not transform the cells When investigators looked for the difference between this nontransforming RSV and the nontransforming strain was missing one gene Investigators named this gene the src gene pronounced sark By convention the names of genes are italicized while the names of their protein products are not Investigators called this src gene an oncogene because it causes cancer quotOncoquot is from the Greek onkos meaning tumor The study and treatment of cancer is the eld of oncology What does src do What does it encode that causes this dramatic change in the behavior of cells As a next step in answering this investigators created a radioactive DNA probe that was complementary to the src gene and probed the DNA of normal cells using a Southern blot to see what they could nd Surprisingly they found a version of src in the genome of perfectly normal cells While these normal genes were not absolutely identical in structure to src they had a lot of homology They were so similar that they to be alleles of one another versions of genes that encode the same trait Investigators called this normal gene a proto oncogene Michael Bishop and Harold Va1mus two recent Nobel winners discovered Protooncogenes Also because they had found very similar genes in both a virus and its eukaryotic target in this case chicken connective tissue cells they needed a way to distinguish the viral gene from the eukaryotic gene Thus they called the viral version of the gene vsrc quotvquot stands for quotviralquot and the euka otic cellular version of the gene csrc quotcquot stands for quotcellularquot Since the discovery of src over 20 oncogenes and their protooncogene versions have been discovered through their presence in retroviral genomes and 159 over 50 oncogenes have been identi ed overall It is worth mentioning that src is a kinase that often phosphorylates growth factor receptors The viral form is about 20 times more active than the proto oncogene cellular form which helps explain why some viruses can lead to cancers As an aside you might be wondering why a virus would contain a gene that causes cancer These viral oncogenes don t appear to confer any survival value whatsoever to the virus In the case of a retrovirus the virus39 direct ancestor probably picked up the gene from a host when it became incorporated into that host s DNA Because retroviruses actually become part of the genome pieces of host DNA can be included in the viral genome fairly easily Ifthe viral genome is transcribed from viral DNA plus some of the anking human DNA the viral genome will contain a copy of the host s gene It is assumed that this is the way human genes get into viruses and when the virus infects the next cell it carries this human gene along with it and incorporates it into its new host39s DNA E Retroviral Genome Non transforming Virus Retroviral Insertion Point gene 1 gene 2 gene 3 gene 4 gene 5 gene 6 iiiiiiIiiiiiIiiiiiiiiIiiiiiIiiiiIiiiiiiiiIiiiiiIiiiiIiiiiIiiiIiiiiiIiiiiIiiiiiIiiiIiiiiiIiiiiiIiiiiiIiiiIiiiiiIiiiiiIiiiiIiiiiiiiiIiiiiiIiiiilililiiiiiliiiiii Eukaryotic DNA Infection iIiIiIiIiIlliIiIiIiIiIiIiIiIiIiIiIiIlliIiIiIiIiIiIiIiIililililililiiiliiililil iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii Eukaryotic DNA Retroviral DNA Eukaryotic DNA Viral Replication iiiiiiiiiiiiiiiiiiiiiiiii Retroviral DNA Eukaryotic Gene Transforming Retrovirus 160 In the case illustrated above if gene 4 is a protooncogene when the retrovirus picked it up in the process of replication it would become a retrovirus carrying an oncogene the de nition of a tumor virus While tumor viruses provide an invaluable approach to the study of cancer we should not get too carried away at this point and give the impression that cancer is caused by little bits of human DNA attached to retroviruses In fact this occurs only in a few cases of animal cancer However these Viral oncogenes have led us to their normal counterparts the cellular proto oncogenes and it is assumed that most cancers are caused when these normal proto oncogenes become mutated thus becoming oncogenes related to the development of cancer Investigators assume that cancercausing mutations are caused by the same mechanisms that cause other mutations such as 1 chemical agents that alter the structure of DNA 2 irradiation e g UV light that breaks DNA or forms inappropriate covalent bonds 3 retroviruses that insert themselves in or near a gene thus changing its proper regulation 4 normal mistakes typos made when the DNA is replicated during cell division While all cancers are caused by mutations not all mutations cause cancer Some for instance cause cystic brosis or colorblindness or a predisposition to heart disease It is assumed that what distinguishes cancercausing mutations from other mutations is that cancercausing mutations occur in protooncogenes Thus investigators have focused intensively on trying to nd out what proto oncogenes do Study Questions 1 Know the name of at least one human virus that is thought to be associated with the development of cancer Explain why it is dif cult to prove that viruses cause cancer in humans 2 What is a tumor virus What is cellular transformation Tumor viruses do not cause most human tumors Given this explain why tumor viruses have been the focus of such intensive research efforts What types of information have we gained about cancer through the use of these viruses 3 What are the characteristics of cells that have been transformed in tissue culture by a tumor virus If you were looking through a microscope at a cell culture what would you look for to determine whether or not you were looking at transformed cells 4 How do retroviruses come to carry human genes 5 Carcinogenic mutations are probably caused by the same agents as other mutations What are these agents How do carcinogenic mutations differ from other mutations Why do these changes cause cancer while other types of mutations do not 161 6 Understand the terms used to identify oncogenes What does it mean when it is preceded by a quotVquot Bya noquot qTOP In order to illustrate some of the normal functions of protooncogenes let s look at some speci c examples of protooncogenes that have been fairly well characterized First let s look at the ms protooncogene rst identi ed in a Qt sarcoma The protooncogene ras encodes a Gprotein Remember G proteins It39s the protein that forms the link between a membrane receptor for a hormone or in this case probably a growth factor and adenylate cyclase or phospholipase C Focused Review p 282 Receptors quot stop at end of page p 2867 Protein kinase cascades stop at Cyclic AMP WWW Reading Gprotein similar to ms with GTP bound RasMol Image The normal ms protooncogene encodes a Gprotein that contains 189 amino acids So far three oncogenic versions of this gene have been isolated from cancerous tissue These oncogenes differ from the protooncogene at only one amino acid at position 12 or 61 The mutations are diagrammed below 162 1 12 61 189 gly 939 Normal ras protein 1 12 61 189 gly Ieu Protein encoded by Hras oncogene 1 12 61 189 gly arg Protein encoded by Nras oncogene 1 12 61 189 arg gn Protein encoded by K ras oncogene Turn to page 224 and compare the amino acids in the different versions of the ras protein Hras has a leucine instead of a glutamine at position 61 Leucine is nonpolar while glutamine is polar This mutation could change the folding pattern of the molecule signi cantly The other two mutations glutamine to arginine in N ras and glycine to arginine in Kras also change the characteristics of the amino acid signi cantly Biochemical studies show that the mutant ras oncogenes encode proteins that cannot hydrolyze GTP to GDP Pi As you will recall the hydrolysis of GTP is the step that inactivates the G protein making it unable to stimulate its enzyme target any longer The ras G proteins therefore are quotstuckquot in the quotonquot position Once they become stimulated by the binding of a growth factor to a receptor and the subsequent binding of GTP to their active site they are permanently on and keep stimulating their target enzyme which keeps making second messenger which keeps signaling the cell to divide The drawing below is an illustration of a computergenerated structure for the normal ras G protein The GTP binding site is on the lower part of the molecule bound to GTP The areas of the molecule that change shape when GTP is hydrolyzed are drawn indicated by a black line These 163 changes in shape represent the quotonquot and quotoffquot conformations of the molecule Oncogenic versions of the ras protein are stuck in the quotonquot conformation Further evidence of the linkage between the ras proteins growth factors and control of cell division comes from intracellular antibody binding studies When antiras antibodies against the normal version of the protein are injected into the cytoplasm of normal cells these cells are unable to divide in response to growth factors Thus the ras protein forms a link between the grth factor signal and the cell division response Study Questions 1 What protein does the ras protooncogene encode What is the normal function of this protein 2 In general you need not remember the exact changes how are the ras oncogenes different from the ras protooncogene How do these changes alter the protein39s function How do these changes lead to the development of cancer 3 Describe the intracellular antibody binding studies that link the ras protein to the response of the cell to growth factors To illustrate the types of growthrelated proteins that can be altered in cancerous changes here is a list of some of the known protooncogenes and the normal proteins they encode As you can see protooncogenes come in four varieties growth factors growth factor receptors signal transducers and nuclear proteins involved in gene expression 164 Type of Protein Product Growth Factor PlateletDerived Growth Factor GF Factor 1 GF Receptor Protein with similar structure to Growth Factor Transducer for into G2 of cell Transducer Nuclear Proteins Factor APl In several cases carcinogenesis is associated with gene ampli cation In this situation the gene is frequently normal in base sequence and may be located on the correct chromosome However hybridization studies show that the gene has been duplicated sometimes hundreds of time and is repeated over and over again in tandem sequences Each gene is active and therefore the protein product of such gene ampli cation is over expressed and therefore over stimulates cell division The oncogenes that cause some types of leukemia and lung skin colon and breast cancers are in this group Please note that oncogenic mutations can be inherited or can arise in the af icted individual In some cases people get cancer because they inherited an oncogene from their parents These types of cancer tend to run strongly in families e g breast and colon cancers For reasons we will discuss below however the inheritance of these does not the J 39 I of cancer The majority of cancers however are probably associated with the development of new mutations in a protooncogene in one cell of the af icted individual This cell becomes cancerous and gives rise to the disease Thus in the case of many of the mutational changes associated with cancer there is no way to test for the presence of the mutated gene since it has not been inherited but rather is present only in the tumor cells and their descendants Study Questions 165 1 Discuss the differences between oncogenes that are inherited and those that arise in the af icted individual In which case can a test be developed for the presence of the gene Explain 2 Describe the four types of protein products that protooncogenes are known to encode Give an example of each Focused Reading p 3446 quotTwo kinds of genes quot stop at quotTreating genetic quot Oncogenes are usually expressed in cells as dominant traits that is only one copy of the oncogene is required for cancerous transformation However while the presence of an oncogene is required for the development of cancer it is not sufficient The cell has a number of tumor suppressor genes that function to prevent outofcontrol cell division Ifthese tumor suppressor genes are functioning normally one oncogene by itself will not produce a cancerous cell Thus at least two genetic changes are required for carcinogenesis 1 changes that create an oncogene from a protooncogene and 2 changes that inactivate tumor suppressor genes See figure 18 18 on page 346 for the steps required for colorectal cancer the system we understand the best The good news about tumor suppressor genes is that usually m alleles at a tumor suppressor locus have to be destroyed before there is loss of growth control A mutation that inactivates one allele will not have an effect that is will be recessive to the dominant suppressive effect of the other allele Often times it is the inheritance of a defective tumor suppressor gene that predisposes us to cancers Study Questions 1 Describe the relationship between tumor suppressor genes and oncogenes What genetic changes must be present in these genes in order for cancer to arise 2 What is wrong with the phrase Some day we may find the cure for cancer NEWS ITEM A group at the Scripps Research Institute inLa Jolla CA have synthesized a bacterial compound called Epothilone A which can kill cancerous cells Like the drug taxol which is extracted from the bark of the yew tree in the northwest part of the US this newly synthesized compound binds to microtubules and prevents chromosomes from separating during mitosis However there are two great advantages for Epothilone A It can be manufactured in the lab and therefore is not dependent upon the slowgrowing yew Secondly it is watersoluble and therefore it will be easier to administer to patients See summary by Robert Service Science Vol 274 2009 1996 WWW reading MAPK Signal Transduction In a urry of scientific papers recently investigators have outlined the entire relationship between the ms protein and cell division By examining this pathway you should be able to get a clearer picture of the link between the development of cancer and changes in G proteins tyrosine kinases growth factor receptors and nuclear transcription factors Note You do not have to memorize this pathway It is presented here simply to help you see how oncogenes might cause uncontrolled cell division 166 Some cells contain receptors for epidermal growth factor EGF These receptors are membrane bound tyrosine kinases When EGF binds to its receptor the receptor autophosphorylates that is it adds a phosphate group to its own tyrosine residues This causes the receptor to change shape This change in shape allows the receptor to bind to a cytoplasmic protein called growth factor receptor binding protein GRB Z This binding activates GRB Z which then binds to the ras G protein and activates it in the classic manner by causing it to bind GTP Activated ras activates a protein called Rafl itself the product of a protooncogene Rafl is a kinase that phosphorylates and activates a protein called MEK MEK is akinase that phosphorylates MAPK Phosphorylated MAPK travels to the nucleus where it activates transcription factors that are necessary for gene expression These transcription factors are encoded by the protooncogenes myc jun andfos These transcription factors may allow the production of proteins such as cyclin that trigger cell division Here is an illustration of this pathway EGF Receptor Plasma Membrane Ras G protein J A 9 MEK gt MEK MAPK gt MAPK Nucleus ea DNA Thus the signal pathway initiated by EGF is filled with the products of protooncogenes Any change in any of these proteins could cause abnormally high levels of expression of cell division signals thus producing a cell dividing out of control Study Questions 1 Given what you know about signal transduction and the cascades used in cellular communication would you be more susceptible to cancer if you had a mutation that A left the EGFreceptor always activated B left the MAPK always activated C left the 167 transcription factors always activated D all of the above E none of the above Be able to explain your answer NEWS ITEMS In June 1996 a team of researchers found a species of voles that is resistant to mutations caused by radiation When they analyzed their cells they found that the voles had elevated levels of IDH which they believe is protecting them from radiationinduced mutations See summary in Science Vol 273 19 July 1996 A new finding by Pascal Goldschmidt Claremont from Ohio State Univ suggests that ras also uses superoxides an oxygen molecule with an extra electron to communicate within the cell They speculate that oncogenic alleles of ms may produce more superoxide than wildtype alleles do You may remember from earlier News Items and our lab work using the Ames test that oxidative damage to DNA can lead to mutations and thus cancer Therefore researchers are looking at antioxidants es the same thing healthfood stores have claimed will cure cancer as potential drugs for treating cancer This can be summarized in one phrase that you have heard all your life Eat your green vegetables which are high in antioxidants See summary by Elizabeth Pennisi Science Vol 275 15671568 1997 2 Let s imagine that there is a wonder drug that is capable of protecting us from cancer causing mutations and it works by stimulating the transcription of the cytoplasmic form of IDH Explain to your grandparents how this drug might prevent cancer NEWS ITEMS In 1998 National Cancer Institute launched a new web site as a part of the Cancer Genome Anatomy Project CGAP This is a spin off of the human genome project and its goal is to sequence all the cDNAs from healthy and cancerous tissues to compare what genes are expressed in each situation Interestingly a Davidson biology major alumnus 97 was one of the first technicians to work on CGAP The URL is lthttpwwwncbinlm nihgovncicgapgt Many people think that the hottest area in cancer research in on the enzyme called telomerase Telomerase is the DNA polymerase that replicates the telomeres of our chromosomes and keeps them from unraveling It appears that normal cells do not have much if any telomerase while cancerous cells have a lot Interestingly telomerase has a lot of similarity to reverse transcriptase so there is some hope that drugs similar to AZT might be effective treatments for cancer A new type of cancercausing mutation was found recently A group at Johns Hopkins found that many people carry a particular allele for a protooncogene involved in the formation of colon cancers For years this sequence variation in the DNA was ignored because it was a silent mutation causing no changes in the resulting protein However they recently learned that this particular mutation made the surrounding DNA susceptible to errors in replication These subsequent mutations resulted in oncogenic mutations Now labs around the world are going back over old data to see if any of these unstable mutations were overlooked see Science Vol 277 1201 August 1997 In focusing on mutations in the genes that control cell division we shouldn39t forget about the second criteria for malignancy the ability to metastasize In order to spread cancer cells must be able to break free from the tissue bed they are in enter a blood or lymphatic vessel leave the blood or lymph and invade a different tissue bed Leukocytes are the only cells of the body that can normally do this Metastasis requires changes in cellular motility most cells don t move they just sit there which requires changes in the cytoskeleton and changes in the secretory products of the cell since they have to digest their way across barriers All these changes are caused by mutations inherited or new in the genes that control the cytoskeleton secrete degradative enzymes and receptors that interact with neighboring cells Thus even if a cell acquires an oncogene mutation and loses some tumor suppressor genes this is Lill not sufficient to cause cancer In addition the cell must acquire mutations that allow it to metastasize which is makes this story even longer 168 NEWS ITEMS A specific protease was identified that enabled breast cells to migrate out of the breast tissue This protease cleaves a protein in the extracellular matrix called laminin5 This may be a target to blockmetastasis Science Vol 277 225 July 1997 We know that tumors become much harder if not impossible to treat when they metastasize We also know that cancer is the result of inappropriate signaling Dr S Wiley Univ of Utah has shown that a majority of cancers remain sensitive to signaling by EGFR a growth factor receptor and that blocking ligand release from this receptor can be enough to block metastasis Drugs that block EGFR ligand release are being tested on tissue culture cells and may provide a way contain cancer qTOP 169 II AIDS Brief Overview Reading Chapters 12 13 17 18 amp 19 Focused Reading p 2401 Viruses reproduce stop at Bacteriophages p 221 quotRNA Viruses quot stop at quotTranscription quot p 2423 quotAnimal Viruses quot stop at quotMany plant Viruses quot Pay special attention to figures 134 and 135 Some De nitions Since its identi cation in the 1970 s Acquired Immune De ciency Syndrome AIDS a preventable sexually transmitted disease STD has claimed the lives of over 2 million people worldwide By 1983 the cause of this syndrome had been identi ed as the Human Immunode ciency Virus or HIV AIDS is the clinical syndrome associated with chronic infection by HIV Just as the u the disease is caused by In uenza the Virus AIDS the disease is caused by HIV the Virus Unlike most Viral infections HIV infected or HIV individuals may be infected for months or years before they become sick with AIDS This is called the latency period of the Virus and it is one reason that HIV is such a dangerous organism People can carry and spread the Virus for many years without having any symptoms of the disease to tell them they have been infected Because blood tests for HIV are not mandatory we have no way of knowing exactly how many people in this country are HIVinfected Who can get infected with HIV In the United States as of 1995 over 500000 AIDS cases had been reported to the CDC and about 300000 of these people have died from the disease According to available information it appears that 56 of AIDS Victims are gay or bisexual men 24 are IV drug users or their heterosexual partners and 6 are heterosexuals Two percent of AIDS Victims over 3800 are children Thirteen percent of children with AIDS contracted the disease from blood transfusions while the remainder contracted it at birth from their HIVinfected mothers In 1997 the United Nations published the following list of estimated AIDS patients 440000 in East Asia 16 million in Latin America and the Caribbean 6 million in South America and South East Asia 720000 in Europe North Africa and the Middle East and 208 million in SubSaharan Africa HIV continues to spread worldwide the estimate of HIVinfected people has risen from 14 million in 1995 to 306 million in 1997 In the United States the epidemic has spread through African American and Hispanic populations at a faster rate than it has through the Caucasian population For example whites make up 80 of national population but represent only 38 of AIDS cases Conversely African Americans comprise 126 of the population but represent 40 AIDS cases Hispanics make up 6 of the population but represent 19 of AIDs cases HIV is spread when bodily uids containing the Virus contact the blood of an uninfected individual The body uids that contain the highest levels of Virus are blood and semen Entry can be gained through any breach in the skin or lining of an organ eg mouth rectum and vagina The breach can be microscopic well below the size one would detect normally High risk behaviors include sharing needles during IV drug use and participating in anal vaginal or oral sex Because semen contains the Virus if semen comes in contact with a small cut or tear HIV can be transmitted Because stretching and tearing of the anus and rectum can accompany anal intercourse this practice 170 is a highrisk behavior Vaginal intercourse is also presents high risks because the uterus and cervical area tend to be rich in blood vessels naturally and abrasion during vaginal intercourse may cause areas of access for the virus Also if an open sore or cut exists in the mouth nasal cavity or esophagus the recipient partner during oral sex is at risk While it is possible for the virus to be transmitted from the recipient partner to the penetrating partner during any type of sex the transmission rate is much lower in this direction However the virus can certainly be spread through any tiny cut or opening in the skin of the penis ngers or hands Finally anyone who comes in contact with blood as part of hisher work physicians dentists emergency medical technicians etc or on an occasional or accidental basis eg helping at the scene of a lab injury where blood is present is at risk Because HIV spends most of its life inside human cells the body uids that do not contain cells also do not contain the virus or do not contain it in a transmissible form These uids include saliva tears sweat and urine Touching hugging kissing and sharing food or drink does not transmit HIV Worldwide the vast majority of HIV transmissions are through heterosexual vaginal intercourse In the United States the disease has spread most rapidly and continues to be predominantly present in the gay male population This selectivity is due to two unrelated factors First it appears that HIV has mutated into different strains some of which adhere to vaginal cells better than other strains do Therefore heterosexual intercourse is the primary means of transmission where this strain predominates currently in Asia and Africa The second factor is because the disease appears to have entered the US via the homosexual population and HIV was spread through gay sexual encounters If the disease had arrived from Asia and entered the country through the heterosexual population still a possibility the impact of the disease would be significantly different It should be noted and understood that the disease is not a quotgayquot disease and makes absolutely no distinctions based on sexual preference Heterosexual women make up one of the fastest growing HIW subgroups In 1992 at the Johns Hopkins Hospital emergency room 30 of women delivering babies were HIV no statistics on their babies The disease is not spread by being in a highrisk group e g gay IV drug user but by doing risky things unprotected sex of any kind sharing needles AIDS is a preventable disease While we do not yet have effective drugs or vaccines to kill HIV we do know precisely how the virus is spread HIV infection can be avoided by avoiding contact with another person39s blood or semen Because HIV can enter the body through cuts or tears too small to detect it is not enough to simply make sure that the blood or semen of another does not come in contact with an open wound Rather only complete protection from contact with the blood or semen of another person will guarantee safety Protection includes abstinence from sexual practices avoidance of situations in which you may come in contact with someone else s blood and protection of yourself with latex barriers condoms rubber gloves rubber dental dams etc Condoms are normally used with contraceptive foam and they may be better at preventing the spread of HIV if the contraceptive foam contains Nonoxyl9 It can be difficult to approach the subject of protection with a partner especially if the sexual encounter is of a more casual nature It is easy to simply let it go to tell yourself that the chances of contracting the disease are small and that it is too much effort too embarrassing too alienating too unromantic too nerdy or too awkward to say anything In heterosexual encounters it is the woman who is at a far greater risk of contracting the disease than the man and traditionally women are taught that being feminine includes being less assertive about sexual matters not being pushy or 171 demanding about the use of protection It is also tempting to tell yourself that everything will be okay because your partner looks healthy is not in a quothigh risk groupquot says heshe has had a limited number of sexual encounters before you or says he she has just had an HIV test that came back negative Even if your partner is telling you the truth none of these is a that you will be When you have unprotected seX or come in contact with someone else s blood you Q at risk of contracting a disease that m kill you You are gambling with your life Even if the risks are low the stakes are as high as they can be Table 1 People Living with AIDS US 2000 Year Ram39n hnicily 1993 19911 111115 19913 1997 1993 1999 Wlnlu nul Hi pur39lil39 Ill1311 352m 511756 93515 1117273 114895 122303 Buick nu Hispunilr 61361395 718133 312117 FEET1 11153013 11126 12994quot Hlsparlc 312215 36524 n1113i392 115191 52121 51113 629135 A ia II Pati c IandEr 1295 141311 11317quot 2145 21154 23113 2130 M barium hdiant lilakn Malian 5131 1362 T113 1103 133 9139 113125 11112 114de 19741 2153916 24011 2133242 2113703 310232 Adapted from Center for Disease Control Surveillance reports httpwwwcdcgov Based on random sampling data the incidence of HIV infection on college campuses has been estimated to be ten times higher than the general heterosexual population Based on these statistics we would expect 23 students at Davidson student population l700 to be infected with the virus There is an especially disturbing trend in the rise in AIDS among those born since 1959 figure 1 Notice that cases for those born before 1960 has leveled off while those born after 1959 have continued to rise Therefore the rise in cases for people under 38 is responsible for the continued rise in reported AIDS cases for the entire country 172 Figure 1 AIDS incidence cases reported per month This is the reported incidence of AIDS for the total population squares for individuals born in 1959 or earlier diamonds and in 1960 or later triangles Table 2 New HIV infection reported July 1999 through June 2000 in 34 areas with confidential reporting Age Male Female Total gt13 years old 111 114 225 1319 years old 327 536 863 2024 years old 1433 6053 7486 25 years old 12820 385 13205 Examining Table 2 you can see that the reported new HIV infection is highest in males above 25 years old and females between 2024 This is a very high rate of new reported infection for the female category if you consider that it only includes a range of 4 years and the high male is for those 25 and up A recent newscast reported that new infection in teenage girls is up 136 The direct cause of this jump is not known but it certainly shows that HIV infection is not only not going away in some age groups like the one you are currently in it s rising There is also a symptomatic difference between the sexes A study from Johns Hopkins School of Public Health revealed that women who have 5000 copies of HIVmL blood show similar AIDS symptoms to men carrying twice the viral load 10000copies HIVmL blood While the development of fullblown AID appears to take the same amount of time in men and women this 173 nding suggests that treatments that are started based on viral load 39requirements may need to take symptoms and gender into account Some History of HIVAIDS In this Unit we will look at what we know about HIV and AIDS As is the case in the study of all diseases we learn an enormous amount of basic biology as we learn about the disease By studying HIV we now know much more about all viruses and we certame know a lot more about the human immune system the target of HIV One difference between this disease and others we have encountered is that AIDS was discovered recently Your professors remember the news about the first cases and the drama that surrounded identifying HIV and we aren t all that ancient honest In the early 1980s investigators at the Center for Disease Control CDC in Atlanta noted that there was a dramatic increase in the number of adult males dying of a mysterious disease that appeared to compromise the immune system severely The immune systems in these men were so weak that they could not fight off infections that are usually no match for a healthy immune system most notably a kind of pneumonia that was often the cause of their deaths In attempting to determine the cause of these deaths the CDC tried to find out what all these men had in common Four characteristics emerged which were called the quotFour Hsquot being a male Homosexual Haitian IV drug user eroine or Hemophiliac Very quickly the investigators deduced that at least in three of these cases the underlying similarity is the increased likelihood of coming in contact with the blood of another person Gay men IV drug users and hemophiliacs were known to be at increased risk for hepatitis B spread by bloodtoblood contact It was later determined that the gay male population first infected with the disease vacationed extensively in Haiti where some of the native population became infected Being Haitian in itself has nothing to do with the disease The disease spread exponentially in these three populations gay men IV drug users and hemophiliacs reaching epidemic levels very quickly Investigators in the United States and France began a frantic race to be the first to discover the presumably bloodbome agent that caused this disease This race did result in the rather rapid characterization of the viral agent that causes AIDS but it was fraught with fierce competition and accusation of foul play Luc Montagnier from the Pasteur Institute in Paris and Robert Gallo of the National Institutes of Health USA share credit for the discovery of HIV as the causative agent for AIDS If you are interested there are countless articles covering the legal and scientific battles between these two men and their associates Study Questions 1 What is the difference between HIV and AIDS What is the difference between being HIv and having AIDS 2 How does the long latency period of this disease contribute to its spread 3 How is HIV spread What are quothigh risk behaviorsquot for contracting HIV 4 Some people believe that the AIDS epidemic has been wrought as a punishment by God against homosexuals Based on the facts of transmission how would you respond to this argument Why is the disease so prevalent among gay men in the United States 174 5 How can the spread of AIDS be prevented Focused Reading p 240244 quotViruses quot stop at quotViroidsquot p 3735 quotAIDs quot stop at end of chapter p 244 Figure 135 WWW Reading Life Cycle of HIV Attachment Structure of HIV From your focused reading you can see we know a great deal about what the virus looks like structure but we still have a lot to learn about how it works function Figure 137 is the best illustration of what HIV looks like but there are a few special features we need to note gp 160 The HIV genome is surrounded by a protein capsid which is surrounded by a phospholipid membrane containing large glycoproteins The lipid bilayer with embedded glycoproteins is called the viral envelope remember it also contains human integral membrane proteins from the infected cell The glycoproteins in the HIV envelope are called gp160 for quotglycoprotein 160quot because its molecular weight is 160 kilodaltons gpl60 is composed of two smaller subunits gp120 large star shape and gp41 the stalk 120 is the rotein that s ecif1call binds CD4 allowin attachment and infection The genome consists of two identical strands of ssRNA which contain nine genes Each ssRNA strand is bound to a molecule of reverse transcriptase RT the enzyme required to transcribe ssRNA into ssDNA Because human cells never do this they do not contain RT and therefore the virus must bring RT along with every virus particle The two identical copies ssRNA are reverse transcribed using reverse transcriptase like what is used to make cDNA into DNA and then inserted into the host genome where it can remain dormant or latent for months or years Human retroviruses were discovered by Robert Gallo well before HIV One more important feature of HIV biology is that when its nine genes are transcribed and translated the encoded proteins are not made individually but are made as a few multiprotein structures all stuck together likethiscompoundword In order for these individual protein components to perform their functions they must be cut free from each other One of HIV s genes encodes for a protease that acts like molecular scissors to cut the multiprotein structures into their proper and functional subsections The critical functions provided by the protease and RT have been the subject of a lot of research and pharmacological treatment of AIDS patients see below 175 Study Questions In general describe the structure of a typical virus 2 In general how do viruses reproduce What molecules must they encode in their own genome Which molecules does the host cell provide 3 Unlike bacteria that will grow on nutrient agar viruses will not What must you supply to support the replication and growth of viruses 4 What special structures do animal viruses contain that allow them to enter and leave animal cells without having to cause the entire cell to rupture Describe this process How HIV Infects Cells We will begin looking at how your cells become infected with HIV by looking at the target of HIV the immune system Brief Overview Reading Chapter 19 Focused Reading p 35864 quotSpecific defensesquot stop at quotHybridomaquot Figures 196 and 1911 p 3648 quotT cellsquot stop at quotMHC molecules are responsiblequot Especially Figures 1918 animated version available at Purve6e site p 3725 quotAn inappropriately active to end of chapter WWW Reading Cytotoxic Tcell Killing Its Target The interactions of the immune system are extraordinarily complex and the subject of one of the frontier disciplines of biology immunology It is well beyond the scope of this Unit to delve deeply into the workings of this system However if you are to understand how HIV produces such a deadly effect in the body you do need to understand a few things about how the immune system works As we discussed in Unit IH microbes are constantly invading your body despite your best efforts to keep them out You wash them away with mucus secretions in the lungs you wash them away by sloughing off the outer layer of cells in the intestine and skin you try to kill them with acid skin stomach vagina with enzymes in tears sweat saliva with antibodies in all the secretions of the body and still they get in Those resourceful creatures that make it through all these hostile defenses are met by an internal surveillance system so precise and deadly that all but the most virulent microbes are completely destroyed Without this system of surveillance and destruction the immune system microbes would overrun your body and kill you fast by this time tomorrow The immune system functions by recognizing and attacking foreign molecular shapes usually due to amino acid sequences that are not quotselfquot that is not part of any of your own personal proteins The cells of the immune system that do this are called lymphocytes Lymphocytes have specific receptors in their membranes for foreign shapes Lymphocytes come in two varieties T cells mature in the thymus and B cells mature in the bone marrow B cells make antibodies the same specific proteins you have encountered in looking at the method of immunocytochemistry or immunohistochemistry These proteins can bind 176 specifically to the foreign substance and trigger a number of responses that destroy it T cells do not make antibodies and they come in two varieties T helper cells T1 and cytotoxic T lymphocytes Tc TC s kill other cells directly by making membranetomembrane contact with them and inserting proteins in the cell s membrane that produce large holes TC s effectively punches holes in the membranes of other cells This makes it impossible for the host cell that contains the pathogen to maintain any ion gradients across its plasma membrane and it dies TC s kill virally infected cells cancer cells and transplanted organs a process called the cell mediated immune response As their name implies Th cells help other cells perform their functions They help B cells make antibodies a process called the humoral immune response The uids of the body are called humors and antibodies were initially discovered in body uids blood plasma In general the humoral immune response neutralizes foreign proteins e g bacterial toxins and bacteria Ths also help TC s become capable of killing Ths perform both helping functions by secreting various lymphokines that provide activation signals Lymphokines function as local signaling molecules binding to specific receptors and triggering cell functions through second messenger systems Because both B cells and Tc s require its help the Th plays a pivotal role in all immune responses Unfortunately the TB is the primary cell that is targeted by HIV Thus by interfering with the function of Th HIV cripples the entire immune capacity of the individual Viruses target certain cells based on specific binding between proteins in the virus envelop and proteins in the cell s membrane For example the in uenza virus binds specifically to proteins on the surface of the respiratory tract the chicken pox virus binds to target proteins in the skin the herpes virus binds to target proteins in the lips or genitals etc These viruscell interactions are specific just as are the interactions of enzymes and substrates receptors and hormones antibodies and antigens transport proteins and transported substances etc Thus viral targeting attachment and infection just like virtually everything else in biology relies on the interactions between molecules with specific threedimensional structure The protein molecule on the surface of the Th cell to which HIV binds is called CD4 Immunologists have complicated ways of naming things so this name doesn39t stand for anything very meaningful HIV will bind to any cell that bears CD4 in its membrane This includes Th macrophages and some supporting cells in the brain However the story is more complicated than this CD4 is necessary for HIV binding but not sufficient For example if the gene for human CD4 is transfected into monkey COS cells HIV will not infect these COS cells During the summer of 1996 several research teams read lots of people working cooperatively in the labs made significant progress in understanding HIV infection Science 272 809 May 1996 Science 2721740 June 1996 Science 274 502 October 1996 There are at least two types ofmolecules coreceptors that are also required for HIV infection CXCR4 and CCR5 such catchy names see figure 2 As shown in the figure below HIV requires cells to have CD4 and either CXCR4 or CCRS in their plasma membranes CXCR4 had been cloned previously and though its function was unknown the cDNA sequence suggested that CXCR4 would turn out to be a G proteincoupled receptor sound familiar for an unknown ligand CCRS is a receptor for the chemokine RANTES Chemokines cytokines and lymphokines are chemical messengers secreted by cells to alert the immune system the significance of RANTES will be discussed later We now know that CXCR4 is a chemokine receptor too What is especially interesting is that there are different strains of HIV that infect different types of CD4 cells at different times during a person s HIV infection One strain infects macrophages during the first phase of infection and another strain prefers Th cells later after the disease progresses As it turns out macrophages express CCR5 and Th cells express CXCR4 It has been known for years that when a person is first infected with HIV macrophages get infected 177 first A plausible explanation is that the strain of HIV that is responsible for initial infection requires CCRS as a coreceptor and not CXCR4 As the infection spreads within a person HIV is able to infect Th cells which means it requires CXCR4 as the coreceptor These discoveries are very recent so their impact is uncertain but they do help explain much about HIV infection Initial Later Infection Infection macrophage A CD4 K quot 39 3 7 5 CXCR4 L m m CCR5 HIV with HIV with HIV with macrophage no TceII type 2 preference preference preference Figure 2 This diagram outlines what we know about HIV infection via its two coreceptors HIV must bind to CD4 but also requires either CCRS during the initial stages of HIV infection or CXCR4 during later stages of infection NEWS ITEM The coreceptors CCR5 and CXCR4 were identified in 1996 and allowed the 39simple39 model described above As of now there are at least 13 known coreceptors for HIV and SIV simian immune virus Many of the co receptors have unknown ligands and are expressed by different cells within the body CCR5 and CXCR4 appear to be central to infection but the jury is still out See Science 1998 Vol 280 p 825 As is the always the case these membrane proteins that bind viruses are not in the membrane for that purpose this certainly would not be adaptive Rather they are there for some other purpose and the virus exploits their presence to gain entry into the cell CD4 is one of the molecules that allows Th to bind to antigen in order to become activated Figure 1918 illustrates the pivotal role of Th CD4 is an integral membrane protein on the surface of the helper T cell and interacts with the Class II MHC Tcell receptor and antigen It stabilizes the interaction of these three molecules Chemokines are secreted by a wide range of cells and they alert immune cells Th cells and macrophages that there is need for immune cells to come to the area of chemokine secretion Study Questions 1 What does the immune system do and in general how does it do it 2 Which arm of the immune system is most effective against protein and bacterial antigens Which arm is most effective against viruses and tumors 178 3 How do viruses target specific cells From an evolutionary perspective explain why a cell would have a viral target in its membrane if this molecule allows the cell to be infected and killed 4 What is CD4 and what does it do How is this molecule related to HIV 5 What are the other two coreceptors and where are they found 6 Which cells of the immune system are primarily targeted by HIV and when Why are these cells so important in immune function What roles do they play in the immune system 7 Describe the life cycle of HIV in detail Understand what happens in each of the steps shown in the WWW reading 8 What is a retrovirus How does it differ from other viruses 9 What is gpl60 What does its name stand for What are the names of the subunits comprising this molecule Which of the subunits is involved in the attachment phase of the viral life cycle How is it involved in this stage NEWS ITEMS For many years it has been known that some people are exposed to HIV but never develop AIDS This led some to hypothesize that HIV is not the cause of AIDS New data have shed light on why a person can be HIV and not get AIDS A group at the National Cancer Institute examined the amino acid sequence of CCR5 in 1995 people They found that there are a variety of CCR5 alleles in the population genetic variation and everyone they found who was homozygous for a mutant allele of CCR5 was not infected with HIV This mutant allele has a 23 base pair deletion note that it is not multiple of 3 which caused a nonsense mutation and the mutant protein never leaves the ER A second study has been conducted with slightly different numbers but both found that the HIVresistant allele was more common in Caucasians 17 and 11 for the two studies than in African Americans 17 or Africans 0 This may help explain part of why African Americans are over represented in the AIDS population Some have speculated that one reason for this might be that HIV is not a new virus and that Europeans have been exposed to this before Since genetic variation can lead to selective advantage survival of an HIV epidemic that would explain why Caucasians have a higher frequency of the resistant allele See summary by Jon Cohen Science Vol 273 17971798 27 September 199639 Samson el al Nature 382 722 199639 or Liu er al Cell 86 367 1996 Recent results have made the mechanism of HIV infection even more complex and troubling A French group has discovered that another molecule called US28 can act as a coreceptor for HIV Unfortunately US28 is not a human protein but a viral one The virus that contains the US28 gene is called cytomegalovirus CMV which is very common It is estimated that 80 of the population has been infected with CMV As it turns out the molecular structure of US28 resembles CCR5 When the researchers put the US28 gene into cells that lacked either CCR5 or CXCR4 these cells that used to be resistant to HIV infection are now capable of being infected with HIV So now the question is whether CMV has an active role in destroying the immune system in AIDS patients For example CMV might be able to infect cells that lack CCR5 or CXCR4 and thus provide a new host cell for HIV See summary by 1Iichael Balter Science Vol 276 1794 20 June 1997 qTOP Treatments for HIV and AIDS So how can HIV s life cycle be inhibited in a way that harms the virus but leaves the HIV infected individual unharmed The major problem in finding effective antiviral agents is that viruses use so many of M proteins in replication e g DNA polymerase RNA polymerase 179 glycosylation enzymes ribosomal proteins spliceosomes etc HIV contains only nine genes encoding nine proteins The simplest retroviruses contain only three genes All of the other proteins required for the viral life cycle come from our cells For this reason it is very difficult to inhibit a virus without inhibiting our own cells at the same time Bacteria on the other hand are freeliving organisms with their own enzymes They have been separated from us by evolution for so many years that their enzyme systems are usually quite different from our own Thus we can treat bacterial infections with antibiotics which function by inhibiting the action of proteins or enzymes that are peculiar to bacteria and not shared by humans Thus you can fairly easily inhibit the growth of bacteria without harming yourself While researchers have had a hard time devising such an agent our immune systems specialize in making such fine distinctions Thus when we become infected with the u mumps measles chicken pox etc our immune systems can usually eliminate the invading virus without harming us in the process However in the case of HIV the Virus attacks the veil cells that are responsible for its elimination Thus it knocks out our defenses leaving us unable to kill the virus or as the disease progresses any other microbe Defenseless against microbial attack AIDS victims are ultimately killed by microorganisms growing out of control in the body Study Questions 1 Why do strategies for producing antiviral agents differ dramatically from those used to develop antibacterial agents 2 In general what are antibiotics and how do they work Why don t antibiotics work against viral infections 3 Why isn39t HIV eliminated from the body in the same way that the viruses that cause colds u chicken pox and measles are eliminated Due to the rush of recent research results many new therapies are under development and at various phases of clinical trials Here are some approaches that are being tested to cure AIDS Focused reading p 375 figure 1925 Example 1 It has been known that TC cells also called CD8 cells are capable of secreting a factor that is capable of stopping the spread of HIV At a meeting in December of 1995 Science 270 1560 several research teams including one headed by Robert Gallo who is helped discover HIV in the first place announced that they had discovered this mysterious and elusive factor With hindsight it s easy to see why identifying this factor was so difficult it is actually three factors that work as a group The factor is comprised of three chemokines RANTES MIPl OL and MIPl B The names are acronyms that stand for RegulateduponActivation Normal T Expresses and Secreted Macrophage In ammatory Protein 1 a and lb Although the mechanism for inhibiting HIV replication is not known the more recent discovery that CCRS is a coreceptor is very exciting because it is known that RANTES binds to CCRS For the first time in years there is a lot of optimism for discovering a way to treat andor prevent AIDS The most obvious explanation is that these three factors bind to CXCR4 CCRS resulting in the inability of gpl60 to bind to CD4 cells There are at least 14 pharmaceutical companies that are developing drugs that will interfere with 180 HIV s ability to bind to CCRS andor CXCR4 For more information see the excellent summary Science 275 12611264 28 February 1997 WWW Reading Life Cycle of HIV Reverse Transcriptase Example 2 Interfering with reverse transcription of viral RNA In a second therapeutic approach which interferes with the viral life cycle about a dozen drugs have been developed which interfere with the process of reverse transcription As of April 1997 the FDA Food and Drug Administration has approved 7 out of 11 of these drugs by for treating HIV infection and AIDS The most popular drug treatment for AIDS is AZT This drug s chemical name is 339azido239 339 deoxythymidine AZT and six other drugs only four of the six have FDA approval are nucleoside analogs One might ask quotWhat is a nucleosidequot Well you know what a nucleoti is because you39ve encountered them over and over in looking at how DNA and RNA are synthesized and in looking at the energy molecule ATP a triphosphonucleotide The basic structure of a nucleoti is this k Nitrogenous Base Thymine Cytosine Guanine Adenine Phosphate urac Group 1 2 or 3 groups Ribose Sugar Nucleotides have three components nitrogenous base a ribose sugar and one two or three phosphate groups ATP GTP CTP TTP ADP and AMP are all nucleotides A nucleo is simply a 39 quot J without anv 39 39 groups It looks like this quot Nitrogenous Base HOCH 2 Thymine Cytosine Guanine Adenine Uracil Ribose Sugar 181 In making the nucleotides it needs to make DNA RNA and the energy molecules the cell takes nucleosides and phosphorylates them Thus nucleosides are the starting material for the manufacture of nucleotides A nucleoside analog is a molecule that looks so much like a naturally occurring nucleoside that the cell mistakes it for the real thing makes it into a nucleotide and then incorporates it into DNA or RNA in the place of the naturally occurring molecule For instance AZT looks very much like the nucleoside precursor of thymidine Below is the structure of AZT Compare it with the structure of thymidine AZT You will notice that the quot base thvmine of both A is identical The ribose of AZT does not have an oxygen on carbon 2 making it this sugar deoxyribose The only difference in the molecular structure between normal deoxyribose p 84 and this deoxyribose is the N3 group the azido group is N3 the same compound we used in the Ames test sodium azide on carbon 3 in AZT Ifyou look at the chemical name of the compound it actually 3 azido239 D3 deoxythymidine It tells you that the molecule is thymidine has a normal thymine base in it that it is deoxythymidine meaning that it contains deoxyribose missing an oxygen on carbons that it also is missing an oxygen on carbon 3 E that it has an azide group there instead Chemical names are exquisitely meaningful if you know how to interpret them They tell you the actual structure of the molecule take organic chemistry to understand biology fully Because the thymine part of the molecule is identical in thymidine and AZT the cell mistakes AZT for thymidine Thus AZT functions as a thymidine analog in the cell While you could certainly make nucleoside analogs for cytosine adenosine and guanosine if you are trying to interfere with DNA replication you are much better off using a thymidine analog because RNA does not use thymidine it uses uracil instead and therefore the normal process of protein synthesis so important to cellular life will not be affected When reverse transcriptase incorporates AZT into the growing DNA strand instead of thymidine no further elongation of the DNA strand can occur In other words AZT stops replication Normally in DNA replication the next nucleotide is added by dehydration synthesis to the OH group of the 339 carbon of the previous nucleotide However in AZT this OH group has been replaced by an azide group and thus the next nucleotide cannot be added no hydrogens and oxygens to dehydrate39 into water You have encountered this concept before in looking at DNA sequencing technology where dideoxynucleotides prevented strand elongation In fact the other two FDA approved drugs in this class are dideoxynucleotides that stop DNA synthesis in the same manner as AZT 182 Because they inhibit DNA synthesis AZT and other nucleoside analogs inhibit the ability of reverse transcriptase to make a cDNA copy of itself This step is crucial to the viral life cycle Ifit is inhibited viral replication will be blocked and the virus will quotdiequot The principle limitations of AZT therapy are 1 it is not a cure for the disease 2 the halflife of the drug is fairly short requiring that patients take tablets approximately every 4 hours 3 its ability to extend the life of the AIDS victim diminishes with time drug quottolerancequot develops 4 the drug does not appear to delay the onset of AIDS in asymptomatic HIV individuals 5 the drug is expensive costing approximately 7000 per year and 6 AZT has a number of toxic side effects including nausea rash insomnia vomiting malaise headache and severe anemia Only 60 of AIDS patients can tolerate AZT therapy for more than one year A problem with the widespread use of AZT is the development of AZTresistant strains of HIV The use of any antimicrobial drug will act as selection pressure on the microbial population evolutionary selection at a microscopic level If a mutation occurs that that allows the microbe to live in the presence of the drug the widespread use of the drug will give this mutant a competitive advantage over nonmutated microbes that were killed by the drug Thus the widespread use of AZT is undoubtedly favoring the development of an AZTresistant strain of HIV Such a strain or strains certainly exist and may be responsible for some of the cases in which AZT has lost its effectiveness in certain individuals WWW Reading Life Cycle of HIV Viral Protease Example 3 is what made helped make David Ho an AIDS researcher Time magazine s Man of the Year for 1996 and Science s Breakthrough of the year 1996 This approach is the second generation based off of the previous example and utilizes a cocktail of three drugs simultaneously AZT a nonnucleoside analog that inhibits RT two of the four available have FDA approval and a protease inhibitor there are four FDA approved inhibitors available As you will remember HIV must have its multiprotein complexes cut into functional pieces in order to survive Once the three dimensional structure of the protease was determined many companies and researchers rushed to develop drugs to block its action This has lead to a significant reduction in the number of HIV particles in the blood of AIDS patients Unfortunately there are still some negative side effect and now the cost of treatment has risen to about 12000 per person per year NEWS ITEM There is an ethical dilemma when it comes to testing drugs As you know from your laboratory work every experiment M have a control When new drugs are being tested you must administer a placebo to a subset of the people in order to see how well they do without any treatment The triple drug cocktail has been so successful that the experiments have been canceled before they were completed because the group getting the treatment was doing so much better than the control group But the fact remains that the experiment was not carried out completely If allowed to continue would the control group have appeared more similar to the experimental group No one knows for sure and when testing a live saving drug it is difficult to watch the control group get worse knong that you might be able to prolong their lives if they were unluckin placed in the control group See summary by Jon Cohen Science Vol 276 520523 25 April 1997 A new compound called calanolideA has been isolated from trees in Malaysia that blocks reverse transcriptase in the lab This is being developed for clinical trials But an even cleverer trick has been designed The problem has been to deliver a deadly compound to lymphocytes that are infected with HIV but not healthy lymphocytes Researchers at the National Institutes of Health have created a virus that expresses CD4 and coreceptors for HIV These engineered viruses should bind to cells expressing gpl60 and infect them If the virus delivered a deadly cargo then only infected lymphocytes would be destroyed Science 277 1606 September 1997 183 Study Questions 1 Explain the mechanism AZT uses to produce its antiHIV effects 2 What is a nucleoside How does it differ from a nucleotide 3 Ifyou are given the structure of 2 deoxythymidine be able to change the structure into AZT 4 Explain how the widespread use of an antimicrobial drug actually stimulates the development of a drugresistant microbial strain 5 What is a protease inhibitor and how does it ght AIDSHIV 6 What drugs are in the triple cocktail drug treatment for AIDS Vaccines for HIV On May 18 1997 as a part of a commencement address at Morgan State Univ in Baltimore President Clinton called for the production of an AIDS vaccine within the next 10 years to be a new national goal for science in the age of biology Earlier the National Institutes of Health NIH named Dr David Baltimore a Nobel laureate to head a new AIDS Vaccine Research Committee NEWS ITEM An example of politics and science Dr Baltimore acknowledged in an interview that he was hesitant to accept the position until after the November 1996 elections Had the Democrats retaken control of the House of Representatives Rep John Dingell CDMI would have become chaired the subcommittee that oversees scientific misconduct Dingell had aggressively accused Baltimore of being a knowing coauthor on a research paper that contained falsified results Baltimore was later proven innocent I certainly did feel that if the House became Democratic I had to come to some understanding with Dingell before I could take the job See Science Vol 274 2005 20 December 1996 In order to understand how vaccines are developed we need to return to the immune system and see how vaccinations protect against disease Focused Review p 360 quotImmunological memory quot stop at Animals distinguishquot Before vaccines were developed the only way for a person to become immunized to a disease was to get the disease and survive it Given the nastiness of most infectious diseases this was a pretty grim prospect and most individuals died in their youth of infectious disease If an individual contacts and survives a disease he she is immune to that disease at least for a while Thus if you survived the bubonic plague you could safely care for other victims and be protected from contracting the disease again This immunity to disease is due to a feature of the immune system called immunological memory When lymphocytes encounter an infectious organism for the first time they are not prepared to fight off the infection and you become sick Slowly through expansion of the antimicrobial lymphocyte population and genetic changes in the lymphocytes themselves you acquire memory for the infectious organism If you survive the first round of illness this memory remains in place and the next time you encounter that same microbe you 184 quotrememberquot it and can ght off the infection before the microbe makes you sick Immunity is speci c for a given microbe Thus immunity to in uenza will not protect you from tetanus Because lymphocytes interact speci cally with foreign antigens they develop speci c memory Because the immune system functions by recognizing foreign molecular shapes it will respond the same way regardless of whether or not an antigen is harmful This immune system characteristic is exploited in the development of vaccines A vaccine is a harmless version of a pathogen that has the same shape as the pathogen but has been altered in some way to make it unable to cause disease Vaccines are impostors they look like dangerous microbes to the body but they are not The body raises an immune response including a memory response against that particular foreign shape and the next time you encounter that shape this time in the form of the real pathogen your immune system will quotrememberquot the previous encounter and destroy the pathogen before it can make you sick Thus you get the immunity without having to contract the disease In the developed world childhood immunizations for many viral and bacterial diseases can be routine We can vaccinate against the viral diseases measles mumps rubella polio rabies yellow fever small pox and hepatitis B and against the bacterial diseases tetanus diphtheria whooping cough pertussis cholera plague tuberculosis hemophilus in uenza type b meningitis and pneumococcal pneumonia The very rst vaccines were surrogate pathogens Surrogate pathogens are microbes that naturally look like the real thing but are not pathogenic The best example of this is the very first vaccine ever developed the vaccine against small pox Small pox was a virulent and deadly scourge that along with the bubonic plague has threatened most of the known world since the beginning of recorded history Edward Jenner an English physician in the 18th century noticed that milkmaids very infrequently contracted small pox even when the disease swept through their villages af icting almost everyone else Jenner noted that cows sometimes contracted a very mild disease that had some of the symptoms of small pox most notably open skin lesions The cow version of the disease was called quotcow poxquot Suspecting that milkmaids were is some way protected through their contact with cowpox Jenner who must have been a very gutsy guy scraped some of the tissue from one of these open sores from an infected cow and injected the material into a young boy He then exposed the boy to small pox from an open sore of a small pox victim The boy did not become sick from small pox Biomedical ethics committees would have you locked up for doing such athing today Thus Jenner discovered a way to protect against small pox He called this potion a quotvaccinequot after quotvaccaquot Latin for quotcowquot This is also an example of how important it is to keep your eyes open and study many different organisms prevention of a lethal human disease can be aided by knowing cow diseases NEWS ITEM Small pox has been completely eradicated from the human population The small pox virus is present in only two places on earth in a vial at the Center for Disease Control in Atlanta and in a vial in a comparable institution in Moscow Scientists are currently debating whether these vials should be destroyed thus causing the extinction of the small pox virus Some argue that the risk of escape is too great and the human population is now largely unprotected Since the 19705 people have not been immunized against small pox because the disease is no longer a threat An escape of the virus now would cause heavy casualties Anyone up for writing an international terrorist novel around this theme On the other hand scientists argue that it is a mistake to cause the extinction of this important virus before we do not really know what questions we would like to ask about its structure and function We know so little about viruses that we don39t even know what questions to ask and if we destroy the virus now we will 185 lose the answers to those questions forever answers that may save lives if applied to the control of other viruses What do you think We have come a long way since Jenner scraped cow sores and injected them into people Today we have a dazzling array of genetic engineering techniques at our disposal in the development of hitech vaccines Since 1986 more than 15 AIDS vaccines have been engineered and tested in humans Here are two examples 1 Live attenuated Virus These vaccines are living Viruses that have been altered in some way to make them nonpathogenic even though they remain alive like removing the fangs of a snake Microbes can be attenuated by treating them in various low tech ways eg adding certain chemicals to their media or high tech ways e g removing a gene that is necessary for infectivity but not necessary for life Live attenuated vaccines give the most Vigorous immunity because they behave like the real thing in the body they go to the same tissues actually invading the body as a pathogen would and are seen by the immune system in the same way as the pathogen However in the case of HIV investigators have been reluctant to use this approach Because the disease is Virtually 100 fatal and because the attenuation process may not be 100 successful the chance of a pathogenic Virus being included in the vaccine is too great Also investigators have felt that with all the other recombinant DNA technology available they should be able to develop a safe effective vaccine without resorting to the use of live attenuated organisms However so far alternative methods have failed to produce a vaccine and in December 1992 a group of investigators reported that they could prevent infection by Simian Immunode ciency Virus SIV using a live attenuated SIV Virus with one gene removed These are the best results to date and may cause the AIDS research community to rethink their resistance to the use of live attenuated vaccines In December of 1995 HIV individuals who have never contracted AIDS were studied In one study all of the individuals had HIV strains that lacked the nef gene which is necessary for a Vigorous infection There is increasing interest in this approach for vaccination NEWS ITEM Dr Baltimore s group has recently shown that the nef protein can actually make HIV undetectable to our immune system It appears that when a cell makes nef it also makes less MHCI molecules the same molecules that help Tc identify which cells are virally infected Maybe this explains why the nef strains of HIV are not as potent as their wildtype relatives See Science Vol 276 11961197 23 May 1997 2 Cloned Envelope Glycoproteins also called Subunit Vaccines because they contain only a subunit of the Virus not the entire organism These are the safest vaccines because there is no Virus present to cause an infection By applying genetic engineering techniques all of which you have encountered already in this course investigators have cloned gp160 and gp120 placed the cloned genes in expression vectors and made large amounts of the glycoproteins The idea of course is that gp160 and 120 are foreign to humans and should elicit an immune response This immune response should then be able to see the natural gp160 or 120 on the surface of a real HIV and target it for destruction thus destroying the Virus These vaccines have been shown to produce an antibody response that reacts with HIV However they are not especially effective at preventing infection by HIV though antibodies can protect us from other Viral infections However these glycoproteins are not being presented to the immune system in the same manner that they would be if they were 186 embedded in the envelope of a virus Thus the immune system may respond with the wrong kind of immunity When foreign soluble proteins such as recombinant gpl60 and 120 are injected into humans an antibody or humoral response predominates Antibodies are effective against soluble antigens because they can bind up and neutralize soluble protein However when membranebound molecules are presented to the immune system they tend to stimulate a cellmediated immune response aimed at killing the cell bearing the antigen This is the arm of immunity that is responsible for eliminating viral infections For that reason research is underway to attempt to bind gpl60 and 120 into more natural membranebound configurations eg binding the glycoproteins into liposomes or into large lipidprotein complexes in an attempt to stimulate the correct type of immune response to protect against viral infection As a result many researchers are looking for vaccines that will stimulate a TC response NEWS ITEM In June 1994 two gp120 vaccines were considered by the NIH for large scale trials but the NIH determined that the data were not strong enough to justify the expense so neither vaccine was examined further The companies took their vaccines to Thailand where there is a severe HIV epidemic and convinced members of the Thai government to conduct a largescale trial Now other members the Thai Ministry of Health want the trial canceled since the data analyzed by the NIH are not strong The manufacturer of one vaccine Genentech has raised 24 million dollars from investors for the Thai trial and calls the attacks myopic They expect the trial to begin in 1998 See Science Vol 276 1197 23 May 1997 While progress toward an HIV vaccine has been slow this degree of difficulty is typical in the development of viral vaccines The vaccine for hepatitis B took 17 years to develop However HIV presents some unique problems to investigators who are trying to develop effective vaccines 1 HIV has an extraordinarily high mutation rate in the genes for its membrane glycoproteins The membrane glycoproteins are really the only part of the virus that immune system will be able to quotseequot since immune cells can only see the outside of structures These glycoproteins mutate at a very high rate Thus a glycoprotein vaccine developed against one strain of HIV may be entirely useless against another as the virus continually changes the shape of its surface glycoproteins In the case of in uenza a new vaccine must be developed by the Center for Disease Control every year because the changes in the surface protein shape caused by viral mutations make last year s vaccine unusable HIV mutates 65 times faster than in uenza Retroviruses tend to mutate at high rates possibly because reverse transcriptase has poor editing abilities Thus the mistakes that are usually fixed by DNA polymerase during DNA replication are not fixed by reverse transcriptase These mutations get incorporated into the viral genome and are passed on to the next generation of viruses 2 HIV is a retrovirus and after it has integrated into the host genome as a provirus it can lie dormant for many years During this period it produces no protein products so it cannot be detected by the immune system Thus the immune system is powerless to eliminate the virus when it is in its latent stage 3 The lack ofa suitable animal model for the disease Because the disease is species specific no animal model can be used to test vaccines in a faster more efficient manner than are allowed by the ethics of human trials Chimpanzees our closest relatives do become infected with HIV but they do not develop AIDS and their use as test animals poses an increasing threat to the already dwindling chimpanzee population While the pharmaceutical 187 industry is pushing the World Health Organization to relax restrictions on the importation of chimpanzees from Africa scientists warn that this could have a devastating effect on wild chimpanzee populations threatening their extinction Some degree of relief to the primate population has come with the bioengineering of a mouse that contains a human immune system called the SCIDhu mouse This mouse normally has a severe genetic immunode ciency disorder that destroys its own immune system A human immune system can then be seeded into the animals at birth While HIV does not infect these animals in exactly the same way it does humans some limited experiments are possible using this model NEWS ITEM With the identification of the coreceptors for HIV many research teams are racing to develop animal models for HIV They can introduce human CD4 CCR5 and CXCR4 genes into animals in hopes that they will be able to be infected with HIV and develop AIDS Unfortunately not even this is a simple as you might think It turns out that mouse cells grown in culture do not support the growth of HIV as well as human cells do However rabbit cells appear to be better hosts so some teams are trying to engineer rabbits instead of mice To give you an idea how specific HIV is for CCRS the mouse CCR5 cDNA has been sequenced at it is 82 identical to the human protein and yet HIV cannot bind to the mouse CCRS See Atehison et al Science Vol 274 19241926 20 December 1996 Study Questions 1 How is immunity developed What is immunological memory 2 How do vaccinations work What features of the immune system make vaccination a viable approach to the prevention of microbial disease 3 Discuss the aspects of HIV infection and AIDS that make it especially difficult to develop a vaccine qTOP Diagnosis of HIV Individuals WWW Reading ELISA for HIV A blood test for HIV infection has been available for over 10 years This test does not actually detect the virus in the blood but rather it detects the presence of antiHIV antibodies in the blood If you are infected with HIV you will make antibodies against the virus thus allowing the detection of the virus through this indirect route Antibodies are found in the serum the uid part of the blood minus the proteins that cause blood clotting and therefore if the test shows that you have antibodies against HIV you are said to be mositive If you do not have antibodies against HIV you are said to be seronegative Ifyou were seronegative but are now seropositive you are said to have seroconverted Because it takes from six weeks to six months for the level of antiHIV antibody to rise to detectable levels you E be HIV but seronegative Ifyou think you may be infected with HIV get a blood test Ifit comes up negative get another blood test six months later Ninetyfive percent of HIV individuals will seroconvert within six months of infection However some investigators have reported that seroconversion may not occur for up to 36 months in rare instances 188 The screening test for HIV is called an ELISA Enzyme Linked Immun0 0rbant Assay invented by Eva Engvall of Sweden This assay is based on the same principles as immunoc ochemist In one version of the assay the HIV virus glycoproteins are puri ed and stuck onto the bottom of the wells in a 96well plate Blood is drawn from the individual being tested The blood cells are removed by centrifugation leaving the uid component called plasma The individual39s plasma is diluted and placed in a well containing HIV protein As in all good experiments especially ones that determine if someone has a lethal disease control wells are included in the test Negative control wells are lled with plasma from a person known to be HIV negative and plasma from the person being tested is put in a well that does not contain any HIV antigen Positive control wells are lled with plasma from a person known to have high concentrations of antiHIV antibody in hisher plasma The next steps should seem familiar After an incubation period the excess plasma is washed off and a seconda antibody is added usually a mouse anti human immunoglobulin that has horseradish peroxidase conjugated to it similar to turnip peroxidase Antibodies are immunoglobulins so everywhere human antiHIV antibody has bound to the HIV glycoproteins lining the well the secondary mouse antibody will bind bringing along the enzyme peroxidase Ifno antibody against HIV is present in the serum nothing will bind to the HIV glycoproteins and the secondary antibody will also have nothing to bind to so it will be washed away along with its peroxidase In the nal step a peroxidase substrate is added to every well This substrate is colorless when added but peroxidase will turn it into a colored product Thus a change in color in a well indicates a positive result The ELISA is diagrammed below lllllIllllllllllll llll39lll llllillillllllllilllll The change in color is measured by a plate reader just like we did in lab and the results are expressed in optical density units OD units A low OD indicates a negative well with no colored product while a high OD indicates the presence of antibody against HIV or a positive test result The ELISA assay is the most inexpensive assay for the presence of HIV antibodies However it is not the most reliable assay available The American Red Cross estimates that the ELISA is accurate 998 of the time In two times out of 1000 however it will give a false negative or false positive reading A false negative is a test that f to detect the presence of antiHIV antibody when it is present in the plasma A false positive is a test that detects the presence of antiHIV antibody when it is not present in the plasma In the case of HIV both types of errors can be devastating Therefore if a blood sample scores a positive result in the ELISA a second test is performed This second test is 189 called a Western blot and it is more reliable than the ELISA is although considerably more expensive due to the time involved You have already encountered the Southern blot in Unit II In this technique restriction fragments of DNA are electrophoresed and then transferred to a piece of nitrocellulose where the DNA is hybridized with a probe Two other types of blots are based on the same idea In the Northern blot RNA is electrophoresed and then blotted and probed In the Western blot protein is electrophoresed and then blotted and probed with an antibody rather than DNA A scientist named Dr Southern developed the Southern blot In naming the Northern and Western blots the developers took advantage of the fortunate coincidence that Dr Southem39s name has three directional alternatives No Eastern blot exists but a scientist with Asian heritage and a sense of humor developed a Far Eastern blot detects protein binding In the Western blot for HIV the virus is highly purified and taken apart into its individual protein molecules These molecules are electrophoresed and separated by molecular weights and blotted to nitrocellulose As was done in the ELISA these Western blots are incubated with plasma from the individual being tested washed and a secondary antibody conjugated to peroxidase is added The blot is washed and soaked in a clear substrate that precipitates and turns dark when acted upon by peroxidase Thus all bands to which antiHIV antibody is bound will turn dark when the substrate is added Dark bands indicate a positive test and because the individual HIV proteins are separated by this technique the test will also show against which HIV proteins the individual39s antibodies are directed If the Western blot results come back positive the individual is considered HIV and is notified of that fact Both screening and confirmatory tests for seropositivity test only the presence of antibody to the virus While it is not used to screen the general public because of it expense there is a test available that detects the presence of the virus inside T helper cells The test is based on a general technique used to make many copies of a specific piece of DNA called the polymerase chain reaction PCR the same method we use in our last two labs This test is used in experimental situations where it is absolutely essential to know whether or not someone is HIVT Focused Reading p 214 quotThe polymerase chain reaction quot to end of chapter p 215 g 1121 WWW Reading Cartoon of PCR Method When PCR is used to clone DNA one can start with a single copy of the human genome In three to four hours over one billion clonal copies of the DNA of interest can be made Because the DNA primers are specific for the HIV gene you wish to amplify in many cases you need not purify the DNA before you begin In using this technique to detect the HIV virus DNA is extracted from the white blood cells which include T helper cells of the individual being tested This DNA is incubated in the presence of a pair of DNA oligonucleotides to act as DNA polymerase primers of about 20 bases which are complementary to a base sequence present only in the HIV viral genome and not humans Thus these primers will begin the process of amplification only if the viral DNA has been incorporated into the white blood cells of the individual The resulting PCR product is electrophoresed to see if the band of the expected size is present The PCR technique is so sensitive that it needs only copy of the viral DNA in order to amplify it and allow its detection Conversely it only takes one stray cell to contaminate the sample Study Questions 190 1 What is seroconversion Why is it called this What is the difference between being seropositive for HIV and being HIV 2 Describe the ELISA as it is used as a test for HIV 3 What is a false negative result A false positive 4 What is a Western blot A Northern blot A Southern blot What do all these blots have in common How are they different 5 Describe the Western blot as it is used as a test for HIV Why is this test used as a con rmation of a positive ELISA result Note Another detection method hat relies on Western blot technology is the home pregnancy test These tests are so reliable that gynecologists now tell women to use them rather than ordering tests from an outside lab httpwwwwhfreemanpurves6e tutorial 192 goes over how they work and how they have been designed to include the all important control 6 Describe the polymerase chain reaction What reagents are required What does this procedure do In general what are the steps in this procedure 7 Describe the use of PCR to detect the presence of HIV Why is this test far more accurate than the Western blot Why is it not used as the routine screening test for HIV Future Directions The major unanswered question is how the virus actually suppresses the immune system Th cells play a pivotal role in the function of the immune system Because HIV infects Th cells it has been assumed that HIV spreads from Th to Th killing the cells as it goes until so few Th cells remain that normal levels of immunity cannot be maintained It is certainly the case that Th cells are destroyed during the progression to AIDS Normal Th cell levels are about 1000 cells per ml of blood By the time of the onset of AIDS these levels have usually fallen to 200 cellsml and may fall to zero by the time of death When the Th cell level falls below 500 cellsml opportunistic infections begin to occur and by the time the cell count falls to 200 cellsml these infections begin to occur regularly AIDS used to be diagnosed at the onset of opportunistic infections However because the disease progresses differently in different individuals and the diagnosis of AIDS brings govemmentsponsored medical benefits to the individual a more uniform guideline for AIDS diagnosis was required Since April 1992 AIDS has been diagnosed when the Th cell count falls below 200 cellsml an 80 reduction This new definition increased the official number of AIDS cases in the United States by 55 191 While we know that AIDS victims have very low Th cell counts and suffer and die from infections that are caused by the absence of a functional immune system we do not know how HIV produces this crippling state There are currently three competing theories for how HIV destroys the immune system 1 HIV kills Th cells directly 2 HIV stimulates other components of the body Tcs to kill Th cells 3 HIV causes Th cells to commit suicide For years theory 1 was assumed to be true However several years ago it was found that at the time in disease progression when the patient is losing Th cells at the fastest rate very little virus was present in the blood This caused several investigators to wonder how HIV could be directly responsible for T cell death However there was considerable resistance among AIDS investigators to the idea that the direct killing hypothesis may not completely explain the disease This hypothesis was vindicated to some degree by PCR analysis of lymph node cells from AIDS patients which showed that virus infects T cells in the lymph nodes and spreads in these organs throughout the course of the disease Thus the quotlatentquot period of HIV infection may not be classical latency at all but rather a period of incubation in the patient39s lymph nodes Despite these findings some troubling contradictions remain unanswered by the direct killing hypothesis For example investigators have known for some time that some strains of HIV are not able to kill Th cells in culture in vitro while others are Yet in experiments using mice with human immune systems investigators found that the noncytotoxic strains were able to deplete Th cells in the animal in viva at a faster rate than the cytotoxic strains It may be the case that the virus makes the T helper cell a target for destruction by Tgs or some other immune system cell Thus according to this theory the virus simply marks the T helper for destruction but does not destroy the cell itself In support of the third hypothesis T helper cell suicide investigators have shown that if you take HIV T helper cells from the body and stimulate them with antigen they will commit suicide a process called apoptosis or programmed cell death Normal cells will begin to divide and differentiate but HIV cells will die Thus according to this hypothesis the virus does not directly kill the T helper cells but rather it programs it in some way to kill itself at a later time Spooky eh Of course these three theories are not mutually exclusive and all three processes may be acting to destroy T helper cells NEWS ITEM It is believed that macrophages or other immune cells with analogous functions eg dendritic cells throughout the body astrocytes and microglia in the CNS are the other central player in HIV infection that needs further study Many believe that the macrophage is the reservoir for HIV Think about this where do all the viruses come from if Th cells are mostly dead Secondly many HIV proteins are neurotoxins and an HIV macrophage kill neurons and lead to the development of neurological symptoms that up to one third of all AIDS patients develop Another factor is how HIV can cross the bloodbrain barrier It is reported that astrocytes can be infected but produce few viruses Finally the reason Th cells die has never been explained but many feel that infected macrophages may induce apoptosis in astrocytes and maybe Th cells as well See summary by Michael Balter Science Vol 274 14641465 29 November 1996 Finally everyone wants to know how HIV can evade cytotoxic T cells so well Activation of the TC requires the interaction of the T cell receptor on the TC with a MHC Class I molecule that is displaying a viral peptide In November 1995 it was shown that viral peptides in MHC I molecules that vary only slightly from the TC recognizable peptide can inactivate or anergize the TC remember the News Item describing the effects of nef on MHC We raise the issue of how HIV causes AIDS to allow you to see that the quotobviousquot answer is not always the right one and it is extremer important to keep an open mind about things even when a 192 dominant theory makes perfect sense For every natural process there are many many explanations that make perfect sense though most are false Truth in science does not depend on the quality of a rationale Rather it depends on the quality of evidence gathered through work at the laboratory bench Major Stories to Follow 1 Dr Mary Klotman at Mount Sinai School of Medicine has isolated a different factor a very small protein that appears to suppress HIV She calls this factor CD8 Antiviral Factor CAF Science V01 276 1197 23 May 1997 2 The late Dr Angeline Douvas at UCLA has found that people infected with a harmless goat virus caprine arthritis encephalitis virus a distant relative of HIV produce antibodies that bind to HIV She will pursue this line of research by conducting epidemiological study to see if people infected with the goat virus have a lower incidence of HIV infection Science Vol 276 1197 23 May 1997 3 Dr Miles Cloyd at UTGalveston has found that HIV was unable to reproduce in about 15 of the Th cells he isolated from randomly chosen individuals The significance of this story is that HIV is able to infect the cells but once inside it fails to reproduce Ifthis story holds up it would suggest a different mechanism is available from simply blocking the entry of HIV Science V01 275 1258 28 Feb 1997 4 Antisense therapies are making a comeback in many areas and AIDS is no exception Antisense technology is fairly simple in theory but has many practical obstacles Since all proteins are derived from mRNA if there were a way to insert a molecular sponge to soak up all the HIV mRNA then you would have killed HIV To do this you synthesize a short piece of RNA or DNA that has the complementary sequence to your target mRNA When these two sequences get together base pair the mRNA cannot be translated and it is destroyed by the cell see p 364 and fig 1613 for more The two big tricks are a which sequence do you choose that will bind to only HIV mRNA and b how do you get these antisense molecules inside cells There as been a great deal of improvement in part b and only trial and error will solve part a STOP 193 III Genetic Engineering There are two major areas of genetic engineering cloning and creating transgenic organisms We will look brie y at cloning and then focus on transgenics Focused Reading p 29699 last paragraph stop at quotGenes are quot WWW Reading How to Clone Your Own Dolly Cloning Organisms Plants are very easy to clone in fact many do it naturally When a plant sends out a runner and establishes a new individual without reproducing sexually that is cloning Cloning means the production of genetically identical individuals When you take a clipping from one plant put it in some water until it has roots and then plant it you have cloned an organism Scientists have learned how to clone more plants by starting with single cells and growing them in tissue culture But the big news in 1997 was cloning a mammal Animals in general and mammals in particular have been more difficult to clone Amphibians have been cloned before but it was not until Dolly stunned the world see the cover of March 10 1997 issue of Newsweek Previously mammals had been cloned naturally and in the lab by separating embryonic cells and allowing each cell to grow into a different individual thus identical twins But Dolly was the product of a mature nucleus and an undeveloped cytoplasm see WWW reference But there are some problems with this type of cloning Your chromosomes are like batteries they are designed to keep going for a set length of time but eventually they do expire Even the Energizer Bunny will die at some point The telomeres of chromosomes are the limiting factor and every time your chromosomes replicate a little bit of the life span of your telomeres is lost So what will happen to Dolly Will she die at an early age If so with a sample size of one sheep can you make any conclusions NEWS ITEMS As of August 1998 sheep cows and mice have been cloned from somatic cells Some wonder if this technology could lead to way to save some endangered species from extinction This leads to a hot debate on where conservation money should be spent but cloners do have a unique argument In species such as Cheetahs where the gene pool is too small for longterm survival there is a need to introduce new alleles into the breeding population Years ago researchers isolated and froze cells from adults Now it might be possible to use the nuclei from these frozen cells to produce new animals with different alleles to be introduced into the population via normal matings Science Vol 276 1329 May 1997 To date cloning is primarily used in plants and certain animals so it is more a novelty at this point compared to the bigger technology of producing transgenic organisms Transgenic Organisms Focused Reading p 3258 quotDNA manipulation quot stop at quotDNA fingerprinting quot WWW Reading A Portable Gene Gun Making Trangenic Livestock Throughout Units II and IV we have introduced the idea that genes can be moved from one organism to another where they can be expressed as the protein product The transfer of genes to 194 expression vectors is an example However this technology can also be used to move genes into more complex multicellular creatures such as laboratory animals livestock and plants Such transplanted genes are called transgenes and the organisms that bear these genes are said to be transgenic organisms In the case of unicellular organisms or cells in culture you simply have to put the DNA in with the cells create conditions that enhance DNA uptake and wait for the cells to take up the DNA However if you want to create and entire multicellular organism that contains the transgene in eve cell of its body you have to put the gene in the embgo of the organism for animals at least see below for plants In that way the transgene will be replicated along with all the other genes of the organism and passed on to every daughter cell This type of genetic engineering is called germ line engineering because once the gene is incorporated into the embryonic cells it is present in all of the cells of the resulting adult including its sperm or eggs Thus the gene is passed on to the next generation of organisms Once you get one male and one female transgenic animal you can have a transgenic strain simply by breeding them to one another To create a transgenic animal you give a female animal fertility drugs which cause her to quotsuperovulatequot that is make many many eggs You then harvest the eggs just before they burst from the surface of the ovary and place them in a dish with sperm collected from the male of the species By the way this procedure is also done in humans and is called in Vitro fertilization In humans so far anyway the purpose is to enhance fertility not to manipulate genes The egg and sperm join and form a zygote At this stage the transgenes constructed with a promoter that will turn the genes on at the appropriate time or in the appropriate cell are microinjected into the zygotes The zygotes are allowed to grow in the tissue culture dish to the 28 cell stage and are then implanted in the uterus of a pseudopregnant female having the hormones of pregnancy without actually being pregnant The investigator administers the hormones When the offspring come to term they are tested to see which of them carry the transgene by either a Southern blot or by PCR Given all the steps in this procedure at which something could go wrong the chances of producing atransgenic offspring are about 1 in 10 births and much lower odds if you count every implanted embryo Plants are a bit easier to work with than animals because in many species the entire plant can be regenerated in tissue culture from a single adult cell Thus you do not have to manipulate the plant embryo You simply have to insert the transgene into an adult cell and then grow the cell under the correct conditions in plant tissue culture A new plant will grow and every cell of the new plant will contain the transgene Plant cells can be infected with a plasmid or virus bearing the transgene or virus The US Department of Agriculture regulates the field trials of transgenic crops and livestock More than 370 permits have been issued in 35 states for field tests of transgenic crop plants Plant T Alfalfa Herbicide virus resistance Insect Virus resistance Herbicide insect resistance 195 Melon Virus 39 Papaya Virus resistance Potato Herbicide tolerance virus resistance insect resistance starch increase and modi cations to make a variety of nonpotato products such as chicken lysozyme Rice Insect 39 modi ed seed protein storage Soybean Herbicide resistance modi ed seed protein storage Sguash Virus resistance Strawberry Insect 39 Sun ower Modi ed see protein storage Tobacco Herbicide tolerance insect resistance virus resistance Tomato Virus resistance herbicide tolerance insect resistance modi ed ripening thermal hysteresis frost resistance Walnut Insect resistance From Kareiva Nature Vol 363 pg 580 June 17 1993 While as you can see most of the rst generation of transgenes tested so far confer resistance to viruses insects and herbicides the longterm hope is also to be able to engineer the plant product such that it provide more nutrition eg higher levels of protein Scientists are working to develop plants that can X their own nitrogen thus eliminating the need for nitrogenbased fertilizers NEWS ITEMS Dr Rafael Palocios and his colleagues from the University of Mexico in Cuernavaca have produced a better nitrogen fixing bacterium They started with wildtype Rhizabium and then added additional Rhizabium DNA to these cells along with an antibiotic resistance gene They then used evolution to find the best transgenic bacteria They gradually increased the antibiotic concentration and selected bacteria that could still survive Then they search for improved nitrogen fixation among the survivors They do not know which genes have provided the improved fixation but this method can be used to other bacteria to produce nitrogen fixation in nonlegume plants Source Daily InScight httpwwweuropeapnetcominscight sponsored by Academic Press and Science research published in May issue of Nature Genetics 1997 A transgenic mouse has been created that develops sickle cell This mouse will be useful for testing treatments and potential cures See Transgenic Mice from the Biol 11 web page for more information The tobacco industry is under re no pun intended and the farmers of North Carolina need to look for alternative crops Their future may be linked to transgenic tobacco grown on pharms It has been shown that tobacco plants can produce functional human antibodies if they are given the correct DNA Likewise they can produce other pharmaceutical products like growth hormone blood clotting factors and insulin look over pages 3245 for how and some examples Instead of a few dollars per bushel these plants may well be worth their weight in gold if not more The primary animal that has been used with transgenics has been the mouse Researchers have been altering the genes of mice for many years and there is a large number of transgenic mice being studied see WWW site for a small sampling One mouse has been made to have a human immune system so we can better understand our immune system Another mouse has been made that has twice the normal amount of skeletal muscles This could be used to understand and perhaps treat muscle diseases like muscular dystrophy In addition now that we know how to make a mighty mouse we could make mighty cattle and produce twice the beef A very popular transgenic approach is called the knockout mouse which means that both alleles at a particular locus have been deleted This allows us to understand the role that the encoded protein plays in a living organism by determining the phenotype of a knockout mouse Pharmaceutical companies 196 are making many of these mice in order to develop new therapies In fact Merck has hired for 8 million Lexicon Genetics of TX to make 150 new knockout mice see p320l and fig 1710 for overview of the knockout technique The existence of transgenic plants and animals is of course troubling to many There is legitimate concern that these genetically engineered species are not tested by natural selection and if they escape and breed with natural populations may confer a defect to the species and threaten its extinction It is also of legitimate concern that the bioengineered species will exert selection pressure on viruses weeds and insects to evolve into forms that can overcome the genetic trait of the transgenic organism As in the case of AZT by giving one species an arti cial advantage you always change the selection pressure on competing species thus changing the current niche and altering their evolution A large and complex area of patent law has arisen along with transgenic technology In 1988 the first transgenic mouse was patented Of course if companies go to all the trouble to produce a transgenic mouse strain they want the proprietary rights to the animal Normally if you make a product and want exclusive rights to its sale you get a patent But no one had ever tried to patent a living creature before This raises all kinds of problems For instance what if I buy a transgenic mouse or hog or goat from someone that holds the patent Then I want to breed this animal and produce my own line of transgenic animals Can I do this Or does the original patent owner own the exclusive rights to breed Here39s another problem What if a transgenic organism is patented and then someone comes along and changes one base pair in the transgene and creates a second transgenic organism that makes an identical protein product Slightly different transgene but identical product Does the original patent cover this transgene Ifyou are interested in biology and law this might be the career for you since it is a good bet that this controversy will be raging for years to come Possibly most troubling however is the capability that these transgenic organisms represent We know from almost a century of biomedical research that our biology is not essentially different from that of other mammals Ifyou can bioengineer the germline of a mouse or a goat or a hog you can bioengineer the germline of a human being In fact as mentioned above we already do one of the hardest steps of this process harvesting eggs and fertilizing them in vitro The Human Genome Project coupled with transgenic technology will mean that we might be able to bioengineer virtually any genetic trait into the germline as an inheritable feature While this could be a great benefit to families with inherited genetic diseases this technology raises unprecedented ethical questions What will be bioengineered Cures for diseases IQ Skin color Classical beauty What do we mean by quotnormalquot What pressures will parents be under to ensure that their offspring are genetically quotnormalquot If you don t bioengineer your offspring will they be able to sue you for negligence What will your family and community think of you if you choose to quotgo natural and conceive your child the old fashioned way Will bioengineering coupled with genetic testing create whole new categories of discrimination People predisposed to cancer would you hire them What about health care costs people predisposed to violence would you want them teaching in our schools people predisposed to forgetfulness would you want them fixing the airplanes you ride in etc What if only the wealthy can afford to bioengineer their children but everyone is genetically tested Right now these questions are the plots of novels but soon who knows Remember the entire field of recombinant DNA manipulation didn t even exist 30 years ago Ifthis area is interesting to you you should take some of the medical ethics courses 197 As was the case with nuclear energy the revolution in biotechnology provides immense power to those who control it Power that can be used for the tremendous bene t of society or in the service of evil We humans don t have the best track record in using power wisely and for the good our fellow humans While we cannot predict what the future holds we can predict that the biotechnological revolution will dramatically change our lives and the lives of our descendants Study Questions 1 De ne what a transgenic organism is and how this differs from the way gene therapy is used to treat cystic fibrosis 2 Give one benefit and one disadvantage inherent in creating either a transgenic animal or plant 3 Describe the techniques used to introduce a transgene into the potential host cell either plant or animal 4 What is a knockout mouse 5 What is antisense technology and how does it work 6 Be able to cite examples of transgenic organisms and the product they are designed to produce 7 How might gene therapy be used to generate a T cellmediated vaccine for AIDs 8 In an attempt to treat people with high blood cholesterol levels Ihave decided to create a transgenic cow that will produce human apolipoprotein C2 APOC2 in her milk APOC2 binds to cholesterol in the blood and so it might be useful as a treatment for people with high cholesterol I would like to employ you as my biotechnology consultant so you could advise me on how to design the transgene What advice would you give me with regards to the best promoter to use and correct targeting of the APOC2 protein In other words how could you get this new protein to be expressed only in the milk and nowhere else 0 V Describe how the famous sheep Dolly was cloned STOP 198


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