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Mutations Mutation any change in the nucleotide sequence of DNA mutations can occur within a gene or outside it do not occur in other biological molecules Can arise from a rare mistake in the base pairing during DNA replication or environmental conditions including exposure to certain chemicals or some types of radiation may increase frequency of errors during replication or directly include changes in nucleotide sequence 0 The environmental factors can affect DNA polymeraseOs ability to proof read or actually change the DNA Most will be caught by some mechanism DNA polymerase of P53 at the G1 checkpoint in the cell cycle Mutations can occur anywhere in the sequence Change will be re ected in the mRNA copy Insertion and deletion mutations Insertion mutation 0 One or more nucleotide pairs are inserted into the DNA double helix Deletion mutation 0 One or more nucleotide pairs are removed from the DNA double helix Starts out with the same sequence at the top but some base is taken out causing the mutation in the copy The effect of these mutation on the protein has to do with how many nucleotide pairs are inserted or deleted If we insert or delete any factor of 3 nucleotides then we will insert or delete an amino acid 0 Depends on which amino acid is deleted or inserted If the active site amino acid is deleted then that would have a large effect As long as pulling an amino out does not affect the structure or function of the protein then insertion or deletion of those codons is not that big of a deal 0 The more amino acids inserted or deleted the larger the effect on the protein If you do not insert or delete in series of 3 nucleotides there can be disastrous effects It shifts how the entire sequence is read All the amino acids after an insertion or deletion will be wrong Also they can cause a stop codon to occur before it actually should occur Basically these proteins do not have a strong chance of being functional Nucleotide substitutions point mutations The replacement of one nucleotide by another in the DNA template strand Just changes one nucleotide in the strand How will a change in a single DNA nucleotide affect the protein that is made leClConverterInpu 3anD7cOfGtxt12222010 521 56 AM In class activity Review questions How could a nucleotide substitution mutation in the DNA sequence of a gene affect a protein that is made It could lack half of its amino acids it could differ in a single amino acid it could be made with no changes in the amino sequence and translation could fail to produce a protein If the DNA sequence of a gene is mutated so that two nucleotides are inserted into a codon for the 10th amino acid in the protein which consists of 50 acids how will the resulting protein be affected Amino acids 1015 will be changed to a different amino acid Biotechnology Any use or alteration of organisms cells or biological molecules to achieve specific practical goals Goals are wide ranging 39 39 00 r d I 0 food with higher nutrition domestication of animals development of better treatment for diseases 39 1 I Not a new practice EX using yeast to produce bread beer wine and cheese using selective breeding to produce plants and animals with desired characteristics both have been going on for at least 10000 years More recent genetic engineering 0 Definition Direct modification of the DNA makeup of an organism o Producing plants and animals with desired characteristics 0 Learning more about how cells and proteins work 0 To develop better treatments for human diseases The use of DNA technology to lea1n more about an individual 0 Paternity issues 0 Solving a crime 0 Do they carry alleles of specific genes Genetic engineering Recombinant DNA technology A set of lab techniques that is used in combining genes from different organisms into a single DNA molecule making hybrids Human insulin protein was the first pharmaceutical drug made by this through the production of genetically modified bacteria Developed in 1982 leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM 0 Type I diabetes sufferers use this to regulate their blood sugar levels 0 Before insulin came from slaughtered cows and pigs problem was that it was different enough that it hurt 5 of diabetic users of insulin also it was hard to get enough volume Bacterial plasmids Used in making the insulin by the drug company Plasmids are small circular pieces of DNA carry small number of genes that give bacterium special properties are abundant up to 100 copies per cell and they are easily taken in through transformation 0 Transformation is the process by which living bacteria takes in plasmids Plasmid is taken in and replicates in the cytoplasm during and between cell division 0 Plasmids carry different properties that make them useful in these plasmids Properties of plasmids have been exploited Generation of recombinant bacteria Start out by taking a bacterium and isolating its plasmid Also we need DNA which encodes our gene or protein of interest 0 If we are trying to make human insulin we will need any human cell to get that DNA because all have the information to make the protein We isolate the gene of interest too Getting the gene of interest into the plasmid double stranded helix so we are going to have to cut through the sugar phosphate backbone in order to do that 0 We need restriction enzymes to do this special class of enzymes which have been isolated from a wide variety of bacteria They are like highly specific molecular scissors They cut the covalent bond between the nucleotides and they do this at specific locations along a DNA depending on the sequence of nucleotides that are present See the examples in the slide show The restriction enzymes and recumbent DNA work together We mix the two cut pieces of DNA so that we can put the gene of interest into the plasmid This allows base pairing on both pieces of DNA Generation of recombinant Bacteria DNA ligase and the modified plasmid are transformed into the bacterium through the normal process Now it is called a recombinant bacterium or genetically modified organism leClConverterInpu 3anD7cOfGtxt12222010 521 56 AM Now this bacterium is multiplied and divided The products can be used in copies of the gene or copies of the protein Products made using recombinant DNA see slide OPharmO animals Using whole animals to produce human proteins These sheep produce a speci c human protein on their mild DNA technology and agriculture Around 50 of food has some genetic modi cation in it In the US in 2008 around 8090 of crops are genetically modi ed Generation of GM plants Uses the natural system of bacterium A tumefaciens which can infect almost all species of plants and carries within the TI plasmid Takes the plasmid out and modify it taking the tumor forming genes out Read steps on the slide show Genetic modi cation using a gene gun Brut force method of getting the DNA into the cells Not very efficient but its easy goal to increase yield shelf life and protect from disease Review questions Restriction enzymes are useful for generating recombinant DNA molecules because they cut DNA only at speci c locations Which of the following statements about plasmids are true They are used to insert foreign DNA into bacteria into bacteria they are present in the environment and can be taken in easily to bacteria they come into contact with they carry genes that encode for proteins capable of inactivating antibiotics Generation of GM plants Elegant process utilizes plasmid from bacteria and inserts it into plants leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM 0 Starts with 2 pieces of DNA the modi ed TI plasmid and the DNA you want to be inserted o 1 cut the DNA pieces with the same restriction enzyme impottant so that we can put them together again at the ends which were cut the bases which were left unpaired it is very important that we use the same enzyme 0 2 Then we miX together the cut gene and the plasmid adding in DNA ligase to seal the DNA DNA ligase holds them together with dehydration synthesis to form a covalent bond between the two nucleotides Makes sure there is a continuous covalent bond along the two strands of the DNA double helix The bases will make hydrogen bonds on their own 0 3 Transform A tumerfaciens bacteria with the recombinant plasmids o 4 Infect plant cell with transgenic bacteria Bacteria enters the plant cell and gene of interest is inseIted into the plant cell chromosome It gets into the chromosome through genes that are present on the TI segment 0 5 Allow single plant cell to grow into a mature plant 0 6 Genetically modified plant contains a new characteristic Brut force method 0 Gene gun shoots the DNA into the nucleus About 10 of the time we get the modified plant we desire Why go through this 1 To increase yield more produce per acre 0 Done through introducing into plants the ability to survive in the presence of a herbicide things that kill green plants so that herbicide can be sprayed onto crops and not kill plants only weeds Also so that plants can produce their own pesticides Finally through introducing genes into plants that make them less prone to common infections which will make them less likely to die 2 To improve nutritional value 0 EX canola and soy bean will produce a healthier oil Also rice which has been modified to have additional nutrients added to them 3 To increase shelf life 0 EX a tomato which has an altered ripening ability The enzyme keeps the tomato from getting too soft as it ripens Herbicide Resistant GM Plants Kill weeds without ha1ming the crop Most herbicides will kill plants by inhibiting and enzyme which is unique to plants leClConveIterInpu 3anD700fGtXt12222010 521 56 AM which synthesizes speci c amino acids that cannot naturally be obtained by the environment The only way the plant can get these acids is by using the enzyme The herbicide preventing the enzyme will keep the plant from producing the amino acids which it needs to function Herbicide resistant plants have been given a bacterial gene that encodes an amino acid synthesizing enzyme that is not inhibited by a speci c herbicide 0 Plants were given a related synthesizing enzyme which was found in bacteria and that is not targeted by herbicide 0 When they encounter the herbicide their new enzyme is not shut off like their natural one so that they can still survive GM plants containing Bt Gene resist attack by insects When the Bt gene is ingested by insects it damages the insects digestive system Bt is from the soil bacterium Bacillus thuringiensis Bt encodes a protein that damages the digested tract of insect pests but not animals or humans Bene ts of GM crops Resistance to herbicides which should decrease the amounts of herbicides we have to spray and which will increase yields Increasing pest resistance can eliminate need to use pesticides which decreases expense and introducing pesticides into the environments where humans can ingest them Nutritional content always a plus Concerns Potential human health risks 0 Potential side effects caused by eating food produced from GM plants 0 Potential allergic reactions to GM plants we cant always predict the possible side effects Potentially hazardous to the environment 0 Potential transfer of genetic material to the wild non GM relatives 0 Potential evolution of resistant pests o Potental loss of genetic variation leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM Genetic testing Testing for presence of a disease causing allele of a speci c gene How does a disease causing allele differ from a normal allele of a given gene 0 Will have a different sequence of nucleotides Sickle Cell Anemia Autosomal recessive disorder caused by a change in just one nucleotide in the gene that encodes hemoglobin 0 Normal sequence in codon 6 is CTC specifying GLU but the altered version which causes Sickle cell is CAC specifying Val o The nature of the amino acid on codon 6 is that it sticks out on the protein in the water based cytoplasm It changes from a hydrophilic acid to a hydrophobic one The single change is within the sequence that a specific restriction enzyme cuts So with the change the enzyme wonOt cut This is a Orestriction fragment length polymorphismO RFLP o A difference in nucleotide sequence at a specific location on a specific chromosome that alters the restriction site for a specific restriction enzyme Exposing DNA to a restriction enzyme Mst II and look at the DNA Then separated the fragments based on their side through Agarose Gel Electrophoresis What are we going to see on the stain 100000 to 200000 bands on the gelEso you need to make sure an Review questions To make insect resistant transgenic plants a Bt gene is first inserted into a modified Ti plasmid Using the stain gel pictured below what can you conclude about these DNA samples The DNA in sample C is longer than the DNA in sample A Sicklecell anemia Caused by a single nucleotide change in the gene that encodes betaglobin Taking the gel that we ran and staining it with a general stain for DNA positive charge which will stick to all DNA in the cell leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM 0 We would see thousands of strands of DNA 0 How do we simplify it so that we are only looking at the region we want to see That is done through an alternative technique comes in blotting 0 Gel is treated with chemicals that break the h bonds between DNA strands Transfer DNA strands onto nylon paper using electrical current 0 Immerse nylon paper which is positively charged so draws the DNA out in the solution containing labeled DNA probes 0 Wash away all DNA probes that diant form h bonds with DNA fragments 0 DNA fragments that base pair with DNA probe are visible in red 0 Use to identify small number of DNA fragments when have a lot on gel DNA probes We can design a DNA probe so that it will be exactly what we are looking for It is a short single stranded DNA molecule that is complementary to the DNA sequence of the region we are interested in The DNA probe is labeled with a colored molecule so that if it base pairs with a DNA fragment we will be able to see it If DNA probe does not base pair with DNA fragment it will not be labeled and we will not see it Identifying of sickle cell Use the blotting technique to allow us to look at specific fragment The probes we will use are shown in blue and are a mixture of 2 different probes One the can make base pairs with the normal allele of betaglobin and the other which can form h bonds with the same region on the sickle cell allele Long DNA is severed into small fragments Then we have to figure out which fragments we will be able to see after blotting o Depends upon which fragments formed specific hydrogen bonds with the pro e o The second and third fragments will form bonds with part of it so we will see those two The first and last ones will not 0 Our gel for an AA will have two bands 0 For Aa it will be 3 fragments one above the first strand on AA 0 For aa it will have one long fragment one at the top leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM Finding out the DNA of a fetus Amniocentesis from skin cells oating around in the amniotic uid Needs to be combined with PCR to make more copies of the DNA around the gene that we are interested in we can then use the RFLP to analyze it Polymerase chain reaction PCR has 3 steps and is trying to make more DNA D the PCR cycle We start with one molecule of DNA and get 2 But we will actually need millions to do the RFLP So normally we do 2030 cycles Start out with original DNA 1 Heat the samples up to almost boiling which will break the hydrogen bonds 2 Cooling allows primers and DNA polymerase to bind 0 Primers are short pieces of DNA that are complementary to the ends of the region you want to make copies of 3 New DNA strands are synthesized Review questions We can determine which fragments of DNA we will see by looking at which are going to form base pairs with the DNA probe Because she is heterozygous we know half of her DNA is normal and half is mutant Using RFLP analysis to determine gene of an indiVidual with respect to a mutation Look at slide to see steps Blotting B because just cutting all DNA will produce so many fragments You need DNA from so about 1000 cells to do blotting Making more DNA Take small amount of DNA and make copies of the region we are interested in Use PCR 0 Start out with original piece of DNA 0 Step 1 D heating separates DNA strands 0 Step 2 D cooling allows primers and DNA polymerase to bind Primers are short pieces of DNA that are complementary to the ends of leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM the region you want to make copies of Made ahead of time temp needs to be lower so that hydrogen bonds can form We use DNA polymerase out of bacteria because it can be boiled and not denature 0 Step 3 D new DNA strands are synthesized Temperature increases to form covalent bonds between the nucleotides Do about 2030 times so that we can get enough DNA to work with 1 million to a little under 1 billion copies Sickle cell anemia D PCR based test Bright green bars are the primers we designed so we will run the PCR reaction using the primers 0 At the end of PCR we will end up with a lot of copies of the DNA that is between those two primers small stretch of DNA Now take the small pieces of DNA which only have one variation between the normal and mutant and cut them with the MST II restriction enzyme Take the fragments that result and put them through gel electrophoresis and stain them we donOt need to use blotting because we only have a few fragments o Sickle cell version will have one fragment 0 Normal cell will have two fragments We expect to see 0 AA D two fragments o Aa D three fragments 0 aa B one fragment Why we use PCR In order to visualize the DNA fragments on a gel or blot you need 1000s of DNA molecules 0 One band of gel 1000s of DNA molecules ofthe same length 0 The more DNA molecules present the darker the band RFLP analysis alone requires 1000 cells minimum leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM RFLP analysis coupled with PCR requires DNA from as few as 1520 cells in practice although in theory only need one cell Alternative to RFLP Diagnostic arrays D comes in handy if there are multiple mutant alleles that will give you the disorder CF autosomal recessive disorder caused by a defect in a speci c protein which transports chloride ions across the plasma membrane Researches have found 1000 different mutations which can cause CF D but 32 alleles cause 90 of cases 0 With RFLP we would have to set up 32 different experiments and assumes that each of the alleles has a mutation which affects a restriction enzyme site Diagnostic array D takes advantage of the fact that all mutant alleles will have a different DNA sequence from the normal allele Diagnostic array Start out with strip of nylon paper which has a potion l strand of complementary DNA which will form base pairs with the normal DNA or other portions of complementary DNA that will form base pairs with one of the mutant alleles If the DNA forms a base pair with that portion of DNA it lights the portion up 1 Isolate DNA from person being tested 2 cut DNA with multiple restriction enzymes 3 separate DNA fragments into single strands 4 attach a colored label to the single stranded DNA fragments 5 incubate with nylon paper with spots of DNA complementary to the normal and disease causing alleles of the gene Examples 0 l is homozygous normal 0 2 heterozygous o 3 homozygous mutant DNA technology and forensics RFLP and forensics RFLPs are not only found between alleles of a given gene They can also be found between non coding DNA sequences Also Researchers have identified several RFLP leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM sites with noncoding regions of our chromosomes that can be used to distinguish individuals EX an RFLP where some individuals have a region that can be cut with a restriction enzyme and some do not have one that can be cut by that enzyme at that site 0 Used to match up DNA of different suspects at the same scene Review questions Look at the slides Diagnostic arrays Most frequently missed on the nal exam RFLPs and Forensics Looking at RFLPs in noncoding regions the sequence of DNA that is in an intron or spaces between alleles of a given gene This stretch of DNA is at a speci c location on chromosome number 4 Looking at the sequence of nucleotides on this location of chromosome 4 You either have a sequence that allows an enzyme to cut or not to cut So we can see if it cuts to see which sequence and individual has In this situation we are going to blot because we would see hundreds of thousands of fragments otherwise 0 So we need to design DNA probes that will form base pairs with the DNA around that specific sequence site In this situation one will have two cuts and the other will have two With RFLP there will only be 3 possible outcomes If there wasnOt much DNA You would do PCR to make more copies of it This example Shows RFLP for one specific site but researchers would do more than one RFLP site Multiple RFLP analysis With looking at multiple RFLPs we can a total pattern of fragments produced which is referred to as a DNA profile or DNA fingerprint Every single band needs to match completely for it to be a match If there is a difference in sequence it means that the DNA does not come from the suspectvictim leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM Short Tandem Repeats STRs The second kind of DNA analysis used in forensics Repeating sequences of DNA 25 nucleotides long which are found at speci c locations on speci c chromosomes Researchers have identi ed speci c ones which all indiViduals have We do PCR normally So we need to design primers which will form base pairs with the repeat region and with the region outside of the repeat region We are expecting a relatively small number of bands example STR DNA pro le The rst person has only one copy The second person has two copies of the repeat The reference strands are pieces of DNA with a known number of repeats Multiple STRS Scientists look at multiple STRs to determine a DNA pro le They use between 1013 to determine matches They uses known STRs because each individual in the population have the same STRs at different places The more STRs that match perfectly the lower the chance that the DNA samples came from different people Probability If we have a perfect match of 1010 then we have a ltrillion chance that it came from different people If we had a perfect match on 13 STRs then there is less that once chance in 200 quadrillion that the DNA is from a different people Codas Database The database of STRs that is of felons who commit crimes Outcomes The number of possible outcomes and combinations in the population is huge So you can be more sure when you have a perfect match Can be used in identifying criminals remains paternity leClConveIteIInpu 3anD700fGtXt12222010 521 56 AM How do you expect a childOs STR pattern to match up with the parents It will be a combination of their two parents But you can never get the exact STR of one parent unless they both have it Embryonic stem cell research The stem cells in embryos are completely unspecialized cells that have the ability to form virtually any of the 200 different cell types that comprise the human body Under different conditions cultured embryonic stem cells can become blood cells muscle cells or nerve cells Certain conditions include growth factors and or signaling proteins These stem cells come from very early stage embryos that are between 5 and 7 days after fertilization They come from donated surplus embryos which were produced as a product of in vitro fertilization procedures They are isolated from donated surplus blastocyst stage embryos Blastocyst are a microscopic group of cells Inside the blastocyst is the inner cell mass which would have eventually given rise to every cell in that fetus Each cell has the potential to turn into any cell in the body 0 We break open the blastocyst take out the inner cell mass and destroys the blastocyst Then we can culture embryonic stem cells These cells can be coaxed to turn into a type of cell and then transplanted into a patient ParkinsonOs disease Common neurodegenerative disease caused by a loss of speci c neurons The technique has worked on lab mice lacking the same type of neurons Other diseases injuries In order for a disease to be a candidate from treatment the defects must effect defects or loss of a speci c cell type Some adult tissues contain stem cells Adult stem cells are held in reserve until tissue sustains damage or cells need to be replaced Adult stem cells are tissue specific they can only give rise to cells of a tissue they are foun 39 0 bone marrow skin gut liver muscle brain Therapy has been used for a long time in bone marrow transplants leClConverterInpu 3anD7cOfGtxt12222010 521 56 AM Bone Marrow Transplants Bone marrow is first collected from the donor processed so that stem cells are isolated and grown and are concentrated Then through cryopreservation stem cells are frozen until needed Patient undergoes high doses of chemotherapy to destroy all cells in bone marrow which are inducing cancerous cells Finally thawed stem cells are infused into the patient through IV You can also take adult stem cells and put them in a culture dish to make them into blood cells Embryonic stem cells versus adult stem cells Embryonic cells have much more potential to develop into any type of cell in the body they are relatively easy to isolate and grow in a culture dish and they must be matched to patient because they can be rejected by a patients immune system adult cells only give rise to a single tissue are hard to isolate and grow but they are immune compatible and will not be rejected by the patients immune system Challenges for Clinical stem cell therapy Need to generate large number of cells to transplant most take millions of cells for it to occur This is more of a problem for the adult therapies Transplanted cells must be matched to recipient to avoid rejection Transplanted cells must survive integrate into the surrounding tissue and function properly for the rest of the recipients life Procedure and transplanted cells must not harm recipient in any way Clinical trials for stem cell therapies using embryonic or adult cells in humans are in the early stages 0 The rst embryonic one started a month ago Embryonic stem cells Seem to have the most potential but the biggest problem is that they must be matched to the patient or else they will be rejected Cloning To generate a very early embryo that is genetically identical to the patient from that early embryo we can obtain the stem cells Somatic Cell Nuclear Transfer SCNT Somatic cells are any cell in the body that is not a sperm or egg We need a somatic cell from the individual we are cloning commonly use skin cells leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM as well as another individuals unfertilized egg We take all of the DNA from that egg and take it out with a pipet This is now called an enucleated egg Next we replace the DNA we removed with the DNA from the somatic cell This is done through placing a cell into the egg and using a electric charge to fuse the two cells together After 57 days we have a culture fused cell which divides to form a blastocyst This blastocyst is placed into a surrogate mother so that the embryo can come to term This is called reproductive cloning It is difficult and inefficient Ineff1cient because the fused cell often dies before it is implanted into the surrogate If it survives to be implanted it often dies during gestation It took 277 tries to get Dolly It is problematic because clones that survive to birth are often born with severe defects and OsuccessfulO clones often have hidden defects like premature aging Therapeutic cloning allows us to get around some of the problems Where the cultured embryonic stem cells are removed from a blastocyst genetically identical to the donor This has not yet shown to be possible for humans In order to get the cells the researchers have to destroy an early embryo Is therapeutic cloning needed to obtain genetically identical embryonic stem cells Some scientists were able to take adult skin cells modify them in a culture dish and turn them into cells that behave like embryonic stem cells This reprograming was achieved by adding 4 genes to the skin cells that encode specific activator and repressor proteins So far these reprogramming of skin cells are called induced pluripotent stem cells and they appear to be equivalent to embryonic stem cells If this can be conformed there will be no need to generate early embryos to obtain genetically identical embryonic stem cells Induced Pluripotent Stem cells Look at the chart How do we introduce the DNA we want to into the DNA of the skin cells Use a retro virus Take viruses and modify them so that they wonOt cause infection or illness Take advantage of the fact that virus uses enzyme reverse transcriptase to insert DNA of leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM the virus into the host cell The virus is modi ed so that the only result of the infection is to add DNA of choice into the host cell 0 Very ineffective only Ol5 will be successful Challenges of iPS Need to con rm that iPS cells are absolutely equivalent to embryonic stem cells Need to nd a way to quickly and easily produce large numbers of iPS from individual patients or it will not be cost effective Need to nd a way to introduce genes that encode the 4 activator or repressor proteins that do not use retroviruses Sometimes they also insert protooncogenes that are overactive Gene therapy Potential treatment focused on individuals who suffer from recessive genetic disorders that are caused by a mutation in just one gene Goal D to X the defective genes of that patient Involves introduction of normal copy of a gene into the cells of a patient who have two disease causing alleles of this gene Two basic treatment approaches are used direct or cell based delivery Direct delivery Use a virus to deliver the new normal copy of the gene First modify the cold virus insert DNA for normal human CFTR gene into virus for treating cystic brosis into viral DNA then introduce the recombinant viruses into the patients respiratory tract Then recombinant viruses enter respiratory cells and deposit CFTR gene so that cell produces normal CFTR protein In limited clinical trials this has been successful but for CF it only alleviates symptoms for a few wee s Cell based delivery SCID severe combined immune deficiency D disorder that causes child to fail to develop an immune system caused by a mutation in a single gene 0 Classic X linked SCID Bubble boy caused by mutation in a growth factor receptor gene 0 ADA de ciency SCID caused by mutations in gene that encode an enzyme Cells are removed from patient modi ed using a virus then put back in leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM 1990s D modi cation of White blood cells from patient With ADA worked but did not last very long 2005 modi ed blood stem cells from patients with ADA successful all patients doing well 2000 modi cation of blood stem cells from patients with X linked SCID but 317 patients developed leukemia completely hypothetical use of gene therapy leClConverterInpu 3anD7cOfGtXt12222010 521 56 AM Biology 121 General Biology Fall 2010 Review Sheet for Exam 1 Caution This is not meant to be an exhaustive list of evaything youneed to study for the exam but should be used in combination with your lecture notes book and the posted powerpoint les for each lecture Scienti c Method Chapter 1 Key terms scienti c method hypothesis prediction control theory 1 What is science 2 What is the scienti c method What are its components How is it used procedure for making observations of speci c phenomena and searching for the order underlying those phenomena hypothesis question prediction materials and methods dataresults conclusion 3 What is a controlled experiment variables are controlled 4 Are all hypotheses tested by carrying out experiments Give examples 5 Does the scienti c method ever have an end Explain your answer no there are no laws just theories science continually grows and improves itself with new experiments to test the same theories Water and Life Chapters 1 amp 2 Key terms atom will not react with other atoms when its outermost electron shell is completely full aka the atom is inert will react with other atoms if its outermost electron shell is only partially full aka reactive atomic number number of protons in the nucleus number of electrons in an atom de ning characteristic of each element atomic nucleus contains two types of proton positively charged subatomic particle neutron uncharged subatomic particle electron negatively charged subatomic particle orbit lighter electron shell restricted 3d spaces where electrons orbit the nucleus the further away an electron is from the nucleus the higher its energy is of the electrons that occupy it polar covalent bond shared electrons spend more time near one of the atoms requiring a slightly negative charge and other electron has positive charge although molecule is neutral its poles are charged examples include water electron shared between each H atom and central oxygen atom shared electrons spend more time near oxygen than near hydrogen atoms nonpolar covalent bond when electrons are shared roughly equally between two atoms in a covalent bond examples include oxygen nitrogen carbon dioxide and hydrogen hydrogen bond attraction between slightly positive hydrogen in a polar molecule and the slightly negative pole of a nearby polar molecule negative electrons bind to positive neutrons polar molecules result when hydrogen atoms form bonds with nitrogen oxygen or uorine ionic bond electrical attraction between positively and negatively charged ions water often breaks ionic bonds attraction between opposite charges hydrophobic waterfearing do not dissolve in water larger molecules with nonpolar covalent bonds fatsoils don t dissolve hydrophilic waterloving electrical attraction to water dissolve in water sugar glucose hydrophobic interactions have no af nity for water but do not have affinity for each other oil water tendency of oil molecules to clump together when dropped into water 1 What are the characteristics of living things 1 reproduction use DNN genetic material 2 complex organization and structure 3 homeostasis maintaining organization and structure 4 acquire use materials and energy from environment convert them into different forms 5 ability to grow 6 respond to stimuli from their environment 7 capacity to evolve 2 What 4 properties of water make it important for life 1 water is cohesive molecules tend to stick together because of hydrogen bonds causes surface tension tendency for water surface to resist being broken 2 water interacts with many other molecules dissolves many molecules like salt water is a good solvent because of its polar nature and ability to form bonds will dissolve any ionic of polar molecules 3 water moderates the effects of temperature changes the higher the temperature of a liquid the greater the speed of the molecules in it takes more heat to raise water temp than same amt of most other liquids must form precise lattice to freeze water freezes more slowly than other liquids 4 water forms ice and ice oats solid water is unusual be it is less dense than liquid water 9 What are the three major types of chemical bonds What properties of an atom determine which types of bond it will form ionic bond an electron is transferred creating pos and neg ions that attract one another occurs between the sodium and chloride ions of table salt covalent bond electrons are shared polar unequal sharing occurs between hydrogen and oxygen atoms of a water molecule nonpolar equal sharing occurs between two oxygen atoms in an oxygen gas hydrogen bond slightly positive hydrogen in a polar molecule attracts the slightly negative pole of anearby polar molecule occurs between water molecules slightly positive charges on hydrogen atoms attract slightly negative charges on oxygen atoms of adjacent molecules 4 What does it mean for a molecule to be hydrophilic hydrophobic How could you determine whether a given molecule is hydrophobic or hydrophilic hydrophilic waterloving electrical attraction to water dissolve in water sugar glucose hydrophobic interactions have no affinity for water but do not have affinity for each other oil water tendency of oil molecules to clump together when dropped into water 5 What determines whether a molecule will dissolve in water water dissolves polar substances and ions water forces nonpolar substances such as fat to form clumps Biological Molecules Chapter 3 Key terms dehydration synthesis chemical reaction that forms by removing water a hydrogen ion is removed from one subunit and a hydroxyl ion is removed from another subunit leaving openings in outer electron shells of atoms in the two subunits hydrolysis to break apart with water splits molecule back into original subunits with water donating a hydrogen ion to one subunit and a hydroxyl ion to the other subunit polymer a small organic molecule monomer chains of polymers carbohydrates polymer of sugars energy source for most organisms CHO ratio of 121 monosaccharide a carbohydrate that consists of just one sugar molecule CH20n n of carbons in backbone examples glucose ring structure forms when molecule hits water molecule fructose C6H1206 galactose C6H1206 short life span broken down linked to monosaccharide to make another molecule disaccharide two linked monosaccharides take away water and form covalent bond to link 2 monosaccharides together shortterm energy storage polysaccharide many monosaccharides longterm energy storage structural material example starch how plants store monosaccharides glucose monomers dehydration synthesis is used to form it glycogen how humans store energy glucose subunits are attached together used with dehydration synthesis but glucose molecules placed in very long chain of glucose monomers linked together cellulose glucose molecules linked together in totally linear chains with no branching at all the most abundant molecule on the planet fatty acid long chains of carbon and hydrogen with a carboxylic acid group at one end oils fats and waxes lipids that contain only carbon hydrogen and oxygen triglyceride chemical name of fats and oils got the name because fats and oils are formed by dehydration synthesis linking 3 fatty acid subunits to one molecule of glycerol a 3 carbon molecule saturated fat carbons of fatty acids joined by single bonds with hydrogen atoms at all the other bonding sites they contain as many hydrogen atoms as possible unsaturated fat fatty acid is unsaturated if there are double bonds between some of the carbons leaving fewer hydrogens transfat nonnatural manmade manufactured cheaper because it uses vegetable oil partially hydrogenated oils steroid composed of 4 rings of carbon atoms fused together with various functional groups protruding from them example cholesterol amino acid all proteins are constructed from a set of 20 amino acids primary structure rst level of protein structure order of acids as linked together describing linear chain of amino acids order dictated in DNA of organism secondary structure second level localized regions of the long chain start folding up on themselves form either the corkscrewhelix structure if the chain twirls around itself or a pleated sheet structure if it bens and fold back into itself tertiary structure true 3d structure of protein most proteins functional and complete at this stage held together by hydrgogen bonds and ionic bonds quaternary structure small number have this additional level need multiple proteins assembled and put together work together as a unit 1 What are the three major classes ofbiological molecules that we have discussed in detail How are these molecules formed What are the subunits monomers that are joined together for each class How are these molecules broken down carbohydrates contain carbon oxygen and hydrogen CHZO monosaccharide simple sugar often C6H1206 short life span example glucose fructose important energy source for cells subunit for polysaccharides disaccharide two monosaccharides bonded together shortterm energy storage molecule in fruits and honey example sucrose principal sugar transported throughout the bodies of land plants polysaccharide chain of monosaccharides usually glucose starch energy storage in plants glycogen energy storage in animals cellulose structural material in plants ber goes through digestive tract in tact and never broken down zero calories alternating bond con guration differs from starch lipids contains a high proportion of carbon and hydrogen most lipids are nonpolar and insoluble in water oils and fats triglyceride three fatty acids bonded to glycerol examples oil fat longterm energy storage in animals and some plants more storage than carbs phospholipid polar phosphate group and 2 fatty acids bonded to glycerol watersoluble heads phosphatenitrogen head that is polar and waterinsoluble tails 2 nonpolar fatty acid tails example phosphatidylacholine component of cell membranes steroids four fused rings of carbon atoms with funcational groups attached example cholesterol cells can make 85 of cholesterol you need to function so intake is not really necessary cholesterol needed for estrogen and testosterone anabolic steroids synthetic versions of testosterone longterm effects very bad proteins formed by chains of amino acids amino acids are joined to form chains by dehydration synthesis may have up to four levels of structure that determine its function storage proteins store energy seeds nuts eggs contractile proteins movement for muscle cells transport proteins move molecule from one place to another hemoglobin red blood cells transport oxygen throughout body defensive proteins antibodies that immune system makes signaling proteins cells must communicate with one another and work together N What is the difference between starch cellulose and glycogen How are the differences between starch and cellulose relevant to your diet p 41 cellulose structural polysaccharide makes up most of cell walls in plants tree trunks e glucose monomers are oriented with every other glucose being upside down while glucose monomers in starch have the same orienta 39on structural material not energy storage molecule starch stored in plants easy to break down glycogen stored in muscles and liver of animals more highly branched than starch 3 What is the difference between saturated and unsaturated fat unsaturated fats contain carboncarbon double bonds unsaturatedbecause they don t have maximum number of hydrogen molecules possible saturate fats contain as manyhydrogen atoms as possible lack double bonds between carbons straight and can pack closely together 4 What are the four levels ofprotein structure primary straight line sequence of amino acids linked together by peptide bonds covalent bonds in proteins sequence speci ed by genes within molecules of DNA secondary maintained by hydrogen bonds between polar portions of amino acids heliX acquired by many proteins pleated sheet repeatedly fold back upon themselves with hydrogen bonds holding adjacent segments of the polypeptide together tertiary folding of the helix results from hydrogen bonds with surrounding water molecules and disul de bridges between cysteine amino aci s folds determined by interactions of the amino acid functional groups with one another and surroundings determined by secondary structure and environment quatemary certain proteins that contain individual polypeptides linked by hydrogen bonds disul de bonds or by attractions between oppositely charged portions of different amino acids hydrogenbonds and ionic bonds hold the structure together t a Primary structure man m iwsuar Aminu acids n h Seenruiary structure L I 39 53 we Ar Fleatedsheet if 41quot m1 39 OHEItiarystruclure Polypeptide iv sinulesuhunii 39 rt Quaternary structure Complete prntein i with tour pulypentine g subunits 5 Why does heating up a protein cause it to loose its ability to mction high temperature causes bonds to diminish and break apart so proteins cannot stay intact fever Cells Chapter 4 Key terms ro aryotic bacterium only smaller simpler in structure DNA concentrated in nucleoid region lacks most organelles besides ribosomes eukaryotic larger more complex structure nucleus enclosed by membrane contains many organelles larger number of cells that are linear ma membrane isolates the cell s contents from the external environment regulates ow of materials in and out of cell allows interactions With other cells and With extracellular environment cytoplasm uid lled space everything in cell except nucleus nucleus Where DNA stored endoplasmic reticulum Where lipids are synthesized Where protein that will go outside of cell are made smooth lipids synthesized by enzymes glycogen broken down drugs detoxi ed no ribosomes rough synthesized proteins that Will eventually be secreted ribosomes attached mRNA attaches to ribosomes protein injected into rough ER goes into vesicles that eventually break off Golgi apparatus involved in making proteins that go outside of cell mitochondria powerhouse of cell glucose broken down to make energy ATP ribosome site of protein synthesis smooth ER rlbosomes raugn ER vesicles plant cell cell wall made of cellulose central vacuole large sac can take up to 90 ofvolume ofplant cell chloroplast produces glucose site of photosynthesis have outer membrane inner membrane thylakoid and granurn stack of thylakoids Where green pigment is plastid storage moleculecompartments mostly store starch lysosome cell s digestive system stays in cell until called upon to digest most common is food particles enzymes packaged into lysosomes lysosomes bud from the Golgi apparatus a lysosome fuses With a food vacuole and the enzymes digest the food cytoskeleton provides cell shape cell movement organelle movement and cell division has intermediate laments medium rnicrotubules largest and micro laments smallest exible and able to react animal cells totally reliant on cytoskeleton for shape vesicle 1 What is meant by the statement cells are the basic unit of life cells are the lower level of organization that exhibit all characteristics of life every organism is made up of one or more cells 2 Why are the cells of all multicellular organisms the same size size doesn t differ just amount they have to be able to communicate with each other 3 How does a prokaryotic cell differ from a eukaryotic cell What do they have in common eukaryotic more complex prokaryotic no organelles except ribosomes less complex no nucleus 4 How does a plant cell differ from an animal cell What do they have in common plant cell plasma membrane has a little extra protection than in animal cell cell wall made up of cellulose central vacuole large sac where water stored chloroplasts site of photosynthesis where glucose produced plastids storage moleculecompartments mostly starch similarities cytoplasm nucleus ER Golgi apparatus mitochondria ribosomes 5 What are major organelles of a eukaryotic cell What functions does each organelle carryout mitochondria produce energy by aerobic metabolism chloroplasts only in plants perform photosynthesis ribosomes provide the sites for protein synthesis endoplasmic reticulum synthesizes membrane components proteins and lipids Golgi apparatus modi es and packages proteins and lipids synthesizes some carbs lysosomes contain intracellular digestive enzymes plastids only in plants store food pigments cytoskeleton gives shape and support to the cell positions and moves cell pa1ts Cells Chapter 4continued 6 A cell wants to make an antibody that needs to ultimately be secreted outside the cell Describe the pathway from making the mRNA copy of the gene that encodes the antibody remember antibodies are proteins to nally secreting the antibody outside the cell any protein that needs to nd its way to the outside of the cell will follow the same pathway ex antibodies 1 antibody protein is synthesized on ribosome and transported into channels of rough ER 2 the protein is packaged into vesicles and travels to the Golgi apparatus 3 vesicles fuse with the Golgi apparatus carbs are added as protein passes through compartments 4 completed glycoprotein antibodies packaged into vesicles on opposite side of Golgi apparatus 5 vesicles merge with the plasma membrane and release antibodies into extracellular uid Lysosome serve as cell s digestive system stay in cell until called upon to digest most common is food particles 4 enzymes are packaged into lysosomes which bud from the Golgi apparatus 5 a lysosome fuses with afood vacuole and the enzymes digest the food 7 The cytoskeleton is considered a eukaryotic organelle Describe its components in general terms What are the functions of the cytoskeleton cytoskeleton a network of protein bers functions cell shape cell movement lamentstubules assemble disassemble or slide past one another organelle movement move organelles from place to place within cell cell division components include three types of protein bers micro laments thin involved in muscle contraction allow changes in cell shape facilitate cytoplasmic division in animal cells intermediate laments mediumsized provide supporting framework support plasma membrane anchor some organelles within cytoplasm attach some cells together microtubules thick allow movement of chromosomes during cell division major component of cilia and agella Membranes amp Transport Chapter 5 Key terms phospholipid receptor protein when activated cell can grow divide increase metabolic rate produce speci c proteins or move toward nutrient source recognition protein cell communication glycoprotein that has a carbohydrate group attached to it functions as an identi cation tag so when 2 cells meet they know where they re fromwhat their function is transport protein hollow pore allows hydrophilic molecules to move across membrane passive transport no energy cost to cell molecules move down concentration gradient includes simple diffusion facilitated diffusion and osmosis active transport cell uses energy to move molecules against concentration gradient bring large molecules into cell by vacuole formation diffusion molecules move from areas of high concentration to areas of low concentration concentration gradient difference in concentration of a substance between 2 parts of a uid or across a barrier facilitated diffusion osmosis 1 What are the functions of the plasma membrane boundary between cell and surroundings controls traf c of molecules into and out of cell allows communication with other cells want some molecules to pass through but only speci c amount to maintain equilibrium 2 How are cellular membranes organized What types of molecules are found in a cellular membrane What roles does each of these molecules play cholesterol make lipid bilayer stronger protein embedded in bilayer proteins surrounded by phospholipids 3 What determines how and in what direction a molecule will move across a membrane concentration gradient difference in concentration of a substance between 2 parts of a uid or across a barrier 4 How is the movement of water across a membrane in uenced by the concentration of dissolved molecules on either side of a membrane Energy amp Enzymes Chapter 6 Key terms exergonic reaction energy released during reaction endergonic reaction energy consumed during reaction for every endergonic reaction that occurs in cells there must have already been an exergonic reaction to make energy reactions occur in diff parts of the cel example photosynthesis ATP adenosine triphosphate see below ADP adenosine diphosphate 1 What is the difference between an exergonic and an endergonic reaction Provide several examples of each ATP CYCLE exergonic break down glucose fatty acids amino acids releases energy endergonic joins amino acids together to make a protein requires energy example photosynthesis making carbs lipids and proteins 2 What is ATP What functions does it serve in the cell energy carrier in cell used for shortterm transfer Within cell exergonic endergonic glucose breakdown protein synthesis lawenergy ADP P lowenergy products reactants 602 H20 amino acids 3 What does metabolism refer to Does metabolism only apply to the chemical reactions associated with digestion sum total of all chemical reactions that occur in an organism almost all metabolic reactions require the assistance of enzymes 4 What general characteristics are common to all enzymes reactanUsubstrate initial molecule binds to active site of enzyme 9chemical reaction very speci c usually assisting a single chemical reaction activity is in uenced by environment 5 Describe the interactions between substrates and an enzyme if that enzyme is assisting in the formation of a covalent bond Describe the interactions between a substrate and an enzyme if that enzyme is assisting in breaking a covalent bond formation of covalent bonds substrates join together with covalent bond change shape reaction occurs bonded substrates leave enzyme enzyme ready for a new set of substrates breaking of covalent bonds substrate enters active site substrate and active site change shape promoting breaking of bond substrate bond broken subunits leave active sites 6 How is the activity of an enzyme be in uenced by its environment salt concentration if its too high too many interactions interfere with amino acids that hold proteins together pH level too manytoo few interactions disrupt ability to hold amino acids together temperature the higher the temp the more the molecules move higher chance of interacting with one another because they bump into each other because they re moving quickly Review Questions 1 why or why do you not think viruses are living things 2 how are ionic and covalent bonds related b ionic and covalent bonds both result in a full outer electron shell for the molecules involved in that bond 3 which best describes a hydrogen bond d a weak bond between two partially charged atoms in two different molecules 4 what is an atom that forms hydrogen bonds easily c water 5 why does ice oat c the water molecules in ice participate in a greater number of hydrogen bonds than the water molecules in liquid wateribecause when molecules are far apart it has nothing to do with the strength of the hydrogen bond just the number hydrogen bond all same relative strength no matter what two molecules are involved 6 why is the linking of two monomers called dehydration synthesis b because the atoms removed from the two monomers combine to form water 7 what is the difference between starch and cellulose a in cellulose the glucose monomers are oriented with every other glucose being upside down in starch the glucose monomers have the same orientation starch not hard to break down cellulose structural material not energy storage molecule 8 what is the difference between saturated and unsaturated fats d unsaturated fats contain carboncarbon double bonds don t have max of hydrogen possible that s why they re unsaturated 9 the process of creating and exporting a protein outside the cell requires many organelles which of the following lists these organelles in the correct order rough ER vesicle Golgi apparatus vesicle Golgi and rough ER are not attached to each other rough ER has ribosomes attached to it smooth ER only involved in lipid synthesis break down glycogen 10 which of the following is not a function of the cytoskeleton to allow cells to stick to each other is a function physically pull the duplicated chromosomes to the opposite sides of a dividing cell enables sperm cells to swim provides shape to cells lining your intenstine and provide a pathway for vesicles to follow to the appropriate destination forms trackshighways to cell vesicles link on and have special tag go to destination 11 if a cell has no proteins embedded in its plasma membrane which of the following will be able to diffuse across the membrane chloride sodium water and glucose all need transport proteins carbon dioxide only one that can freely cross membrane without transport protein 12 what will happen to your cells if you drink seawater Seawater contains 35 salt NaCl Your cells contain l salt NaCl water will ow out of your cells and cause them to shrivel dehydrated BIOLOGY EXAM STUDY GUIDE NUMBER THREE from parents to offspr1n Genes Alleles and Chromosomes Chromosomes contains a DNA double helix together with proteins that help to organize and regulate the use of DNA Genes a series of nucleotides ranging from a few hundred to a few thousand that determine inheritance traits Encode the information needed to produce proteins cells and entire organisms Part of chromosomes9genes are physicallylocated on the locus ofa chromosome Both members ofa pair ofhomologues carry the same genes located at the same loci however the nucleotide sequences ofa given gene may differ in different members ofa species or even on the two homologues ofa single individual Alleles alternate version ofa gene at a given locus n though a pair ofhomologues may carry the same genes the nucleotide sequence ofthe gene may differ resulting in alleles Alleles arise as mutations that slightly change the sequence of nucleotides a that is similar in appearance and genetic informationto another chromosome with which itpairs during meiosis also called homologue homologues have the same allele at a given gene locus 9the same pair of chromosomes Homozygous carrying two copies ofthe same allele ofa given gene Ifa chromosome is homozygous for the dominant allele it displays the dominant trait PP ifthe chromosome is homozygous for the recessive allele it displays the recessive trait rr Heterozygous chromosomes carrying two different alleles ofa given gene Heterozygous chromosomes displays the dominant trait Rr Genetics The study of how specific traits characteristics or disorders are passed on g Gene luei Dominant l Ilumnlnguus chrumnsnmes P a ecssswe allele Genotype PF llnmnzygaus Hnmnzyguus Hetemzygnus lnrlhe ur he dnminanl allele recessive allele Mendel39s Law ofSegregation The two alleles ofa gene separate from each other during meiosis so that each gamete receives only one allele Mendel crossfertilized truebreeding white owered plants with true breeding purple owered plants he called this parental generation denoted by the letter P when he grew the resulting seeds he found that all of the first generation offspring produced purple owers wondered what happened to the white color in the F1 generation he allowed the F1 owers to selffertilize and planted the seeds the following spring where the results produced threefourths purple owers and onefourth white owers This showed that the capacity to produce white owers did not disappear it was just hidden in the F1 plants recessive trait The dominant allele may hide the expression of the recessive allele the recessive allele however is still present and can appear in future generations ifit mates with another organism with the same recessive trait Mendel s Law of Segregation The pairs of alleles on homologous chromosomes separate from each other during meiosis As a result of the Law of Segregation each gamete receives only one allele of each pair when a sperm fertilizes an egg the offspring receives one allele from the father and one from the mother Truebreeding organisms have two copies of the same allele for a given gene and therefore are homozygous for that gene all of the gates from a homozygous individual receive the same allele for the gene Hybrid organism have two different alleles for a given gene and are therefore heterozygous for the gene half ofa heterozygote s gametes will contain one allele for that gene and half will contain the other allele Inheritance of a Single Trait Phenotype an organism s physical trait Pp dominant trait is what we see PP homozygous dominant pp homozygous recessive Genotype an organism s genetic makeup Pp even though the recessive allele doesn t appear in the physical trait the recessive allele will still be passed down to future generations and if bred with another recessive trait it will appear in the physical makeup of an organism Punnett Square allows you to predict both the genotypes and the phenotypes ofa specific cross Assign letters to different alleles capital letter for dominant alleles lower case letter for recessive alleles Determines the expected offspring of mating two traits or individuals one individual goes across the top and the other goes down the side Test Cross cross between an individual that expresses the dominant trait and an individual that expresses the recessive trait This method is used to determine the genotype of an individual expressing the dominant trait For example P x pp 9 the genotype for the plant with one dominant allele could be either FF or Pp the cross allows us to see the outcome of each possibility Parental Genaanon P V X H Pp Pp First Genemuan O spnnq rm gamerasr m Parann P p E Phenmypi Ratio 1 P PP PD a purple39 1 while a E Genotyplc Ratio 2 9 Pp pp lPF39EZF39pllpp punnen Square Inheritance of Two Traits After determining the inheritance patterns of single traits Mendel looked at a more complex questionithe inheritance of multiple traits He began by cross breeding plants thathad more two traits ie seed color and seed texture From the crosses of plants with these traits Mendel already knew that the smooth allele of the seed texture gene S is dominant to t e wrinkled allele s and that the yellow allele of the seed color gene Y is dominant to the green allele y He crossed a truebreeding plant with smooth yellow seeds SSYY to a truebreeding plant with wrinkled green seeds ssyy9 the SSYY plant produced only SY gametes and the ssyy plant produced only sy gametes Therefore all of the F1 offspring were heterozygote39s genotypically Sst with the phenotype of smooth yellow seeds In order to determine the genotype of an individual with a dominant phenotype for both traits cross it with an individual that is recessive for both traits Mendel found that from the F2 generation the ration was about 1 smooth yellow wrnk yellow smooth green wrinkle green 9 mnemanceor 9 WUTrHils mm u 7 The Law of A Hm quott39 The alleles of quot39 quot quot to the gametes independently of the alleles for other genes e inheritance of one trait has no effect on the inheritance ofa second trait a long a thegene are on different chromosome his rquot 39 39 behavior on during meiosis9determines what the genotype of the sex cells will be 7 The alignment of chromosomes during metaphase 1 is random ifa person begins heterozygous they will have four different genotypes in their sex cells and they are all equally likely 7 When following two trains and both parents are heterozygous there is a 9331 ratio Inheritance of Sex Chromosomes Females have two identical chromosomes X chromosomes while males have one X chromosome and one Ychromosome The other chromosomes that have identical appearance in both males and females are autosomes 7 Sex7linked genes 7 X hromosome over 1000 genes but only a small number of the genes play a role in female reproduction the rest encode tmits that are important to both sexes ex The ability to see a full range of colors 7 Yr 39 ts m 39 0U gene and most of these genes play a role in male reproduction 7 The male phenotype is dicmted only by the dominant allele on the X chromosome9ex Color blindness is more prevalent in males because only one allele has to have the gene whereas in order for a woman to have color blindness it must appear on both alleles R red e as r white eyes H mm mm we xv m For gems an m x chmmniomu Mal WWW 5 nanquotmum by he allcln an mm mm x cmmvwsamz Do the Mendelian rules of inheritance apply to all traits Assumptions using Mendelian Ru es One allele is completely dominant to the other e e We have to extend these assumptions ofrules ofinheritance because most traits are in uenced in more varied and subtle ways Incomplete Dominance When the heterozygous phenotype is intermediate een the two homozygous phenotypes offspring is intermediate oftheir parents Ex A red pigment and white pigment produce a pink pigment Heterozygous vs homozygous if there is one copy ofa gene we will see a little bit of red pigment n I I u I r gives us a range of severity for this disorder LDL packages cholesterol and sends it through the bloodstream LDL receptors encoded by H gene breaks down cholesterol most of us are HH meaning we have functional LDL receptors H39H heterozygous mild version of hypercholesterolemia not all or nothing H39H39 homozygous severe version of the disease individual will not produce any functioning LDL receptors Rim pigment produoed w R39nc red plgment produced R R sun have 3 Dussmle genotype oul axsn have 3 0055mm phenotypes Ry Codominance and multiple alleles ofa single gene combination phenotype of two possibilities A single gene has three alleles that encodes enzymes to add sugar molecules to proteins that are sticking out of red blood cells There are 3 different alleles in a population but an individual actually only has 2 alleles or only one if they are homozygous In blood types A and B alelles are completely dominant to 0 and A amp B are codominant to each other s L L L L t i 39 iiniiai traits controlled by multiple genes h ater number of genes that contribute to a single trait the i a L c L 4 E among t em f a polygenic tra Example o human height weight eye color and skin color they all have a variety ofdiFferent phenotypes and are n r es easily divided into de ned oatego 39 individuals quot L v H r a aiti uiai tiaiti inherited in a dominant recessive or sexlinked pattern n 39 ee 39 and 39 ar ma 1an qua 39 an n39 39 139 391 quot 39 traitif th uuai i 39 39 L quot39 quot the trait ifthe square or circle is half shaded the individual is aknown carrier t square or circle we cannot determine the genotype from this pedigree l l Maw w mud IIL lgrLus Munitions inmalc Mnmls i lmg shuwsimli K L unlinelnnwnsn a 7 known r1mcmxyguiellm L Q recesslvzl u L org 7 urinal amrmmnmngnnnxypnlmmmls min Autosomal Recessive Disorders 39 quot J 39 39 there i typically an affected individual every 0 her generation and the 394quot l all arrim T e on yway an autosomal recessive disorder would not skip a ifboth parents are affect d or carriers Unaffected parents can have a dau te or son that is es ofautosomal recessive disorders include albinism sickle cted cell and cystic brosis Fealures cl Fedlgles l ollen sk lps a generallon 2 unelleeled parsan can have daughter nr son hat ls allected r AutosomalDommantDlsorders e In autosomal domlnantdlsorders at leastone ofthe parents ofthe affected lndlvldual IS affected 7 Thls type ofdlsorder rarelysklps a generauon lines not skipu generutl39u e Examplesofautosomaldomlnantdlsorderlnclude dwarflsm lluntmgton39s dlsease and Alzhelmers dlsease No cameS Fealures of Pedlgree t al least one 0 parents dl aneeled mdwldual ls alloclcn 12 rarely sklps a generahon llnked recesslve dlsorders often sklp a generatlon r Unaf ted parents can have sons matare affected and daughters that are carrlers mostaffected lndlvlduals are males e rmal male normal female 0 eelen male Xe rlrrmal female semen x a ecled female IV O Fenlums of Pcdlgruc m i C ll often sk ane allun V I C Q ips a 9 2t una eclsd paman can have sun Iha ls af oclsd 3 mcsl mauled lndlvlduals are male DNA lmtd and prot s em DNAls a Nuclelc Acld Composed ofNucleotldes through dehydration synthesis Nucleotides are a subunit ofwhich nucleic acids are composed a phosphate group bonded to a sugar deoxyribose in DNA which is bonded to a nitrogen containing base Adenine guanine Qytosine or Ihymine in DNA9listed in bold are the 3 components that make up nucleotides Nucleotides are linked through dehydration synthesis forming a strand of nucleic acid by bonds between the phosphate of one nucleotide and the sugar of the neXt nucleotide Nucleic acids provide the instructions for building proteins DNA Nucleotides vs RNA Nucleotides Deoxyribose nucleotides is a subunit ofDNA and the bases include adenine guanine cytosine and thymine Ribose nucleotide is a subunit of RNA and the bases include adenine guanine cytosine and uracil AZT and HIV HIV Human Immunodeficiency Virus RNA attaches to a receptor on the host s plasma membrane its core disintegrates and viral RNA enters the cytoplasm RNA enters the cell Viral reverse transcriptase produces DNA using viral RNA as the template if we could block viral reverse transcriptase it would block the virus from spreading Viral DNA enters nucleus and mixes with host chromosomes it is transcribed into mRNA and more viral RNA which move to the cytoplasm viral RNA continues to spread Viral proteins are synthesized using mRNA the viral protein infect the cell but they don t kill the cell Viral proteins and RNA assembled Viruses bud from the plasma membrane AZT AntiHIV Drug AZT binds to the viral reverse transcriptase in the place of thymine and stops DNA synthesis essentially blocking the reverse transcriptase once AZT is added the neXt nucleotide cannot be added9AZT binds to the viral reverse transcriptase which blocks the reverse transcriptase not allowing DNA toform AZT doesn t interfere with replication of host cell DNA because it binds to cellular replication enzymes for weakly AZT is very specific and doesn t bind very well to enzymes in your cells Can t add another AZT after one AZT because it causes incomplete DNA which causes a crippled virus Structure of DNA Chargaff s Rule 1947 Within a given species DNA contains equal amounts ofAamp T and C amp G always paired together XRay Diffraction Technique used by Maurice Wilkins and Rosalind Franklin to study the DNA molecule Wilkins and Franklin discovered through XRay diffraction that a molecule ofDNA is long and thin with a uniform diameter DNA is helical and DNA molecules consist of repeating subunits Chemical and Xray diffraction data didn t provide enough information for researches to determine the structure of DNA James Watson and Francis Crick used the data and their knowledge of how complex organic molecules bond together and proposed a model for the structure of DNA Structure of DNA WatsonCrick Model Watson and Crick determined that the structure of DNA followed a double heliX model A single DNA molecule consists of two strands strands are a series of nucleotides that are covalently bonded to each other Within each DNA strand the phosphate group of the nucleotide bonds to the sugar of the neXt nucleotide in the same strand this bonding pattern produces a backbone of alternating covalently bonded sugars and phosphates The nucleotide bases protrude form this sugar phosphate backbone The two DNA strands are held together by hydrogen bonds between nitrogen bases not covalent bonds AT a QC complementary base walrs 2 strands ofDNA have to be opposite of each other in order to form the DNA covalent bonds on either side face opposite directions phosphate base sugar Complementary Base Pair in nucleic acids bases that pair by hydrogen bonding In DNA adenine is complementary to thymine and guanine is complementaryto cytosine in RNA adenine is complementaryto uracil and guanine to cytosine DNA Replication The Purpose ofDNA Replication the copying ofthe doublestranded DNA molecule producing two identical DNA double helices Replication of DNA is a critical even in the cell cycle because before cell division the parent cell must synthesize two exact copies of its DNA9creates exact copies of genetic information The base sequence of each strand contains all ofthe information needed to replicate the other strand DNA replication does not happen all ofthe timebecause it happens exclusively during the S phase ofthe cell cycle9when replication is complete 2 DNA molecules are atached by the centromere sister ch ro matids and they re main attached until Anaphase Semiconservative Process of DNA Replication means it concerves one parent strand andproduces one newly synthesized strand parental strands serve as tem lates Replication Fork the intersection between wou nd and unwound DNA veplxcahon mm The Process of DNA Replication First the DNA double helix must be opened up so the base sequence can be read9hydrogen bonds must be broken so they can bring in free nucleotides in orderto form base pairs with the parental group Second new DNA strands with base sequences complementary to the two orignial strands must be synthesized the new DNA strands are synthesized in relatively short pieces 9this is carried out using the enzyme DNA polymerase Third step ofthe process is to stitch togetherpieces to form a continuous strand9new double helix is composed ofone parental and one new strand DNA replication begins at a replication bubble where replication forks are apparent 9du ring the process of DNA replication the replication forks move in opposite directions 9once DNA replication is complete two separate strands are formed and there aren39t bubbles or forks Dllgm n Imam m rellllca nn HHTMBNA39 lepllczlinn VAYAYKYxVY AYAYRVAYu each nhmmosume a DNA double hellx 01lean replication mmmm 1 IDnuumwslrin 0 mm 4 doc I rm Replcailu39i VvV w I Wquot 39VAV U39Vlww ViVVV w I 39 wuwx quota alwhlu DNA maleculls sismrahmmall s DNA polymerase will bring in T for A and G for C wants to make an exact copy A T A T A r A 39r A T CI 5 c c c a c 39 G c e a 39 c 6 r c 6 G c 5 c 4 A 139 A T A 1 A 139 T V T Nucloolidos A T A Y Pmaulnl Bulh pnronlnl Two idonll col molecule slmnds sewn duughier mulncules 1 nuiom ma OIDNA DNA Polymerase Proofreads and Fixes Mistakes DNA polymerase inserts an incorrect nucleon39de once every 1000 to 10000 base pairs but the actual number oferrors seen in the DNA molecule after replication is one every 100000000 to 1000000000 base pairs thus there is a much lower error rate in replication rate than in the actual enzyme The reason for a lower error rate in replication rate than in the actual enzyme is due to the fact the the DNA polymerase checks its work identifies and fixes mistakes so we see very few mistakes in the DNA molecule Gene Expression and Regulation Flow of Genetic Information Transfering Information DNA to Protein 1St Step Transcription making copy ofinformation to make a single protein and convert it into RNA 2 Step Once processed it is mRNA 3 71 Step Translation information used by iibosome to make protein Basic relationship between transcription and translation Transcripn39on genetic information is in the language of the nucleotide Translation genen39c information must be converted into the language of the amino acids Ribosomes convert the information DNA Encoding Genen39c Information There are only 4 simple nucleotides in DNA information is reliant on combinations Chromosomes have thousands ofnucleou39des so DNA has no problem keeping information in the sequence ofnucleon39des Transcription and Translation 3 nucleotides together in RNA equals a single codon M 1 ip odons can code for the same amino acid 5 is the mo Special Codons an UAA UAG or UGA will always be the stop codons in a series ofamirio ids Transcription P 39 39 u e DNA 39 i i a a Initiation L 39 39 39 39 39 39 single gene is the the instruction to create a protein RNA polymerase needs to Promoter9 near th 39 39 39p 39 rocess o cule breaks hydrogen bonds s on the bases and separates the two stran 39 7 L l quotMom WWW Wequot i l base inRNA Elongation hrin bonds with DNA NA polymerase travels along the DNA template strand unwinding nNAi iii i 44 the ofribose nucleotides into an RNA molecule Nucleotides in the RNA are complementary to the template strand of the DNA As RNA polymerase moves to the right atrail of RNA gros in the back rewinds DNA behind it u NA complementary strand H 39 Termination the point when the RNA polymerase detaches fromthe DNA and releases the RNA molecule At the end ofthe gene RNA polymerase encounters a DNA sequence termination signal at this point the RNA polymerase separates from the DNA and releases RNA molecule Termination signal signals to RNA polymerase to stop making RNA because it has reached the end of the gene will break the remaining hydrogen bonds so the RNA molecule is free RNAlgzwz elase 39 termlpalion b q vqna n N ullil39l l Hum Promoter In order for transcription to begin the RNA polymerase must find the beginning ofthe gene near the beginning of every gene is an untranscribed sequence called the promoter a specific sequence nfDNA at the beginning nfa gene in which RNA pnlymemse binds and starts gene transcriptinn A promoter consists of a short sequence of bases often TATAAA that binds RNA polymerase and one or more other sequences often called transcription gactor binding sites or response elements Template DNA strand VS Complementary DNA stran Template DNA strand is the parent strand the the strand of the DNA double helix from which RNA is transcribed The complementary strand is a single strand of RNA with bases complementary to that ofthe DNA strand mRNA Processing Exon coding segment he exon is expressed in the protein and contains the information for aking the protein codes for amino acids Intron noncoding segment The intron is part ofthe intervening sequence and does notcontain information for making the protein Cap and Tail aditional nucleotides Help move the mRNA through the nuclear pore Help the mRNAbind to a ribosome Prevent enzymes in the cell from breaking down mRNAbefore it can be used to make a protein m 44M a l Ehffil39l39f umu m In mum I wlmu V I quot39 m39 l lnllnvsrlmwni quotquot l 1 ammmuummm 1 gt cymg39vnm 7 4 mm mRNA Splicing During mRNA splicing the introns are removed and the exons are joined together9all ofthe unessecary noncoding information is removed in order to put the important coding information together w W on m quotmquot 5quotquot c pllun A aquot mum nmu um mm tiff V quotmm 1 Inwnxnmnued quotn f l Emmwllud MEDIAquot mm 392 cn lnl nqulnu Translation Three Types of RNA are Required for Translation mRNA messenger RNA the base sequence of mRNA carries the information for the amino acid sequence ofa protein groups ofthese bases codons specify the amino acids carries the codefor the amino acidsequence ofa protein from DNA to the ribosomes which synthesize the protein specified by the mRNA base sequence rRNA ribosomal RNA comines with proteins to form ribosomes the small subunit binds mRNA the large subunit binds tRNA and catalyzes peptide bond formation between amino acids during protein synthesis The ribosomes help form peptide bonds between amino acids to form peptide covalent bonds to make proteins tRNA transfer RNA each tRNA carries a specific amino acid to a ribosome during protein synthesis the anticodon of tRNA pairs with a codon of mRNA ensuring that the correct amino acid is incorporated into the protein tRNA translates between the language of nucleotides and the language of amino acids It is a translator molecule responsible for reading the sequence of nucleotides and translating it into a sequence of amino acids tRNA holds anticodons that are complementary to codon codons are MADE during transcription and are USED during translation Process of Translation Purpose of Translation the goal of translation is to build a protein made of amino acids linked by dehydration synthesis covalent bonds9 decodes the sequence of an mRNA into the amino acid sequence ofa protein Initiation tRNA will form a base pair with start codon AUGmethionine which will form a base pair with the anticodon UAC which will basepair with the start codon AUG of the mRNA The large ribosomal subunit binds to the small subunit methionine tRNA binds to the first tRNA site on the large subunit Elongation Repetition of adding amino acids to form peptide bonds Energy to form peptide bond between 2 amino acids come from ATP Remove empty tRNA and entire ribosome moves down 3 nucleotides tRNA forms a covalent bond with amino acids then shifts down Empty binding site attaches to complementary tRNA and ribosome shifts to the neXt codon to be read Summary of elongation empty tRNA detaches ribosome moves one codon to the right a new tRNA is brough in and a new peptide bond forms this process takes place until it comes to a STOP codon Termination No tRNA binds to STOP codons when stop codon is reached proteins called release factors bind to the ribosome forcing it to release the finished protein and mRNA Release factors cause everything to fall apart causing the finished protein to be released Translation summary build proteins by covalently bonding amino acids where mRNA is carries the recipe for this process DNA is not directly involved in translation Universality of the Genetic Code We are able to see that the genetic code is universal because we can take DNA out of any organism and put it in another organism s chromosome and the plant will grow For example ifyou take DNA out ofa fire y and put the DNA in a tobacco plan ts chromosome the plant will grow tobacco plant has a fire y gene inserted into one ofits chromosomes Difference in Cells Cells are made different by how they use information in the DNA all cells have the same DNA but they have difference functions and different proteins Not every cell makes every protein they only make those that help carry out their specific jobs and functions Red Blood Cells vs Stomach Cells Red blood cells transcribe the hemoglobin gene and make hemoglobin protein which binds to and carries oxygen Stomach cells transcribe pepsin gene and make pepsin protein which helps break down food These cells both have the same DNA however they don t need to use all of it at the same time to perform their important functions Some proteins are made in all cells Housekeeping Proteins These proteins are present in every cell to carry out day to day functions For exmaple we need ribosomal proteins in order to put together a functional ribosome Biology Study Guide Unit 4 Mutations 000 O O O 0 Any change in the nucleotide sequence of DNA They don t originate anywhere but the DNA Can occur within a gene or outside it Can arise from I A rare mistake in base painting during DNA replication by DNA polymerase I Environmental conditions including exposure to certain chemicals or some types of radiation may increase frequency of errors during replication or directly induce changes in nucleotide sequence Xrays UV light chemicals smoking Protein p53 looks for mutations and brings enzymes to fix Insertion Mutation I Original sequence9TA nucleotide pair inserted I One or more nucleotide pairs are inserted into the DNA double helix I Can affect protein produced I 9extra pair9now a natural part of the sequence affect on protein depends on how many are added9codons9amino acids 3 nucleotides I Amino acids still need to be able to fold properly otherwise the function is affected I gt 1 nucleotide disastrous effects Deletion Mutation I Original sequence9C G nucleotide pair deleted I One or more nucleotide pairs are removed from the DNA double helix I Can affect protein produced I FrameShift Mutation everything after the deletion is the wrong amino acid can cause premature STOP codon Nucleotide SubstitutionsPoint Mutations I TA AU nucleotide pair I The replacement of one nucleotide by another in the DNA template strand I The ideal unless it is on the STOP or START codon I how will a change in a single DNA nucleotide affect the protein that is madesometimes there is no effect Biotechnology 0 O 0 Any use or alteration or organism cells or biological molecules to achieve specific practical goals Not necessarily a new practice I Use of yeast to produce bread beer wine and cheese I Use of selective breeding to produce plants and animals with desired characteristics Genetic engineering is the direct modification of genes I To produce plants and animals with desired characteristics I To learn more about how cells and proteins work I To develop better treatments for human disease 0 Use of DNA technology to learn more about an individual I Do they carry any disease causing alleles of specific genes I Are they the father of specific child I Were they present at a crime scene 0 Recombinant DNA Technology I A set of laboratory techniques for combining genes from different organisms into a single DNA molecule I First product human insulin protein produced by genetically modified bacteria 1982 0 Bacterial Plasmids o Plasmids I Small circular pieces of DNA I Carry small number of genes that give bacterium special properties I Abundant up to 100 s of copies per cell I Easily taken in via transformation process by which living bacteria take in plasmids I Plasmid replicates in cytoplasm 0 Generation of Recombinant Bacteria 1 Plasmid isolated 2 DNA isolated 3 Gene inserted into plasmid 4 Plasmid put into bacterial cell transformation 5 Cell multiples with gene of interest I 9Copes of gene 0 Gene used to alter bacteria for cleaning up toxic waste 0 Gene for past resistance inserted into plants genetically modified organisms contain one or more genes from a different organism I 9Copies of protein 0 Protein used to make snow form in higher temperature 0 Protein used to dissolve blood clots in heart attack o Recombinant DNAplasmid l 0 Products made using recombinant DNA Technology gardasil vaccine human insulin human growth hormone o quotPharmquot Animals I Sheep genetically modified to produce a human protein in milk 0 Restriction enzymes 0 A specific restriction enzyme they are specific to a certain sequence of bases binds to the sequence and then cuts the DNA creating DNA fragments with quotsticky ends 0 Same restriction enzyme is used so that there is the same cut so that they can fit back together 0 Cuts the covalent bonds not the hydrogen bonds 0 Restriction Enzymes amp Recombinant DNA 0 There is a gene of interest and a plasmid 0 Cut both the gene and the plasmid with the same restriction enzyme so that the ends match up 0 Mix DNA with gene of interest and plasmid 0 Add DNA ligase to join DNA fragments together 0 Recombinant DNA DNA in which genes from two different organisms are combines into a single DNA molecule 0 DNA Technology amp Agriculture 0 Researchers estimate that over V2 of all foods in the US stores contain at least small amounts of GM foods 0 Generation ofGM Plants 1 Cut DNA containing gene of interest and modified Ti plasmid with the same restriction enzyme 2 Mix together cut gene and plasmid add DNA ligase to seal DNA 3 Transform A tumefaciens bacteria with the recombinant plasmids 4 Infect plant cell with transgenic bacteria Bacteria enter plant cell and gene of interest is inserted into the plant cell chromosome 5 All single plant cell to grow in a mature plant 0 Genetically modified plant contains a new gene and has a new characteristic 0 Genetic Modification of Plants using a gene gun 0 Microscopic particles coated with DNA from gene of interest are quotshotquot into plants cells 0 Why generate genetically modified crop plants 0 Increase the amount of produce increase the nutritional value increase the shelf life 0 Resistance to herbicide I Herbicide will kill the weeds and not the crop plants higher crop yield I Herbicide can t produce all 20 ammino acids dies because can t create proteins I HerbicideResistant GM plants 0 Most herbicides kill plants by inhibiting a plantspecific enzyme used to synthesize certain amino acids plants can t obtain from the environment 0 Herbicideresistant plants have been given a bacterial gene that encodes an amino acid synthesizing enzyme that is not inhibited by a specific herbicide o llRoundup Ready GM plants I Ex Corn cotton flax potato rice soybean tomato I GM Plants produces natural and bacterial still works when the herbicide is there O O O O 0 Resistance to pests Crop plants suffer less damage from insects higher crop yield Ex Cotton plants Bt gene encodes a protein that damages the digestive tract of insect pests coats the insides of the insect but not animals or humans To make a Bt gene is first inserted into a modified Ti plasmid Ex Corn cotton potato rice soybean Loss of genetic variation unforseen disaster970 crops gone Resistance to disease Sterile Plants are less prone to infection by viruses bacteria or fungi higher crop yield Ex Papaya potato squash Transgenic plants cannot cross with wild varieties they are safer for the environment and more economically productive for the seed companies that produce them Ex Chicory corn Altered oil content Oils can be made healthier for human consumption or can be made similar to more expensive oils Ex Canola soybean Altered ripening Fruits can be more easily shipped with less damage higher returns for the farmer Ex Tomato 0 Concerns about GM crops 0 O 0 Genetic Testing 0 O O Potentially hazardous to humans Potential side effects caused by eating food produced form GM plants Potential allergic reactions to GM plants Potentially hazardous to environment Potential transfer of genetic material to wild nonG M relatives Potential evolution of resistant pests Potential loss of genetic variation Test for the presence of diseasecausing allele of a specific gene How does a diseasecausing allele differ from a normal allele of a given gene Ex Sicklecell Anemia Exam Question Autosomal recessive disorder caused by a single nucleotide change in the gene that encodes encodes Bglobin Hydrophobic outside of protein change of shape People with sicklecell disease are resistant against malaria O Mst cuts the strand 3 times for a normal Bglobin allele and only 2 times for a sicklecell Bglobin allele 0 Example ofa Restriction Fragment Length Polymorphism RFLP o A difference in nucleotide sequence at a specific location on a specific chromosome that alters the restriction site for a specific restriction enzyme DNA probe would be specific to the section of DNA where the Mst does not cut for sicklecell Identification of Sicklecell allele 1 Obtain blood sample 2 Extract DNA from white blood cells 3 Cut DNA with Mst 4 Gel electrophoresis and blot 0 Results 0 Homo normal 0 Hetero I A combination because one copy is normal and one is diseased o Homo sicklecell PCR Based Test 1 Obtain small amount of DNA 2 Only make copies of a very small specific section of DNA 3 Make primers that flank altered Mst site 4 Run PCR reaction 5 Cut resulting DNA with Mst 6 Gel electrophoresis and stain 0 Results 0 Homo normal 0 Hetero o Homo sicklecell Mst makes many fragments need to simplify Gel Electrophoresis Mixture of DNA fragments of different sizes is put into the gel at the negative end DNA is negatively charged all DNA has the same amount of negative charge so the DNA fragments want to travel toward the opposite positive end The shorter fragments have an easier time traveling so they travel further down towards the positive end than the larger fragments The gel is then stained to help visualize the fragments A power source is required for this process 0 9quotStainquot 0 DNA quotbandsquot visible 0 Visualize all DNA fragments by immersing gel in DNA stain Use if expect small number of different DNA fragments on gel 0 9Blotquot 1 Gel is treated with chemicals that break the Hbonds between DNA strands Transfer DNA strands onto nylon paper using electrical current 2 Immerse nylon paper in solution containing labeled DNA probes 3 Wash away all DNA probes that didn t form Hbonds with DNAS fragments 4 DNA fragments that base pair with DNA probes are visible in red and the rest of the DNA is not visible 5 Used to identify small number of DNA fragments when have a lot on gel 0 DNA Probes Polymerase Chain Reaction PCR Heat separates the two DNA strands Cooling allows the primers and DNA polymerase to bind 1 2 o s 0 Short single stranded DNA molecule that is complementary to the DNA sequence of the region are interested in o The probes are specific to a certain sequence DNA probe is labeled with a colored molecule so if it base pairs with a DNA fragment we will be able to see it If DNA probe does not base pair with a DNA fragment that fragment will not be labeled and we will not be able to see it They are specialized and they can be hand selected depending on what you re looking for I Primers short pieces of DNA that are complementary to the ends of the region you want to make copies of New DNA strands are synthesized Each PCR cycle doubles the number of copies of the DNA 30 cycles l billion copies Why PCR I In order to visualize DNA fragments on a gel or blot you need 1000 s of DNA molecules length One band on the gel has thousands of the DNA fragments that are the same I The more DNA molecules present the darker the band Diagnostic Arrays O Isolate DNA from person being tested 0 Cut DNA with multiple restriction enzymes 0 Separate DNA fragments into single strands 0 Attach a colored label to the singlestranded DNA fragments 0 Incubate with nylon paper with spots of DNA complementary to normal and disease causing alleles of the gene 0 DNA and Forensics o RFLP s and Forensics I Comparison of DNA sequence between 2 individuals for the same noncoding region I RFLP s are not only found between alleles of a given gene but also between noncoding DNA sequences I Researcher have identified several RFLP sites within noncoding regions of our chromosomes that can be used to distinguish individuals 0 Multiple RFLP as produce a DNA profile I Forensic examine as many as 10 different RFLP s at the same time I The total pattern of fragments produced is referred to as a DNA profile of DNA fingerprint 0 Short Tandem Repeats STR s I Each STR is a repeating sequence of DNA 25 nucleotides long found at a specific location on a specific chromosome usually in introns or noncoding regions I Researchers have identified several different STR s scattered throughout the human genome I Different people may have different quotallelesquot of an STR where each quotallelequot is different number of copied of the same short sequence o If STR differs between people it differs in the number of times it is repeated I Primers used 0 Multiple STR s Produce a DNA Profile I If it is a perfect match of 10 or more STR s the chance that it is from different people is very slim but there is a still a chance I The more STR s that match perfectly the lower the chance that the DNA samples came fr4om different people 0 STR s and Paternity I A child s DNA is a mixture of the parents DNA The children get each one of their quotlinesquot from one of their parents I Only 2 STR s can t know for sure 100 if they are the parents Though we might be able to tell with certainty who is not 0 Embryonic Stem Cell Research 0 Embryonic stem cells can be turned into any type of cell based on the culture conditions growth facts and signaling proteins 0 Embryonic stem cells are specialized cells that have the ability to form virtually any cell type that comprises the human body 0 Where do the embryonic stem cells come from All human embryonic stem cell lines that are being used for research today were isolated from donated surplus blastocyst stage embryos produced by In Vitro Fertilization procedures Sperm and egg In Vitro Fertilization occurs in a petri dishgtfertilized egg all to develop 57 days in a petri dish blastocyst early embryo9cultured embryonic stem cells the removal of inner cell mass cells will destroy the Blastocyst Blastocyst 0 A blastocyst is a microscopic group of cells that is small enough to fit into Roosevelt s eye on the face of a US dime Potential embryonic stem cell therapies o Cultured embryonic stem cells specific culture conditions specialized nerve cell transplant specialized cell into patient 0 Ex Parkinson s Disease 0 Common neurodegenerative disease 0 Caused by los of specific neurons 0 Technique worked in mice lack the same type of neurons 0 Ex Diabetes Heart Disease and Spinal Cord injuries in mice 0 Could help cure a variety of diseases that all involve defects in or loss of a specific cell type 0 Adult Stem Cells 0 Some adult tissues contain stem cells Adult stem cells are held in reserve until tissue sustains damages or cells need to be replaced Adult stem cells are tissuespecific they can only give ride to cells of tissue they are found in Adult stem cells can be isolated from some adult tissues or organs grown in culture and induced to form a limited number of specialized cell types Although keeping adult stem cells alive in culture is difficult and they are not as easily directed to form specialized cell types outside the body Muscle blood bone marrow skin gut liver brain 0 Bone Marrow Transplant 1 P9 5quot Collection bone marrow is collected from donor Processing stem cells are isolated from bone marrow grown and concentrated Cryopreservation stem cells are frozen until needed for transplant Chemotherapy patient undergoes high dose of chemo andor radiation to destroy all cells in bone marrow including cancerous cells Infusion thawed stem cells are infused through IV 0 Embryonic Stem Cells vs Adult stem Cells Embryonic Stem Cells Adult Stem Cells 0 Can give rise to any cell type in body 0 Can only give rise to cells of a single tissue 0 Relatively easy to isolate and grow in 0 Rare difficult to isolate and grow in culture dish culture dish 0 Must be matched to patient can be 0 Immunecompatible will not be rejected rejected by patient s immune system by patient immune system if obtained from patient 0 Challenges for Clinical Use of Stem Cell Therapies 0 Need to generate large numbers of cells to transplant 0 Translated cells must be matched to recipient to avoid rejection 0 Translated cells must survive integrate into the surrounding tissue and function properly for the rest of the recipient s life 0 Procedure and transplanted cells must not harm recipient in any way 0 Clinical trials for stem cell therapies using embryonic or adult stem cells in human are in the early stages 0 Cloning o Frogs1950 s o quotDoy 1996 o Stomatic Cell Nuclear Transfer SCNT 1 Get the unfertilized egg from donor and remove the nucleus using a quotgunquot 2 Get the stomatic cell from the individual that you are cloning culture stomatic cell anything other than gamete 3 Electric pulse stimates the fusion of the stomatic cell with the unfertilized egg 4 57 days later the culture fused cell so divides forming blastocyst 5 Implant the blastocyst into uterus of a surrogate mother 6 Surrogate give birth to the clone of the stomatic cell donor 0 Reproductive Cloning I Inefficient o Fused cell often dies before implanting into surrogate o If survives to be implanted often dies during gestation o Took 277 tried to get Dolly I Problematic o Clones that survive to birth are often born with severe defects 0 quotsuccessfulquot clones often have hidden defects like premature aging 0 Therapeutic cloning I Cultured embryonic cells removed from blastocyst genetically identical to dono o Is therapeutic cloning needed to obtain genetically identical embryonic stem cells In nov 2007 two teams of researchers reported they had turned adult human skin cells into what appearto be embwonic stem cells This reprogrammingwas achieve by adding4 genes to the skin cells that encode specific activator and repressor proteins So farthese reprogrammed skin cells referred to as induced pluripotent stem cells iPS appearto be equivalent to embwonic stem cells If additional experiments confirm this then will not need to generate early embwos to obtain genetically identical embwonuic stem cells Induced Pluripotent Stem Cells An alternative to Therapeutic Cloning nnlnu smn mmapmiwm m nus ia n iF Scellsl i i r llmwulinulll 7 l l m u mm mm nutka we r lRenovlm5aciassnlvimses Mm Wm mm mm mm DNA by veve m I MM au mm quotnew Wm verkumnml UN allhe haslcsll Relmvims used is madman so man my resull of Median i5 is m mum mm am mm m and DNA who 0 has new m m quotml 9mme mm mm m i mu39vuml mm mm W m calls urs cells 0 Challenges for Clinical Use of iPSbased Stem cell therapies Gene therapy 0 Potential treatment for 39 Need to confirm that iPS cells are absolutely equivalent to embwonic stem cells Need to find a way to introduce genes that encodes the four activator or repressor proteins that does not use retroviruses Need to find a way to quickly and easily produce large numbers of iPS cells from individual patients orwill not be cost effective Hum recessive quot by a mutation in a single gene 0 Involved the introduction of a normal copy of a gene into the cells of a patient who has two diseasecausing alleles ofthis gene 0 While there has been some success in clinical trials there have also been setbacks and gene therapy remains highly experimental 0 Two basic treatment approaches are used direct delivew and cellbased delivew Direct delivew of gene therapy 0 EX Cystic Fibrosis o In limited clinical trials this treatment has been successful but CF symptoms have only been alleviate for a few weeks 1 Modify a cold virus 2 Insert DNA for normal human CFTR gene into viral DNA 3 Introduce recombinant viruses to patient39s respiratow tract 4 Recombinantviruses enter respiratow cells and deposit CFTR gene so that cell produces normal CFTR protein 0 Cellbased delivew of gene therapy 0 EX Treatment for SCID o SCID Severe Combined Immune Deficiency is a rare disorder in which a child fails to develop an immune system 0 There are several forms of SCID each caused by a mutation in a single gene 0 2 common forms L Cells are removed 2 In me Iabnrawrg ram palieni a virus is Altered 50 um it cannot r repro ce 7 m genellcailg mend cells malice me desired pralein 3 gggm m the virus 5 s The altered cells are 3 in jeciea into me yaliem 391 The altered Virus 15 mixed with cells from the patient 5 The cells from the patient become geneeicallg alienea 1 non um ml 39 WW 39 sum ml all patients doing well 39 39 Ru nns 117 palienis had developed leukemia 0 Completely Hypothetical use of gene therapy 0 Current technology only allows doctors to camout genetic tests on early embwos provided they were generate using In Vitro Fertilization techniques 0 Can remove one cell from an 8cell embwo isolate the DNA and use it for genetic testing Could then select healthy embryos for implantation into mother39s uterus 0 quotDesigner Babies where specific traits are manipulated or enhanced remain in the realm of science fiction parents with a genetic disease quot fertilized egg with g a defective gene embryo with a genetic defect cell removed up and cultured therapeutic treated culture A t 39 39 539 viral vector genetically corrected e99 Ge WithOUt a nucleus cell from culture genetically corrected egg cel lt o gar genetically corrected clone of the original 39 embryo healthy baby