Genetics Exam 5, Final
Genetics Exam 5, Final 85033 - GEN 3000 - 002
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85033 - GEN 3000 - 002
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This 28 page Study Guide was uploaded by Toni Franken on Monday April 25, 2016. The Study Guide belongs to 85033 - GEN 3000 - 002 at Clemson University taught by Kate Leanne Willingha Tsai in Summer 2015. Since its upload, it has received 168 views. For similar materials see Fundamental Genetics in Biomedical Sciences at Clemson University.
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Date Created: 04/25/16
GEN 3000 – Final Exam Exam 5 2016 Study Guide Dr. Tsai, Clemson University Exam 5: Chapters 17 – 19, 21, and 22 Modeled around Dr. Tsai’s recommended subjects to study – color coded/organized by chapter. Chapter 17: Recombinant Technology General Chapter 17 information: o Some examples of results of Recombinant Technology: Jellyfish fluoresce naturally under UV light. Genes from them have been used to create other fluorescent animals. A green monkey was created for Huntington’s disease studies. A pig with green in hooves and nose was made to study stem cells A cloned cat with the addition of red fluorescent protein. Ruppy cloned puppy with red florescent protein Brainbow – three different colors added to neural tissue – neurons turns out a different combination of colors. o Transgenic Animals: Culmination of recombination technology approaches. o Recombinant DNA Technology: Without it, we wouldn’t have the work we did in crime scene forensics, transgenic plants/animals, pharmaceuticals, whole genome sequencing. All of this also falls under the umbrella term called Biotechnology. o Biotechnology: “Any technological application that uses biological systems, living organisms, or derivatives thereof to make or modify products or processes for specific use.” First really identified in 1971 with the identification of restriction enzymes. Restriction Enzymes: Also called Restriction Endonuclease (RE) o They recognize a specific nucleotide sequence called a restriction site, where they make a doublestrand cut. We can isolate these enzymes and use them to cut whatever DNA we want to cut. There are different types, but we focus on type II that cuts within the recognition site it recognizes specific bases. These restriction enzyme sites are small, about 4 – 8 base pairs long. They’re usually palindromic (In the English language, the same order of letters forward or backward). In DNA, we’re looking at double stranded DNA. Read 5’ to 3’ on each strand – the strands are palindromic to each other. HAVE TO READ THE COMPLEMENTARY STRAND. Example: (3’)GGATCC(5’) (5’)CCTAGG(3’) o When the enzymes cut, they have a defined spot where they cut – varies according to the enzyme. Some enzymes cut early or late in the sequence. In the above example, an enzyme might cut between the G and the A on the 3’ end of each strand. This leaves overhanging pieces of the strands, creating “cohesive” or “sticky” ends. This leaves bases that are able to form hydrogen bonds with another strand. Other enzymes cut straight across the middle of the restriction site, which gives you a “blunt” end. (Such as AluI). o Restriction enzymes do not care whether they are cutting bacterial DNA, eukaryotic, mouse, human, etc. If they see their recognition site, they can bind and cut. This means we can cut DNA from 2 different sources (for instance, a mouse and a human) with the same restriction enzyme, and we’ll get complementary enzymes. This allows us to combine DNA from more than one source. Vector properties: Gene Cloning – We can take pieces of DNA, and amplify them. We often use bacterial DNA to make a lot of copies of the genes in question in a very short period of time. o We have to have a way for this “foreign DNA” to get into the bacterial cell, and be stable in the cell (not destroyed by the cell). So, we use cloning vectors. If the DNA can’t replicate within the cell, we have a problem. o We often use plasmids as vectors. They replicate independently of the bacterial chromosome, and we can construct them to have all the traits that we need. Plasmids have an origin of replication. The plasmids chosen to use as vectors often have certain characteristics. Ampicillin resistance (or other antibiotic resistance): This allows us to determine if the bacteria actually underwent transformation successfully. Resistance markers. Multiple Cloning Sites: We don’t want a certain enzyme to cut everywhere else in the DNA also. Our fragment of interest may have a different cloning site (restriction enzyme site) than another piece. A plasmid with multiple recognition sites gives us options. Allows us to pick and choose based on fragment of interest we want to incorporate into the plasmid. o How can we use plasmids in gene cloning: We take our DNA of interest, cut both the DNA fragment and the plasmid using the restriction enzyme to get the plasmid to open up, and then bring in the fragment of interest. Hopefully the fragment of interest is stably incorporated into the “sticky” open ends of the plasmid. If this fragment was placed into the plasmid, ligase will come in and seal it into the plasmid. If the fragment doesn’t get incorporated, the plasmid may re seal upon itself. This is why the resistance marker, and other markers, are important. Blunt ends don’t do this as well. The nice thing about blunt ends, however, is that you don’t need the same enzyme to cut and paste. Efficiency drops, but you have a wider variety of possibilities. We like sticky ends better – more efficient. o We now need to know if the plasmid actually took up the fragment of interest – Blue/white screening is a common screening for this. Often use the lacZ gene – if it is present, it will cause a blue pigment to be made, which indicates that the additional DNA was not added, and the vector closed back on itself. If the new DNA is present, then it interrupts the lacZ gene, and blue pigment is not made. o There are lots of different vectors you can use. Will not be asked specific questions about the different vectors. The goal as they created more vectors was to increase the size of the insert. 15000 base pairs wasn’t big enough. Need a vector that will hold more, and more, etc. BAC (Bacterial Artificial Chromosomes) will be studied further later. o The eukaryotic system is more complex. We can amplify a piece of DNA, but you can also put in a gene that is intact so that it will both make lots of copies of the DNA, but so that it will also express that DNA. o Expression vector – allows protein production – has additional sequences that allow transcription and translation to occur – prokaryotic and eukaryotic host cells. Have to consider that if you need it to express a eukaryotic gene, you need it to get spliced to get introns out and leave the exons. Need to know regulatory things – promoters, etc. A lot more things to think about if you want the gene to be expressed. o Ti plasmid (tumor inducing) – allows you to get genes into plants. Ti plasmid will actually let you get foreign DNA INTO THE GENOME OF THE PLANT. Unlike a bacterial plasmid, which stays separate from the chromosome. NOT GOING THROUGH HOW THIS WORKS. DNA Libraries: Collection of clones containing all the DNA fragments from one source, which may be a tissue type, cell type, or an individual. o Types: Genomic Library: Collect DNA, collect a blood sample for instance, and you cut the entire genome into tiny fragments, and try to get all of these fragments cloned into host cells so they are available to you to go back and query. Lots and lots of clones. This should include everything from telomeric sequences, centromeric sequences, genes, exons, introns, regulatory genetic sequences, etc. Theoretically, regardless which of the tissues from a person you use, the genomic library should all be the same from a single individual. THEORETICALLY. May be mutational differences. Should only have to make one – one library should be able to tell you everything you should be able to know. cDNA library: very different – some people call it copy, or complimentary. Start with RNA as our starting material, which is not as stable, so we reverse transcribe it to get the DNA. With cDNA, since we started with RNA, we only have what is being transcribed at that point in time. Snapshot of what is happening in that cell at a particular time. All different tissues will give a different cDNA library. Different genes get expressed in different tissue types at different times. The goals of both libraries are that we don’t just have one strand of DNA. The hope is that the many fragments are all represented multiple times within this library. How do we use this? o How to make – basic: See above for specifics: Genomic: Take DNA, split into fragments, clone into host cells where they can be replicated. Contains clones of every piece of DNA. cDNA: Take RNA present at some point in time in an individual, reverse transcribe it to DNA. o Use: In order to use a DNA library, the library must be screened. How do we figure out where our sequence of interests are within an entire library? We can take a little piece of a gene of interest, and see if it exists in the library. Need a probe: This probe must be complementary to our gene of interest, and must be marked so we can find it. Can be DNA or RNA – the probe is complementary to what we’re looking for (hybridization – will stick together). How do we use this probe? Have library where we have thousands and thousands of bacterial cells that have a fragment of that DNA in it. We can take many plates of cells growing (on giant square plates, not what we use in lab), and we want to use a similar process to replica plating. Have a membrane transfer (usually nylon), which we want cells and DNA to stick to. The cells are then lysed, and the DNA is denatured. We then add in the probe, and see where it hybridizes. The probe has to be labeled so we can detect it – fluorescent, xrays, etc. This colony on the ORIGINAL plate has live cells that contain a DNA fragment that holds onto our probe – holding a fragment of our DNA of interest. PCR: Polymerase Chain Reaction: Process discovered by Kary Mullis (probably under the influence of LSD, but it still won him a Nobel Prize) that takes everything a cell does for replication to amplify and clone genes we want to use. This is done without bacteria, which speeds up the process. This is a very powerful process, but is limited to what you know about the DNA you want amplified, and must be careful to avoid contamination, because PCR cannot distinguish between the desired gene and a contaminated one. In addition, PCR may be more errorprone because polymerase is outside of the cells. PCR can also only really accurately replicate short strands of DNA, though this is improving with time. o What is needed: Gloves: To avoid contamination of the DNA. Single Stranded DNA (ssDNA) – The DNA that you want replicated has to undergo heating to dissociate it into single strands. This acts as the template to the gene we want to amplify. Primers: Required to allow for identification of the target gene to be amplified, and provides a 3’ OH end to replicate off of. These are typically human designed to direct polymerase where to go. Nucleotides: The required building blocks of any DNA strand. Polymerse: Required enzyme to synthesize DNA. Typically use a polymerase from bacteria that can live in high temperatures. Before they used the thermotolerant polymerase, more enzyme would have to be added to every single round of PCR due to the denaturing step breaking it down. The thermotolerant polymerase was discovered by Tom Brak. o Steps: The three steps of PCR are performed repeatedly to achieve rapid accumulation of the desired DNA strand. Every time a round is completed, there should be a doubled amount from the round before it. Denaturation: Double Stranded DNA template must be separated through heating so that it can be used for replication/amplification. This usually occurs at the highest temperature of the process. Annealing: The primer must be bound to the nowsinglestranded DNA. Occurs at the lowest temperature in the process. The low temperature is required to allow hydrogen bonds to form between the primer and the template strands. Extension: Synthesis of new DNA strands through DNA polymerase. Occurs at a medium temperature of the process. o More information about PCR: Reversetranscription PCR: You begin with an RNA template. You start with a first step of reverse transcribing – going from RNA to DNA, then you can use that to undergo the full process of PCR. Realtime PCR: Measure gene expression levels. This lets you monitor how quickly things are amplifying in the tube in real time. Goes through the cycle, takes a picture, and tells you how much DNA is there. Monitors amplification in each cycle. Originally used to measure gene expression levels – if we want to monitor how much a gene is being expressed, we can measure how much RNA of that genetic sequence is going to be present. There should be lots of mRNA strands to act as a template. After 1 round of amplification, every present strand can act as a template and give us our product at the end of the cycle. We expect, if there is more gene expression, the level of highest product production will be reached more quickly. If there is a low level of expression, then it takes longer for the max product to be produced – start with fewer transcripts. o This is also being used for copy sequence – how much copy you have. Also use for heterozygosity analysis. The more copies there are in the genome, it will resemble high gene expression. Low number of copies will express like low gene expression. o These PCR methods have been incredibly useful in various scientific parameters. Diagnostically, they are used for genetic testing, identification and forensics, parentage testing. Did not go over tiny details of the picture – don’t worry about for the exam. Gel Electrophoresis: A laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores. o Size: Larger fragments move slower, don’t get as far. Smaller pieces go further. Use in conjunction with PCR. Small move faster to the bottom. Large fragments stuck to the top, closer to the well. Blots (Southern, etc.): A blot is essentially a modified form of Gel Electrophoresis. We know the fragment of interest, and it may be found in several genetic libraries. We can look at different organisms in the same species, and see if we have the same thing across their genetics. o What is the source: Southern Blot: Created by Edwin Southern utilizes DNA. The DNA gets cut up with restrictive enzymes, put into a gel, and separated by size. Each lane can have a different bunch of information. Can then take the DNA from the gel, stick it to a membrane, and use a radioactive probe (DNA or RNA) to add to the membrane to see where it sticks. We can use this to compare what is different from one sample to the next. Northern Blot: Same process as the Southern blot, but with RNA as your source. Western Blot: Similar process (not quite the same), but using protein instead of genetic material. In situ Hybridization (ISH): Have a probe that sticks to a specific spot on a specific chromosome, which can be used to determine chromosomal location. o If labelled with florescence, we have FISH (Fluorescence ISH). We can use it to probe tissues for a particular mRNA, a particular chromosome, or RNA expression. For instance, through the different developmental time points of an organism’s life, we can monitor gene expression. One gene may be expressed differently as an infant than as an older individual. o Chromosome painting (spectral karyotyping). Can make multiple probes – very useful in identifying translocations. Can also get a banding pattern to see the different gene sequences. Big deal in cancer genetics, determination of chromosomal counts in pregnant mothers. Can use chromosome paints, and apply them across different species. Called ZOO FISH. Sanger Sequencing: Uses dideoxyribonucleoside to enable us to determine the actual sequence of nucleotides. We want to be able to go in and say, at this point we have an A, then a T, then another T, then a C, then a G, etc. Want to be able to know the genetic sequence and identify the bases. There were two main methods used. Sanger Sequencing became the biggest. o Have a regular Deoxyribonucleoside – has a 3’ OH to bring in the new base/primer. A Dideoxyribonucleoside lacks the 3’ OH group. This removal of the oxygen will prevent elongation from continuing after it. For instance, if there was a normal sequence of CCATG, and at the A, a T dideoxyribonucleoside was added, that is where elongation would stop. KNOW HOW TO READ A SANGER SEQUENCE GEL!!!!!!!!! o Reading a Sanger Sequence: In this process, we have a normal template, normal primer, normal DNA polymerase, all in one tube. We take that tube, and split it into four different tubes. In each one, we add Dideoxy versions of A, C, G, and T. We allow for Anealing and extension to occur. Because we’ve added in the dideoxy versions, the polymerase may add in either the deoxy or the dideoxy version of a base. If it adds in the dideoxy, elongation will stop due to lack of OH group. We are able to use each individual tube, and the varying lengths of the DNA that stop elongation due to the dideoxy bases, and see where each piece falls. We know that each segment ends with whatever the dideoxy base we added in was. DNA has to be made in the 5’ to 3’ direction. The very first base that the polymerase puts in is on the 5’ end of the fragment it is making. Dr. Tsai could ask what a sequence was based on a Sanger Gel. Or, she could ask what is the sequence of the TEMPLATE. Have to make it antiparallel and complementary to the strand it made. (The 5’ end is on the bottom of a gel if you’re reading the sequence of the created strand). o We can use fluorescence in addition to the use of dideoxy bases, separate by size, and see what happens. o Nextgeneration Sequencing: Beginning to develop newer and newer chemistries to expedite genome sequencing. Not going into the details of this. We have recently started with third generation sequencing, but is not as readily used at this point in time. Chapter 18: Genomics –omics definitions: There are a wide array of –omic fields created regularly, but the following definitions are those most pertinent to our studies. o Genomics: The field of genetics that attempts to understand the content, organization, function, and evolution of genetic information contained in whole genomes. o Structural Genomics: Studies the organization and sequence of genetic information contained within a genome o Bioinformatics: Emerging field consisting of molecular biology and computer science that centers on developing databases, computersearch algorithms, gene prediction software, and other analytical tools. We often rely on bioinformatics to compare results and evaluate findings. Genetic Maps vs Physical Maps: o Genetic maps: Based on recombination frequencies. More of an estimate. o Physical Maps: Based on the direct DNA sequence – the exact location of the bases within a genome. This is not an approximation. However, Sanger sequencing is pretty limiting – typically can only do about 500 bases at a time. Would take a great deal of time and work to evaluate a whole genome. Mapbased sequencing: Useful for repetitive genetic sequences. Shotgun approach was much faster, but had gaps in repetitive sequences. Wholegenome shotgun sequencing Whole Genome Sequencing: o Human genome research project: International project to unlock the entire human genome. Decided to use a mapbased approach. They decided not to go in randomly and sequence only 500 bases at a time using Sanger Sequencing due to the time commitment that would require. o Mapbased vs Shotgun: Mapbased: They made a map of the genome using molecular markers, using genetic maps with genes in order, and began attaching them to the physical location of genes along the chromosome. This was very time and labor intensive. Many beginning years were spent making maps. Molecular markers acted as road signs that told them what part of the chromosome they were looking at in any given point in time. They separated chromosomes to just look at one at a time. They would take a single chromosome and cut it up with restricted enzymes to get fragments. They lined this up according to maps. Started out with larger sequences, and they’d get really big pieces of DNA, and made a library specific to the chromosome. Then they took the processes of screening, and used those road signs to see what clones had the sequence they were looking for. Able to go through and put clones in order along the chromosome. The benefit of this approach is that you automatically could order clones along the chromosome. Were able to anchor the map information – anchored each clone – useful with repetitive sequences. Shotgun approach: Doesn’t use maps – An original researcher on the genome project didn’t want to use maps, so he developed his own method. It became a race to see who would finish first. The shotgun approach was developed where you separate the chromosome out and sequence it. Went straight to cutting up the fragments, and put them back together. Did it first with a virus that showed it worked. It turned out that he needed bioinformatics. Relied heavily on a computer. Contigs are overlapping fragments – look and see where one 5’ to 3’ fragment lines up with a 3’ to 5’ piece. Relied heavily on computer programs that could go through and find the overhangs and make sure they matched up correctly. Both approaches were relatively successful. By the end of the game, both groups of people were using information from each other. The genome research project switched and began using more shotgun approach. Where it is repetitive, the mapbased approach is much stronger. This was a huge undertaking. Had rooms full of sequencing machines. Probably had 384 different sequencing reactions in each plate. The human genome research project pushed bioinformatics, improvement of machines, etc. Genome Comparisons: Compares similarities and differences in gene content, function, and organization among genomes of different organisms. o Prokaryotes – the amount of DNA ranges from 490 kb to 9105 kb. There is about 1 gene per 1000 base pairs in prokaryotic organisms. If there are about 4000 bases, there are probably about 4 genes. The bigger the genome, the more genes prokaryotes typically have. o Eukaryotes tend to have larger genomes than prokaryotes, but there is no relation between genome size and complexity. You CANNOT make any assumptions based on genome size. Eukaryotic genetics tend to keep genes that maybe aren’t necessary. Eukaryotes are more ok with polyploidy – many copies of genomes. As people began looking across eukaryotic genomes, you can’t really assign numbers of genes to numbers of base pairs. This is because there are gene deserts – areas of the genome that have no known genes. There are some gene rich areas of the chromosome, but some areas that have no known genes. The point of these have been hypothesized in multiple ways: may be remnant of chromosomal rearrangements in the past, might just not be able to be scientifically understood. Transposable elements: We have approximately 20,000 genes in the human genome, but it was estimated that we had close to 100,000 because of how many proteins we make. Alternative splicing, alternative cleavage sites, and protein modifications after translation are all used to have only 20,000 genes, but make over 100,000 distinct proteins. o There are often groups of genes that we don’t necessarily know what they’re doing, but we know they’re involved in particular processes. o Can also use comparative genomics to look at all different animals compared to the human genome and see sequence similarity. There are a lot of forms of conservation of protein coding genes. Start looking at how genes are expressed. Depends on what’s between the genes, how expression occurs. o Most mammals seem to have around that 20,000 gene mark. o There aren’t as many differences between very different creatures as you might expect. Intron length seems to be correlated to complexity. Humans seem to have more introns that are much longer (in base pairs) than worms, flies, etc. This might be due to enhancers, silencers, and other genes used for regulation purposes. It might be that intron length could increase alternative splicing. The purpose is unknown at this point. o Human genome: don’t have to memorize the table. With 20,000 protein coding genes, we use about 2% of our genome to make proteins. We have so many regulatory elements that are essential to our genome, even though they don’t directly make proteins. When the human genome was sequenced, they also created a reference genome. A reference genome is a compilation of genome from multiple animals of a certain species. We can now use the reference genome to tell us about every single human’s genome. If you compare between individuals, the genome is 99.9% the same, regardless of characteristics. Most of those differences, that .1%, comes from molecular markers, such as a single base change. Microsatellite – different lengths of repeats. Those differences aren’t even in the protein coding genes. o Functional Genomics: Looking at what genes actually do. o Trancriptome: All the RNA molecules transcribed from a genome. o Proteome: All the proteins encoded by the genome. o While we know a good portion about genes, the largest part of the genetic sequence is unknown. Expression arrays – goal: o Microarrays – Monitor gene expression in different tissues or under different conditions – is a gene expressed in a certain tissue at a certain time, to a certain level. This procedure is very much like the northern assay, but on steroids. Have a glass slide – instead of asking about a single probe at a time, put probes in specific locations for thousands of different genes. You’ll still use nucleic acid hybridization – the complementary strands of DNA and RNA want to stick together. If there is a black dot on the slide, no probe showed up in that spot. Higher glowing = higher expression of RNA – more of the probe stuck. You’re still asking what is being expressed, but you’re asking it about thousands of genes at one time. This can let us look at gene expression related to diseases. Competitive ones can be used for cancer analysis. There are lots of questions you can ask with different types of experiments. The basis, though, is if the gene is being expressed and at what level? o Proteomics: Study of the proteome (complete set of proteins found in a given cell. For proteomics, there are many different ways you can do this. Mass spectrometry helps you identify what proteins are present in a cell or tissue type. Can use a twodimensional gel electrophoresis – separates proteins based on their charge (isoelectric point) and then size. Mass spectrometry might be weaker due to the modification of proteins. Can use both methods to compare between tissues, find what’s the same and what’s different, and identifying what is there at all. There are Protein microarrays: Can be used to analyze protein protein interactions. You can put antibodies on the slide and analyze many proteins at a time. o So, how does this all work together? Take a systems biology approach – make bigger connections – can miss some relationships if you focus in on the details so closely. Bioinformatics is really useful here. It takes tons and tons of data. How good can you make your program? Can it make connections based on what you’re telling it? Chapter 19: Biotechnology Genetic Engineering: Altering an organism’s genome – makes the creature a Genetically Modified Organism. Biopharmaceuticals: When talking about biotechnology, pharmaceuticals have benefited strongly from this. Insulin was first human gene product manufactured by recombinant DNA technology. Before this was developed, pig insulin was often what was used, and many people developed a reaction to it. So, researchers set out to modify bacteria to create human insulin to reduce this issue. o Humulin – approved by FDA In 1982. Originally, insulin required two strains of E. coli to produce the human insulin, and put those two products together to form the marketable insulin product. This isn’t the same process done today. o Biopharming is the production of therapeutic proteins in GMOs (involves animals and plants). There can be limitations to protein production in prokaryotes. Using transgenic animals is usually done to produce the human version of a product. One of the things they’re trying to do for a lot of protein product, they try to get the protein to be expressed in a specific way. Such as having protein expressed in the milk of a cow so that you can simply milk the animal, and get the product noninvasively. o Many vaccines are considered a part of biopharmaceuticals (attenuated/weakened or killed) – exposure so that your body can recognize and amount a more potent immune response. People suggested only taking one or a few proteins of the pathogenic organism, and inject them into people to eliminate all the extra stuff you’d be exposed to if injected with the whole organism. This is called a subunit vaccine: HPV vaccine is an example. There are still often issues of getting the vaccine to certain places, and we can have trouble keeping it stable. So, people are now working on an edible vaccine. Trying to get into food source, the production of the single or few proteins. For instance, get proteins expressed in a banana, so when someone eats the banana, they become vaccinated/immunized against the pathogen. This enables easier transportation, and bigger stability. You have to start thinking about things like dosage. Will amounts be variable across fruit pieces, how many bananas would someone need to eat, would it be different between children and adults. o There are genetically engineered biopharmaceutical products available to the market, or are under development and getting close to that point. Agricultural biotech: With genetic engineering, it is important to realize that we were doing genetic engineering before recombinant genetics existed. We’ve been selectively breeding plants and animals for years. The very first time we interfered, we started modifying the genomes. It just took much longer than recombinant genetics. Designed crosses produced corn. It is important to know that these selective breeding practices have been very beneficial to humans. Can get more efficient food, produces more, takes less time. Selective breeding is efficient, but it takes more time. Recombinant technology is the idea of skipping the years in between. o We can improve growth characteristics and yield, and increase nutritional value of produce. Golden rice is a good example. Areas that have a heavy reliance on rice in the diet often have Vitamin A deficiencies, especially in children. It can lead to blindness. Therefore, golden rice was developed – has higher levels of beta keratin, which is the precursor to vitamin A. Transgenic animals: Most transgenic animals have been made to study a gene to figure out how changes to the gene will impact the phenotype in the animal. It is less of creation for agricultural benefit like there is in plants. We would like for the permanent change to the DNA to be in the germ line. Originally, transgenic animals were attempted to be created through microinjection. However, this was a very difficult process. Only about 10 – 30% of the eggs survived the injection of the copies of a transgene into its pronucleus. It is very labor intensive, and takes a lot of time. This was used to prove that the SRY gene is a maledetermining factor in mammals through using mice. o Definitions: Transgenic: the genome has been permanently changed by the addition of DNA. We have added in something that wasn’t in the genome before. You can also take genes out, but it is more commonly an addition. It MUST be a permanent change. Transgene: Itself, the foreign DNA that gets stably incorporated into the genome. The foreign DNA in a transgenic organism. We have made crops that are resistant to environmental changes, herbicides, resistance to insects. o Knockout vs –in: As this process has gotten better and more refined, we went from hoping that the transgene would get passed to every cell in the germ line, to being more specific. Knockin: This is another specific method – want to get a specific sequence in place. Want to exchange the transgene for the gene that is in the genome. This is often looking at a specific allele. Example is replacing a mouse allele with a human allele. Goal is to see how a human gene would respond to a testing method. Knockouts: Targeting a specific gene in place. Want homologous translocation to take place. The point is to remove the gene, disabling it. What is that gene supposed to be doing that it can no longer do that activity. o Examples: Use of Ti plasmid: It stably incorporates into the genome. This means that whatever is put into the genome is the transgene, and the organism it is in is a transgenic organism. Roundup: Have plants that are resistant to roundup so you can treat your plants with roundup so weeds will die, and the plants you want to survive will do so. Golden rice: Made to improve nutritional value. Transgenic Atlantic salmon: Overexpress a growth hormone gene – weigh an average of 10 times more than nontransgenic strains. Usually this process isn’t very successful, but it was in Salmon. Grow much faster and larger. There have been transgenic cows (not in main stream food production): Mastitis is a big issue in the dairy industry. Mastitis causes about 2 billion in losses to the dairy industry per year. The major cause of mastitis is Staph aureus, and it causes a lot of inflammation and tissue damage. So, there is a protein called lysostaphin that kills staph aureus. Transgenic cow mammary gland expresses this protein, which kills the S. aureus. Builtin antibiotic. Also have daisy: transgenic cow that produces hypoallergenic milk. Also have “can we make this milk more nutritious?” Talk about making a cow without the prion gene – cannot develop mad cow disease if they aren’t making that protein. If we can make cows resistant to other pathogens – removing possibilities of bioterrorism. Transgenic Pig: EnviroPig – can break down dietary phosphorous, cuts down on smell, and better on the environment. GloFish: First transgenic pet approved by the US. They are little fish that glow. These started out for a different reason. The goal was to use them for a bioassay – hook the color up (red fluorescent protein for instance) to a promoter that responds to a heavy metal. So, if there was concern that water had been contaminated with heavy metals, the fish would have their color gene activated, and the fish would glow if contamination was there. Molecular Markers: o Uses: Biotechnology has led to the development of many different tests, and is used to carry out a lot of different genetic tests. This often involves genetic markers such as RFLP, VNTR, and SNP. These are spread throughout the genome, and can act as little road signs. o Types: RFLP: Restriction fragment length polymorphism. These are changes that happen in the DNA sequence that modify restriction enzyme recognition sites. There are two recognition sites from the enzyme. In a person, if there is a genetic mutation, one site is no longer recognizable, so the enzyme will not cut that segment. If we have identified the base change (for instance, a base change that causes a muscular disease), can set up PCR that amplifies only that region. If it cuts twice, then we get 3 fragments. Just once, get a big and small fragment, we know that person has that base change. This lets us know that the mutation has occurred without using sanger sequencing. VNTR: Variable number of tandem repeats – differences in copy number. SSLP, Minisatellites, and Microsatellites are all examples. SNP: Single nucleotide Polymorphism – a single base change is made. Modified gene expression microarrays – can do types of testing, where we make it competitive. Have a normal cell, label RNA one color. Tumor cell, label its RNA a different color. Put both on a slide together, and see where color occurs. Only normal cell, only tumor cell, or if both express it equally. Expression rates can also be used to take time points – say a mouse is exposed to a pathogen. We get a signature of what genes are targeted, and which genes get expressed. Trying to understand what is making someone sick after exposure, it could be a long process. This could severely delay treatment, or make treatment less specific. The use of this expression assay can narrow down pathogen possibilities, and narrow treatment options. Can also start saying, when we’re exposed to this, we need to get these genes up and going. So, we need those gene products, so can we give those products to use as treatment to jump start the process to get rid of the illness? GWAS: Genome wide association studies. o On the news, you might hear of new regions of genomes associated with autism, or Alzheimer’s. This means we are using molecular markers spread throughout the genome (which we know the location of). Typically use SNPs, but can also use microsatellites. This allows us to see, in this location, the majority of people either have this base, or this one. We can go in and probe those. Those probes are used to determine one allele from another. With the genomewide association study, the goal is to find a region of the genome that is inherited in the same way as your genome in question. Do we see all of these gene expression in those affected, and do we not see them in all of the control group. This Does NOT require the use of a genomic library of each individual. Can use those assays. o Can also look at Haplotypes: A group of alleles. SNP #1, SNP #2, and SNP #3 are most often inherited together. Allows us to say that we have these alleles on each piece, and get inherited together from one parent or the other. This is linkage. o GWAS is a very powerful technique. Pituitary Dwarfism in the German shepherd – able to find the causative mutation using only 4 dwarf dogs and 39 control dogs. Able to see a peak in the chromosomes. Looked at 68,426 SNPs. Each SNP represented by a dot. How often do I see that my cases have this genotype, and all of my controls have this genotype? This is telling where to look for a mutation. It is not saying that this snip is the mutation causing it. Through time, as the dogs have been bred, can see an allele marker inherited at the same time as your mutation or your phenotype. Some of these might start being lost due to crossing over. o Manhattan plots – GWAS plots of SNPs look like little buildings – skyline of a city appearance. Variation in humans might be due to the differences in people demographics, and there may be different causes for disorders. o Goal: The point of the GWAS is to tell you WHERE to look. So after that, you have to go in and see where that SNP is located. Is there any mutation around it that could be causing the phenotype? Gene Therapy: o Biogenetics is often used in hopes of curing diseases, not just treating it, through gene therapy. Therapeutic technique that aims to transfer normal genes into a patient’s cells. The hope is that giving one treatment will keep them from having to have routine treatments. With this, you’re not trying to make an entire transgenic organism. Can I get some cells into this person that are making what they’re missing? o Somatic vs germline: Right now, gene therapy is limited to somatic gene therapy, because it will only affect the person undergoing therapy. If it is put into the germ line, it will affect the children. The idea of making this decision for future generations is controversial. In the UK, there is mitochondrial therapy undergoing. Injecting wild type mitochondria into an oocyte to limit mitochondrial diseases. This mitochondrial change will be inherited by a female child. o Also, gene editing technologies have been coming out lately. Scary and cool. o For gene therapy, the first disease treated was SCID. Basically, they have no immune system, they get a common cold, and it is lethal. This disease has been treated with gene therapy. Used a technique where they got some patient cells expressing the missing gene to make the deficient protein, and inject them back into the patient. About 20 children have undergone successful gene therapy for SCID. But, the idea of gene therapy was put on hold for a while due to some of the things that happened with some of the patients. A big part of gene therapy involved viruses – modified those viruses to get material into the cell. There was a group of patients that had their initial disease cured, but they also later developed leukemia due to the viral activation of a protooncogene. One person had a huge immune response to the vector itself – lethal. So, gene therapy was stopped for a while. There have been a few limited studies. People have continued to improve vectors and delivery system. There are over 1000 gene therapy trials that are at, or almost at, clinical trial stage. o Gene silencing using RNA interference – allows the gene to be turned off temporarily. People have started using this to treat macular degeneration – goal is to get that gene to make the microRNA to trigger RNA interference. This is turning off a gene that is being over expressed. Can essential knockdown the overexpression of genes. o There are lots of issues to consider. Much of that is going to fall onto the shoulders of our generation. Crisper Cas – gene editing – new systems where making these transgenic animals could take much shorter amounts of time instead of years, getting it into place could take so much time. Policies will have to be made. Scientific world in general is very good at selfregulating. But that doesn’t mean there shouldn’t be anything in place to help guide. Many politics are based on fear, and we are responsible for looking at what is happening and deciding what is fact, and what is fiction. What should we be making our decision based on? Chapter 21: Quantitative Genetics Quantitative vs. Qualitative: So far we’ve focused on qualitative characteristics – height, color, etc. These are discontinuous characters. These are what Mendel studied. o Quantitative characteristics: Become much more complex. These are called multifactorial – they have many factors involved. Polygenic – result from the action of many genes. We generally see continuous variation within a population. Within a range, you can fall anywhere within it. Classifications: o Discontinuous Characters: Qualitative characteristics. Easy to determine with crosses and calculations. o Quantitative (Polygenic) Characters: Much more complex. You can get the same phenotype from multiple different factors. They can yield similar phenotypes even if the genotypes are very different. This type of character can vary continuously within a population. Additive alleles: When you have an additive allele, you add something on. For instance, adding on additional height, or another dose of pigment. For this, though, if you look at the phenotypes, you might have a plant that is 12 cm tall, you have a whole group of genotypes that could give you this height. o + signs indicate an additive allele. – signs indicate a nonadditive allele, which is not helping your phenotype. A zero may be used, or no superscript at all. o With the environment, when you look at quantitative characteristics, the environment has an effect no matter the genotype. So, if we just look at one particular gene only, there is a range of phenotypes where you can land. The environment will determine where within the range you fall. For example, a plant’s height might change in the range according to if it gets fertilizer or not. The phenotypic ranges of different genotypes can overlap – so how do we tell if the difference in phenotype is due to genetic sequence, or due to environment? o Calculations to determine # of genes/phenotypes: Distributions: o The number of different phenotypes are unlimited with a continuous characteristic (at least between two extremes). Meristic characters – they are quantitative because multiple genes are involved, and there is interaction in the environment. For example, dog litter size. Can’t have 4.5 puppies, you get whole numbers only. o Also threshold characteristics – A disorder is often either present or absent, but there is a threshold at which this happens. For many complex disorders, there is a range in which people are gaining predisposing alleles, but you don’t necessarily see the disease until you get all the way over a certain point. There is a threshold point where we can try to define how something is inherited and affected by the environment. o In the early 1900s, everyone was rediscovering Mendel’s work. When we start looking at models with tons of different phenotypic variation, do Mendel’s models no longer work? Does this apply to every gene in the genome. o NilssonEhle: Worked with wheat kernels that had different pigments. He got very lucky – he was looking at a parental cross that was homozygous with no pigment, and a homozygous that had a very dark purple color. He crossed them together and got an intermediate phenotype. He then crossed those, but instead of getting a defined set, he got a very large variation in pigmentation, ranging from white, to light red, to dark red, the intermediate red, to purple. So, we have some continual variance. He got very lucky that it was only caused by two genes, the effect was additive, the environment had no role, and the loci weren’t linked. Was this happening in the way Mendel described? Yes, because we were still seeing that 1/16 of the resulting offspring were the homozygous parents. Able to break down each color and their ratio to see allele inheritance. The difference is that the genes are working together to give the same phenotype. Each allele is contributing more to each phenotype. If you get two copies of the additive allele, you get two doses of the color. Four doses gave the purple. One dose gave the light red. No doses gave the white. This led to the multiplegene hypothesis. The multiple gene hypothesis: The more genes involved in a phenotype, the more phenotypic characteristics you will see. o First equation: 1/(4 ) where n
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