Genetics Wk 5 Notes
Genetics Wk 5 Notes Bisc 336
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This 10 page Class Notes was uploaded by Anna Ballard on Friday September 30, 2016. The Class Notes belongs to Bisc 336 at University of Mississippi taught by Ryan Garrick in Fall 2016. Since its upload, it has received 20 views. For similar materials see Genetics in Biology at University of Mississippi.
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Date Created: 09/30/16
Lecture 14 9/26 Learning Goals • Describe applications of genetic engineering (GE) to pharmaceutical and vaccine production • …and applications to crop plants (e.g., herbicide and pest resistance, nutritional enhancement) ** will be exam questions about the Research Experiment we read and studied Ch. 19 –> Applications and Ethics of Genetic Engineering and Biotechnology Modified Organisms • Genetic Engineering: alteration of a genome (adding/removing genes) using recombinant technology - E.g., insect resistance/pesticide in crops (Bt cotton) • Biotechnology: use living organisms (and their biochemical reactions) to “manufacture” a product - E.g., synthesis of pharmaceuticals by GE species - Number of jobs in this field is constantly increasing - Allows rapid growth of a product Insulin-Producing Bacteria • Biopharmaceutical: therapeutic proteins produced by an organism (now usually has recombinant DNA) • Insulin: hormone produced by the pancreas, regulates carbohydrate and fat metabstism • …1 human gene product made by a GE organism - Can make large quantities of insulin in a pure way • Insulin has 2 polypeptide chains (A and B), bonded - Originally produced A and B separately and then fused them together • Human genes encoding A and B in different vectors • Inserted genes are next to the bacterial lacZ gene • Transcribed and translated, creates a fusion protein - Promotor region: where polymerase is binded - lacZ is bacterial gene not relevant to producing insulin - insulin gene is important and of eukaryotic origin - white colonies are the ones that have taken up the plasmid - fusion protein – string of amino acids encoded by insulin gene A • Fusion proteins extracted • A/B chains then are separated from lacZ - first step • Subunits spontaneously unite and bond… becomes active insulin molecule - first widespread application of genetic engineering for human health - no risk of passing on diseases Transgenic Animals • Bacteria useful, but don’t always work well - can’t have successful translation of mRNA transcript generated by electrolytic gene when happening with prokaryotic machinery o bacterial machinery for transcription and translation doesn’t always work well when dealing with mRNA transcripts from eukaryotic DNA • Not able process/modify eukaryotic proteins - May have reduced activity or be completely inactive • So can use eukaryotes as “bioreactors/bio-factories” Vaccines from GE organisms • Stimulate the immune system to produce antibodies • Normal vaccines are injections of weak but still living (attenuated) or a dead (inactivated) pathogen - Heat kill a virus – virus still has all its surface proteins • GE organisms now used to make subunit vaccines - Contain only surface proteins of the pathogen - Still stimulates immune response - This approach can slow the rate at which a virus evolves Subunit Vaccines • Gardasil: protects against sexually transmitted HPV, including strains that are the major cause of cervical cancer • Provides immune protection prior to infection, but not against existing infections • FDA recommend vaccination before adolescence, nut some strong political resistance - Political in sense that in some states that STDs aren’t a problem for young kids - FDA argues it’s better safe than sorry - Argument of possible side effects – are birth defects possible? GE and Agriculture • Genetic engineering – bringing gene from different species into an organism • Different phenotypic variants occur naturally • Selective breeding: cross individuals that share a desirable phenotype - Corn –> thousands of generations of selective breeding causes shift of phenotype • Farmers have been doing this for 1000’s of years • Recombinant DNA technology can fast-track what could have been done through selective breeding… • Can also create GE organisms could not have ever arisen via selective breeding, irrespective of duration • GE food plants and/or animals: insect resistance, herbicide resistance, nutritional enhancements GE Crops are Global • > 300 million acres planted with GE crops in 2008 • roughly 800 GE crop trials, mostly corn and cotton Herbicide-resistant crops • Traditional herbicides (e.g., sprays) often kill weeds and crop plants, whereas some GE crops unaffected • Roundup = effective herbicide with nice properties (use in low concentration, effective, rapid degradation) - Inhibits photosynthetic pathways so must be put directly on weeds not the crop • Roundup inhibits EPSP synthase production - Shuts down a gene crucial for photosynthesis • Create Roundup-resistant fusion gene (EPSP syn. From E. coli, viral promotor) - Agrobacterium from soil goes into roots of plants and spreads throughout; genes that reside on chromosome in bacteria are translocated into the main chromosome of the plant’s nucleus o How EPSP is reintroduced into the plant cell • Make recombinant plasmid, transport into crop plant via infective soil microbe - Increase amount of EPSP in plants so that when they are sprayed with Roundup the crops don’t die because they have more of those EPSP - Problem: Roundup is not as harmless as originally thought – female frogs turned into male frogs in fields sprayed with roundup; lots of human health problems as well linked to Roundup - Aka should not be spraying lots of Roundup anyways - GE did exactly what it needed to do but the consequences of the herbicide caused problems Plant Resistant Crops • Bacillus thuringiensis produces a protein that kills some insect herbivores when ingested (crystallizes) • Previously needed recurrent spraying, but now many GE crops have Bt genes (= continually protected) - Cotton growers - Cause of declines in Monarch butterfly (non-target)? o Pollen plants produced travel far and wide but the protein is still on the pollen, killing butterflies due to the GE cotton o Accidental non-target kill Lecture 15 9/28/16 Learning Goals • Describe applications of genetic engineering to agricultural crop plants and livestock • Recall how crossing-over frequency relates to physical distance between a pair of genes Ch. 19 –> Applications and Ethics of Genetic Engineering and Biotechnology Recap • First biopharmaceutical (Humulin) produced by expressing insulin A and B in bacteria… fusion p/ts - Know process of how • Subunit vaccines (e.g., Gardasil), surface proteins only • Herbicide resistant crops… over express EPSP synthase gene, tolerate spraying with Roundup - Extra copies of EPSP gene introduced into genome of crop plants - Negative consequences of overuse of Roundup NOT crop GE • Pest resistant cotton.. express bacterial (Bt) gene which makes proteins that crystallizes in gut of insects when injested - Protected 24/7 because they contain a bacterial gene to protect it from insect attack - Non-target kill of insects that do not try to feed on crop o Monarch butterflies killed off due to pollen that also had the bacterial gene Nutritional Enhancements • Vitamin A deficient human diet in much Asia and Africa, preventable permanent blindness, >500M people/year - Malnutrition is a worldwide problem • Rice irdstaple food in these regions but has no vitamin A - 3 world countries have lots of vitamin A deficiencies • Golden rice engineered to have enhanced ß-carotene (A precursor) - ethical consideration: companies that put a lot of money in research and development want to recoup their expenditures o farmers need to buy this golden rice specifically from one company every time o paten on genes unethical? o Using humans as research subjects for effects of golden rice is not good Transgenic Cows • Mammary glands susceptible to infection by Staphylococcus aureus, block ducts/contaminate milk - Also leads to death of cow itself • introduce lysostaphin gene from S. simulana, expressed in milk - Natural enemy of staphylococcus aureus because it produces a protein detrimental to the growth and production of protein • This enzyme breaks down S. aureus cell wall • Acts as a natural antibiotic in milk, wards off infection - Helps in longevity of dairy cows Genetic Engineering in the News • Species – homo sapiens • Habitat – everywhere • editing genes in human embryos: using CRISPR/Cas for introducing mutations that are resistant to HIV - Shouldn’t really be messing with genomes of humans - Global leaders got together and agreed to not use this CRISPR technology for modification of humans - Some Chinese researchers defy the ethics and experimented by editing human embryos; adding a mutation that damages a gene linked to natural HIV resistance o Moderate success in the possibility of producing fetuses immune to HIV CRISPR-Cas System • Bacteria create a “database” of viral infections and use it as a form of prokaryotic immunization • database: a set of spacer DNA regions on the bacterial chromosome, matching parts of viruses genome • immunization: targets and modifies those parts of the virus DNA sequences (editing), renders it inviable - Releases enzymes that edit DNA of virus that is attacking bacteria, making that virus ineffective - Can specify what kind of editing Ch. 7 –> Linkage and Mapping in Eukaryotes Physical Linkage • Genes on the same chromosome (i.e., basic unit of inheritance) may not show independent assortment (Mendel’s 4 Postulate) • Recombination (the outcome of crossing over in prophase I of meiosis I) can decouple linked genes • Probability of decoupling depends on physical distance between a pair of genes on a chromosome Basic Expectations of No Linkage FIG 7-1a • Genes are on different chromosomes (independent) • Together, gametes have different combinations of alleles from the 2 genes • No crossing-over required What is being represented here? A) 2 individuals, 2n = 4 chromos each B) 1 individual, 2n = 8 chromos C) 1 individual double heterozygote D) 4 pairs of homologous chromos Basic Expectation: Strong Linkage FIG 7-1b • Two Genes on the same chromosome., extremely close • Very unlikely to be affected by crossing-over • Gametes have the same combos of alleles that were seen in the parent Basic Expectation: Weak Linkage FIG 7-1c • Two genes on the same chromosome, very far apart • Very likely to be affected by crossing over • Some gametes have combinations of alleles not seen in the parent - outside chromatids unaffected Linkage and Recombination (FIG a, b, & c on slide) Unlinked - genes on different chromosomes - Gametes have all possible combos (AB, Ab, aB, ab) Fully Linked - On same chromosome, no recombination - Gametes without all combos (just AB, ab) Decoupled - On same chromosome with recombination - Gametes have all possible combos Recombination and Mapping • As distance between 2 genes increases, frequency of crossing-over between them also increases* ** need consider multiple meiotic events; not just 1!** • can be measured in terms of the number of recombinant v. parental gametes that are formed • This information can be used to determine the distances between a pair of genes (mapping) Hypothetical example: - Pair of homologous chromosomes; same size, same genes, heterozygous - Physical distance - 8 separate meiotic events – 8 different points of crossing over per chromosome - crossing over effects top portion of chromosome • Probability a cross-over point lands over certain genes is based on distance between those genes - the further apart the genes are, the probability of cross-over points increases Lecture 16 9/30/16 Learning Goals • Recall how crossing-over frequency relates to physical distance between a pair of genes • Run through a worked example (old exam Q) • Know the difference between single- vs. multiple cross-overs and how it affects mapping accuracy Ch. 7 –> Linkage and Mapping in Eukaryotes Linkage and Mapping Recap • Genes on same chromosome segregate together, linkage alters phenotypic ratio - not 9:3:3:1 • Gametes can be parental (unchanged chromosomes) vs. recombinant (cross-over chromosomes) • At least 50% of gametes produced by meiosis are parental (non-crossover) gametes - unmodified chromosomes • % of recombinant (crossover) gametes depends on distance between genes • the ratio of parental vs. cross-over gametes reflects physical distance separating 2 genes - proportion increases the further apart they are and decreases the closer they are ** Need consider multiple meiotic events, not just one** Linkage and Recombination Following Meiosis, several outcomes possible: • Only parental gametes: 2 genes are very close together, a cross-over point unlikely to fall between them • Mostly parental gametes: 2 genes are fairly close together, some crossing over occurs, but rare • 1:1 parental vs. recombinant gametes: 2 genes are far apart, always decoupled by crossing-over Mapping • Chance of a crossing-over point repeatedly forming between 2 genes that are close together is very low • This principle can be used to map the locations and order of genes on a chromosome - How often the 2 traits appear to “separate” from one another • Perform crossing over experiments and record number of recombinant genes • Distances measured in “map units” (mu), where 1 mu = 1% recombination between a pair of genes • Alternate name for mu = centiMorgen (cM) • Chromosome map: estimated locations of genes on a chromosome (incl. distances between them) Worked Example A cross is made between a male AaBb mosquito and a female AABB mosquito (shown below), and the genotypes of all of their offspring were then determined… if these two genes occur on the same chromosome, which of the following gametes have an allele combination that can only be produced by crossing over? A) Ab B) ab C) aB and Ab D) aB and AB E) AB • a B crossing over with A b causes a b and AB - AB is recombinant but not unique because it already existed - ab is recombinant and unique • A B crossing over with A B causes A B and A B - AB is recombinant but already existed so it is not unique From which individuals would gametes that can only be produced by crossing over have originated from? (first one is male, second one is female) A) Female parent only B) Male or female parent C) Female offspring only D) Male parent only E) Male offspring only Single Crossovers During prophase I: • Amount of crossing-over is limited - When A and B X a and b are close to each other, segments of two non- sister chromatids are exchanged, but the linkage between the A and B alleles and a and b alleles is unchanged • Only 1 pair of non-sister chromatids are involved • Locations of cross-over points are random - Could cross over at any point along the chromosomes Multiple Crossovers During prophase I: • Double crossovers also possible - Can occur concurrently during same round of meiosis o Aka simultaneously at 2 locations at the same time • Detectable only when following inheritance of alleles at 3 genes - Minimum 3 genes (A, B, and C) • Again, only 1 pair of non-sister chromatids involved • In a double cross-over, 2 events are independent and simultaneous (i.e., apply the product law) - Takes probability of each event and puts it together –> smaller probability • Probability of a particular double cross-over is less than each of its components (2 single cross-overs) • In mapping experiments, many offspring needed to determine the location and order of neighboring genes Mapping Accuracy • Ideally, crossover frequency is directly proportional to distance between genes (linear relationship) • Deviations can arise from double (or multiple) exchanges that restore the original state - AB X ab –> non-detectable recombinant bc it essentially restored original state - Assume they are close together but in reality they are quite far apart because double cross-over did occur o Leads to decrease in accuracy • These undetectable events are a source of error; lead to underestimation of distance between genes • This error is most pronounced for pairs of genes that are very far apart (more undetected dbl. crossovers) • Solution: avoid long distance mapping…focus on genes that are fairly close - When distances are small, the theoretical v. actual relationship are pretty spot on, but when distances are larger, the relationship can be skewed
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