Genetics Wk 6 Notes
Genetics Wk 6 Notes Bisc 336
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This 13 page Class Notes was uploaded by Anna Ballard on Friday October 7, 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 13 views. For similar materials see Genetics in Biology at University of Mississippi.
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Date Created: 10/07/16
Lecture 17 10/03/16 Learning Goals • Describe Lederberg and Tatum’s classic experiment • Contrast conjugation and transformation • Recall the steps involved in phage reproduction • Describe how conjugation, transformation and transduction can be used to map bacterial genes Ch. 8 –> Genetic Analysis and Mapping in Bacteria and Phage Bacteria and Phage Bacteria = Prokaryotes • Single celled, 1 haploid chromosome (often circular), +/- plasmid(s) - May or may not have a plasmid Bacteriophages = Viruses • Infect bacteria, DNA, or RNA - Original source of Restriction Enzymes (used as a defense mechanism) - Genetics in the news – CRSPR gene editing system isolated from bacteria – natural mechanism for protecting from viruses Bacteria, Mutations, and Growth • DNA mutations are spontaneous, constantly arise (inducement by environmental agents not required) - High rates of mutation in human genomes in places like Chernobyl due to high radiation, etc. • Primary source of new genetic variation - Why mutations important in evolutionary process – what natural selection acts on - Ex: beach mice - Usually detrimental to the individual • Haploid, so genotype = phenotype (new mutations are expressed, not ‘masked’ as in diploid organisms) - No dominant allele overriding recessive • Growth medium – liquid broth or semi-solid agar, nutrient content can be experimentally manipulated - Important: minimal medium has just bare essentials - Enriched media – has lots of nutrients Synthesizing Compounds Different strains of same species of bacteria: • Prototroph – just needs carbon and ions to make all necessary compounds (AAs, sugars, vitamins, etc.) - Can biosynthesize its necessary amino acids • Auxotroph – unable to synthesize one (or several) organic compounds required for growth, usually due to a mutation (designated his- if can’t make histidine) - Have sustained these mutations - Growth medium must be spiked so that they get the right nutrients they need • Only prototrophs can grow on minimal media; auxotrophs require enriched (=complete) media - Enriched media spiked with whatever amino acids are needed Recombination via Conjugation All inform us about gene order • Conjugation – process in which one bacterial cell passes genetic information to another - Happens within their lifetime - Recipient cell gains genetic variance • Followed by genetic recombination – replacement of one or more genes (NOT reciprocal exchange) - One chromosomes cuts out a set of its genetic material and puts in a new set - One remains unmodified whereas in crossing-over, both homologous chromosomes are modified • Still leads to ‘mosaic-like’ novel chromosomes… altered genotypes, and altered phenotypes - Changing genetic makeup of chromosome in recipient cell - The giving cell shares only one of the two complimentary strands so it does not lose any information when sharing with recipient cell Lederberg and Tatum’s Experiment • Strains A & B are both auxotrophs (several null allele mutations each) - Those non functional in strain B do not match those that are in strain A • Grown together for several generations, some (not all) descendants = prototrophs • Gain-of-function mutations unlikely… • + functions like it should • - non functional • Two different strains, both auxotrophic but in their own ways • Mixture of strain A and B - Minimal media - Strain A or B alone –> no growth which means no prototrophs - No growth in Strain A or B o Gain of function mutation –> ruled out o Assume it must be something about the mixture of the 2 autotrophic strain to create the prototrophic strain Did they need to come into contact with each other? Q: Why are gain-of-function mutations unlikely? - Mutations strike randomly, should also be seen in controls - Need a particular and complex combo of gains (product law) Davis’s Experiment • Q: release of DNA directly into the media? • A: No… bacterial cells must come in contact with each other - Not enough for the two strains to be in close proximation –> must have contact Conjugation, Donors, and Recipients • Unidirectional transfer of DNA between strains • F+ bacteria = donors of genes, F- = recipients of genes (i.e., become recombinants) • Physical contact between F+ and F- strains involves the formation of an extension of the cell (F pilus) - Recombination and sharing of material to take place • F+ cells are able to donate genes because they carry an F-factor (a mobile DNA element, or plasmid) F-factor Transmission 1) Conjugation occurs between F+ and F- cell 2) One strand of F factor is nicked by endonuclease and moves across conjugation tube - Outside strand that was nicked is unwinding a single strand of the DNA and moving into recipient cell 3) The DNA complement is synthesized on both single strands - Occurs concurrently in donor and recipient cells Following conjugation: • A copy of the F-factor is always passed on • The F-factor is also retained in original F+ • Recipient (F-) cells become donors (F+) - FIG 8-4 The F-factor is a Plasmid • Plasmids: separate units of inheritance, autonomous • Circular dsDNA, carry one or few genes - Usually involved in resistance • Name indicates function - F-factor –> fertility - R plasmid –> resistance - Col plasmid –> colicin protein • In lab, this produced super weird results Becoming a Hfr Strain… • F-factor enters and integrates, converts cell to Hfr cell - Hfr (high frequency recombinant cell) • The new Hfr cell and “normal” cell conjugate and restriction enzyme cuts F- factor • RE cut site creates origin - Origin – location where RE nicks DNA (binds to DNA then gets cut - Cut out internal portion and clean the rest • Transfer of ssDNA via conjugation tube, starting with genes near origin • DNA replication in Hfr cell (replace donated material) and in F- (copy received genes) - Conjugation did not happen lnon - F- cell • Conjugation interrupted prior to complete transfer • A & B genes got in, can recombine with homologous region of host chromosome • F-factor did not make it, recipient cell remains F-/ - Changes in allelic composition of recombinant - Tells us ordering of genes as well as physical distance btetween them o Distance –> amt of time it takes to get new allelic variant Hfr Bacteria and Mapping • Interrupting conjugation between Hfr and normal strains showed ordered transfer = a map of gene order - (first) …azi –> ton –> lac –> gal… (last) Lecture 18 10/05/16 Learning Goals • Contrast Conjugation, transformation, and transduction • Describe how they are used to map bacterial genes • Recall key experiments that determined DNA (not protein) is the genetic material **Make own conjugation, transformation, and transduction compare/contrast chart to study** conjugation: http://highered.mheducation.com/sites/dl/free/0072835125/126997/animatio n6.html transformation: https://highered.mheducation.com/sites/9834092339/student_view0/chapter 28/bacterial_transformation.html transduction: https://www.youtube.com/watch?v=VX1Ze5edmkE Ch. 8 –> Genetic Analysis and Mapping in Bacteria and Phage Conjugation: Recap • Mutant (Hfr) strain, has F-factor plasmid integrated into main bacterial chromosome - once have strains, allow them to come in contact with F- cells and conjugation takes place - FIG 8-8 Conjugation and Mapping: Recap • Recombination in recipient, no change in donor • Interrupting conjugation between Hfr and normal strains showed ordered transfer = a map of gene order - (first)…azi –> ton –> lac –> gal…(last) Transformation • alternative approach to getting bacteria with recombinant chromsomes - haploid bacterial cell with main bacterial chromsome - complement bacterial cell has receptor on surface • Recombination mechanism; introduce foreign DNA into a bacterial chromosome • Small extracellular dsDNA enters a competent cell via receptor site on the surface - receptor site: where DNA from bacteria can be taken up • One DNA strand digested, one ssDNA strand remains • Foreign ssDNA aligns and pairs with its homolog in the bacterial host chromosome • Excision and replacement - ligase “glues” new into place • Heteroduplex formation (i.e., complementary DNA strands with different origins) - does not last long • Transforming DNA often carries novel mutations • After DNA replication: - 1 recombinant chromosome - 1 original chromosome • After cell division: - 1 transformed cell o TGC with ACG Recombinant cell - 1 untransformed cell o TAC with ATG Transformation and Mapping • Fragments of transforming DNA ~10-20 Kb long, so carry several neighboring genes - Only relatively short pieces can be taken up and the length of the heteroduplex is quite constrained - Can start to identify genes that are close together on a chromosome • Two independent and simultaneous transformation events are very rare (product law • Close genes move together, distant genes do not • Compare transformed v. untransformed cells to map linkage groups First…azi –> ton –> lac –> gal… (last Those close to each other co-transform a lot (ex: azi –> ton) Those further apart rarely do (ex: azi & lac) Those furthest apart NEVER do (ex: azi & gal) ** time is our measure for how far apart genes are in Conjugation ** - No heteroduplex - Single stranded DNA moves into recipient cell ** in transformation, frequency with which they co-transform is our measure distance** - Heteroduplex - Double stranded DNA moves through receptor site T4 Bacteriophage (Transduction) • Virus that infect bacteria • Genetic material is DNA, ~150 genes total • Tail fibers have binding sites, recognize E. coli T4 Lytic Cycle • Attaches via adsorption (receptor-protein binding) - Recognizes its appropriate host… likes to attack E. coli bacteria • Phage DNA crosses cell membrane and replicates - Squats and injects DNA into cytoplasm of bacteria cell - Cascade of changes in bacterial cell o Bacteriophage chops up main bacterial chromosome o Uses cellular machinery to replicate its own DNA within the cell • Viral molecule synthesis and assembly …. Lysozyme breaks open host cell - Must reassemble the newly formed bacteriophages within cells - These new bacteriophages lyse and break out of host cell Transduction • Genetic recombination in bacteria can be mediated by the phage that infect them! • During the lytic cycle, defective phage package bacterial DNA in their head, then exit via lysis - Defective phage: head capsules packaged with fragmented bacteria chromosomes instead of DNA from virus • These phages find another host and infect with bacterial (not phage) DNA… host gains new genes - Small proportion of defective viruses are now able to find new host cells • Adsorption of phage, infect host with DNA • Replicate and assemble (some phage defective) • Bacterial genes injected and integrated Transduction and Mapping Genetic composition of defective phage: • (1) phage DNA + bacterial DNA (few genes) • (2) only bacterial DNA (several genes) Standard principles apply: • Close genes move together (co-transduction) • Distant genes only move via 2 independent events … transduction segments represent linkage groups Ch. 9 –> DNA Structure and Analysis Genetic Material Storage and Replication • Complete genome in cells • Copied during cell cycle (mitosis and meiosis) Expression • Only part of the genome “on” at any given time • On-off switches drive information flow Evidence for DNA (not protein) • Avery et al. experimented with 2 strains of Diplococcus (staphylococcus) bacteria: IIR (non virulent) and IIIS (virulent) • Built on the observation that IIR became virulent when mixed with heat- killed IIIS (transformation) • From heat-killed IIIS cells, they removed one component at a time before mixing with IIR FIG 9-2 Time: 31:55 More Evidence • Hershey and Chase experimented with bacteria and phage (E. coli and T2) • Built on the knowledge that a component of a phage entered the host cell and directed viral reproduction • Radioisotopes differentially labeled E. coli DNA ( P) and Protein ( S)… Q: Why not stop after eliminating protein? A: Could have been something other than DNA that the experimenters did not think about - Experimental procedure also could have been messed up and allowed transformation in all of them • First allow T2 infection of the _________ • Then allow T2 progeny to infect ________ • Determine type __________ Indirect Evidence in Eukaryotes • Prediction 1: there should be more DNA in ________ cells • Prediction 2: absorption spectrum of DNA should match action spectrum of UV (mutation inducer) …. Predictions validated Direct Evidence in Eukaryotes • Recombinant techniques: ___________ synthesized by bacteria ____________ • Not just bacteria, also with transgenic animals (e.g., human genes inserted and expressed in mice) RNA (NOT DNA) in Some Viruses • Retro-viruses use reverse transcription to convert their RNA back into DNA once in the host cell • The newly-synthesized DNA is incorporated into the host cell’s genome, codes for production of viral RNA Lecture 19 10/7/16 Learning Goals • Recall key experiments that determined DNA (not protein) is the genetic material • Describe the observations/data that led to Watson and Crick’s proposal of the DNA helix model • Know how mode of DNA replication was determined Ch. 9 –> Structure and Analysis Chargaff et al. determined DNA base composition of many different species. They repeatedly found that: A) Amount of A ≠ T B) Amount of C ≠ G C) Amount of (C+G) = (A+T) D) None of the above ** Needed to account for the fact that there were similar amounts of A&Ts as well as C&Gs More Evidence (DNA, Not Protein) • Hershey and Chase experimented with bacteria and phage (E. coli and T2) • Built on the knowledge that a component of phage entered the host cell and directed viral reproduction 32 35 • Radioisotopes differentially labeled E. coli DNA ( P) and protein ( S)… - Protein contains sulfur but not phosphorus - DNA contains phosphorus but not sulfur OVERVIEW: experiment involved a focus on viruses on whether they had protein or DNA for genetic material - Start off with 2 experimental lines o Viruses placed in jar of E. coli host cells living in medium of radioactive phosphorus o Viruses placed in jar of E. coli host cells living in medium of radioactive sulfur - Progeny phages become labeled and infect unlabeled bacteria - Separate viruses from bacteria and see what it was that made it into the bacterial host cell o Whatever the genetic material is, is the stuff injected into the bacterial host cell - Now have differentially labeled viruses that will interact with host cells that are unlabeled - Labeled phages infect unlabeled bacteria - Centrifuge to get rid of virus “ghosts” still stuck to outside of cell (like a cicada lol) o Infected bacteria are labeled with 32P o Phage “ghosts” are labeled with s5S o Infected bacteria are unlabeled • First allow T2 infection of the radio-labeled host • Then allow T2 progeny to infect unlabeled E. coli • Determine type of radioactivity Indirect Evidence in Eukaryotes • Prediction 1: there should be more DNA in diploid than haploid cells • Prediction 2: absorption spectrum of DNA should match action spectrum of UV (mutation inducer) …. Predictions validated • Also observed… - Know that haploid gametes produced from organisms must have half the genetic material than parent cells o No difference between amount of protein, but amount of DNA is halved Consistent with idea that it is DNA is the genetic material - Also know that genetic material can be mutated with exposed to UV radiation o Whichever of the two is the true genetic material should absorb the most UV light DNA absorbed UV light most efficiently with more mutations produced Direct Evidence in Eukaryotes • Recombinant techniques: - eukaryotic gene produces synthesized by bacteria following DNA insertion • Not just bacteria, also with transgenic animals (e.g., human genes inserted and expressed in mice) RNA (NOT DNA) in Some Viruses • Exception to the Rule - RNA injected into host cell is then converted to DNA via Reverse Transcription - HIV is a retrovirus so virus itself uses RNA not DNA • Retro-viruses use reverse transcription to convert their RNA back into DNA once in the host cell • The newly-synthesized DNA is incorporated into the host cell’s genome, codes for production of viral RNA DNA Molecules • Nucleotides – the basic units of DNA molecules, composed of a nitrogenous base, sugar, and phosphate • Types of bases – purine (A, G); pyrimidine (C, T = U) - RNA replaces T with U (uracil) - One other difference: DNA tends to be double stranded whereas RNA is single stranded DNA Structure • Chargaff et al. determined DNA base composition in diverse organisms, showed repeated patterns: - Amount of A = T and C + G; amount of (C+G) ≠ (A+T) o AKA not necessarily equal proportion between C&G and A&T C goes with G and A goes with T • Franklin’s x-ray diffraction (i.e., images of scatter patterns) suggested a helical structure of DNA … indicated overall arrangement (even though she didn’t recognize it at the time) The DNA Model • Watson and Crick formulated a model to account for earlier observations (published and some unpublished) • Proposed that base complementary & pairing provides a mechanism for DNA replication - Double hydrogen bond holding A and T together - Triple hydrogen bond holding C and G together • Recognized that this could help us realize how DNA is replicated The DNA “Soap Opera” The race to solve the structure of DNA was intense… and ruthless! • A critical piece of Franklin’s data was revealed to Watson without her permission, or knowledge • “Stolen” X-ray diffraction data were critical to Watson and Crick’s model No Prize for You, Rosalind • The 1962 Nobel prize was awarded to Watson and Crick only • Franklin could not share it… she had died of ovarian cancer (working with X-rays has its risks) RNA Structure and Function • Usually, single-stranded, except when folded back on itself, or in some animal viruses - Loops back on itself and hydrogen bonds between complementary bases can occur causing it to look doubled (stems) with single parts (loops) o Stems and loops • U replaces T and sugar ribose replaces deoxyribose DNA codes for main 3 types of RNA: - rRNA –> a structural component of ribosomes - mRNA –> key to expression of gene products - tRNA –> carries AA’s to ribosome, assembling protein Ch. 10 –> DNA Replication and Recombination DNA Replication • Main function of DNA is storage and replication • Replication is critical for maintaining genetic continuity across generations (cells, and organsims) - when replication happens, either of the 2 strands both become a template - synthesis of a complementary strand happens with lots of accuracy via DNA polymerase • Requires extremely high copying fidelity, although some errors still do occur (the basis for evolution) • Many enzymes and proteins involved in DNA copying and synthesis of new strands - DNA polymerase Animation: www.yourgenome.org/video/dnareplication Mode of DNA Replication • Semiconservative: each strand of the helix is a template for synthesis of a complementary strand - New helix = 50/50 mix of new and old DNA Other Possible Replication Modes • Conservative: after replication, the old strands re-associate and newly synthesized strands pair-up - 2 new strands separate separate and associate with one another o original template goes back together • Dispersive: helix fragmented (cleaved); after replication, each strand consists of varying amounts of old and new DNA - each of the 2 DNA strands serve as a template for a new complementary strand for each - chopped up and arranged into mosaic like structure helix - along the length of a new single strand you have old + new mosaic like double helix Meselson-Stahl Experiment • 3 alternative modes make contrasting predictions about helix composition (proportion of old vs. new) - realized that the 3 alternatives make different predictions 15 14 • DNA = nitrogen-containing molecule; N (heavy) distinguishable from N (light) via centrifugation - incorporates nitrogen and these versions were available to them - after centrifuging, you can distinguish between the two • First grew E. coli on 1N media, then transferred to 1N media and isolated DNA from cells each generation - each generation of a bacteria, they looked at proportion of heavy vs. light that had been incorporated into the chromosome o all of DNA incorporates heavy nitrogen and falls into bottom of tube if semiconservative – all bacteria would have DNA that were one light nitrogen and one heavy nitrogen together - found that it had a different weight and formed a band - repeated cycle of 1 round of replicaotin of bacteria and centrifuging Predictions • After 1 generation, a DNA band of intermediate density - semi-conservative - 2 helixes that are both light and heavy 50/50 • After 1 generation, 2 DNA bands; heavy and light - conservative o one helix that is heavy and one helix that is light • After 1 generation: a DNA band of intermediate density - dispersive o mosaic like structure o 50/50 heavy and light • After 2 generations, 2 DNA bands: intermediate and light • After 2 generations, 2 DNA bands: heavy and light • After 2 generations, a DAN band of intermediate density Predicted • if semi-conservative mode is correct Observed • CsCI gradient centrifugation
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