1Reading:“Transposable genetic elements move from place to place in the genome”pp508-514 (Chp 14)Also: p548 (insertion sequences in bacteria)A practice problem set dealing with mutant categoriesis available on the website.Answers will be posted next Monday.Lecture schedule: Wed 3/14: genetic screensFri 3/16: sensitized screensMon 3/19: genetic mosaics in screens(hence: no RNAi and one less sex lecture)Radiation (the first experimental mutagen discovered)non-ionizing (lower energy)ionizing (higher energy)UV lightexcision repair(error prone)photochemical reaction glues adjacent thymines together in cis:A G G C C T C T T C AT C C G G A G A A G TT A G G C C T C T=T C AT C C G G A G A A G TDNA repair machinery called out:light-dependent repair(accurate)replication blockX-rays, γ-rays, cosmic raysGenerally makes single base-pair changes, or at most very small deletions ---in contrast to:ionizing (higher energy)X-rays, γ-rays, cosmic raysgenerates free radicals: chemical bull in the china shop…among other damage, can induce double-strand DNA breakscell must repair this damage at any costto avoid dyingafter next cell division!!…but what repair template available?If none can be found (or found in time), just stick the DNA together blindly.Hence,ionizing radiation causesjust about every genetic change imaginable.(double helix, not sister chromatids)Transposable DNA elements as mutagens…and as general genetic workhorsesCategories of transposable genetic elements:RetroposonsTransposonsInsertion sequences (IS in bacteria, Fig. 15.6)transposes via anRNA intermediate (actually a virus)transposes via a DNA intermediategenerate transposons, some of which areresponsible for ultimately producing multiply drug resistant pathological bacteria)…and as general genetic workhorses“all natural” mutagenic agent (like many of the most potent carcinogens)responsible for half of all spontaneous mutations in “the fly”responsible for generating much of the raw material of evolution (eg. chromosome rearrangements, duplications, deletions, etc.)they are ubiquitous mobile genetic parasites (“selfish DNA”) can insert into DNA causing damage“stripped down” virus -- or real virus in some cases?when “tamed”, have been used to generate the most powerful tools in modern molecular geneticsTransposable DNA elements as mutagenscontrol their mobilitytamedTransposableTE = 12.5% of fly genome;just two of many different in humans (SINEs & LINEs) = 7% of human genomeGenetic analysis of unstable genes“jumping genes” in corn (1948 Rhoades and McClintock)History of transposable element studies:unstable fly mutants in 1920s (crosses between “races”)unstable bacterial mutantsLed by rediscovery of unstable fly mutants in 70’s…the phenominon of “hybrid dysgenesis”molecular explanation in early 1980s…let to Nobel prize for McClintock 1983showed generality (and importance) of mobile genetic elementsMolecular characterisation ultimately related all threekey breakthrough: discovered how to control instability)2Initial evidence for mobile genetic elements in Drosophila:single copymiddle repetativehighly repetative80 different types, ~5 kb, x50 = 12.5% totalin situ DNA hybridization to polytene chromosomes:copia class of middle repetative DNA cloned and characterized30-50 sites of hybridization,but many differences among wildtype flies…but positions seemed relatively stable in individual lab strains)first tobe cloneddifferent classes of DNA based on abundance:…most don’t move often and in most cases,don’t know why/when they moveMobileFig. 14.24 --once again, polytene chromosomeswere central to the finding)white+whiteapricotcopia element at band 3C2NO copia element at band 3C2 (w)One important copia difference between two fly lines:white+whiteapricotcopia element at band 3C2NO copia element at band 3C2 (w)use the copia DNA to fish out flanking white gene DNAnow in a position to molecularly characterizeany mutant allele of whitewhite was one of the first single-copy fly genes cloned (and transposons had made it possible)Morgan’s mutantthat started it alla white- mutantgenerated by hybrid dysgenesisStrain AStrain AStrain BStrain Bw12 was generated by “PM hybrid dysgenesis”Sved and Kidwell’s discovery of strange incompatibilitybetween wildtype races of flies:normal normalnormaldysgenic:(1) hi mutation rate(2) chrom. rearrgmts.(3) sterility(degenerategonads)Female parent in cross:Male parentin crossFemale parent in cross:Male parentin crossnormaldysgenicM strain Strain XStrain XP strainmaternal fordysgenesispaternal fordysgenesisthen Strain X must be MdysgenicCharacterisation of any new strain with respect to P or M identity:Female parent in cross:Male parentin crossnormaldysgenicM strain Strain YStrain YP strainmaternal fordysgenesispaternal fordysgenesisthen Strain Y must be Pdysgenic3Female parent in cross:Male parentin crossdysgenicM strainP strainproduced w12Origin of w12Female parent in cross:Male parentin crossdysgenicM strainP strainw12 unstable(reverts to w+)Odd behavior of whdM strainP strainw12 stablew12 stablew12 stablewother hypmrph stableWhat was wrong with w12 ?It had a P-element transposon in it [2.9 kb DNA (if intact)](generally the P inserts areinternally deleted “defective” elements)What is the difference between P and M strains?This is the key to the value of P-elements: “virgin” strains exist that have NO P elements(not true for any other fruit fly transposons)it looks as if:PM hybrid dysgenesis drove a P element into white+ partially destroying its function (the element moved!)P strains have intact P elementsM strains have no P elementsP-element transposon has a very typical (simple) transposon structure:2.9 kb DNAtransposase geneInvertedterminalRepeats(31 bp)transposaseprotein can work in trans (on IR ends)to move DNAIR ends needed in cis for mobility(DNA between them is moved)transposase geneyour favorite gene(s)transposase geneAn “autonomous” element:can move by itself(has everything it needs)A “nonautonomous” element:can move only if it gets transposasefrom somewheretransposase geneA defective element may bethe source of an “antitransposase”(repressor of transposition)responsible for the P cytotypeP-element (ends) serve asa “vector” to move DNAof our choice(say…from a test-tube into a chromosome to make a “transgene”)(like white+ to “mark” the element)favoriteMaking a more useful nonautonomous element:An
View Full Document
Unlocking...