Reading Transposable genetic elements move from place to place in the genome pp508 514 Chp 14 Radiation the first experimental mutagen discovered non ionizing lower energy UV light photochemical reaction glues adjacent thymines together in cis Also p548 insertion sequences in bacteria A practice problem set dealing with mutant categories is available on the website Answers will be posted next Monday Lecture schedule Wed 3 14 genetic screens Fri 3 16 sensitized screens Mon 3 19 genetic mosaics in screens hence no RNAi and one less sex lecture ionizing higher energy X rays rays cosmic rays generates free radicals chemical bull in the china shop cell must repair this damage at any cost to avoid dying after next cell division but what repair template available If none can be found or found in time just stick the DNA together blindly A G G C C T C T T C A TCCGGAGA AGT DNA repair machinery called out light dependent repair accurate excision repair error prone Generally makes single base pair changes or at most very small deletions in contrast to ionizing higher energy X rays rays cosmic rays Transposable DNA elements as mutagens and as general genetic workhorses Categories of transposable genetic elements among other damage can induce double strand DNA breaks double helix not sister chromatids replication block AGGCCTCTTCA TCCGGAGAAGT Retroposons transposes via an RNA intermediate actually a virus Transposons transposes via a DNA intermediate Insertion sequences IS in bacteria Fig 15 6 generate transposons some of which are responsible for ultimately producing multiply drug resistant pathological bacteria Hence ionizing radiation causes just about every genetic change imaginable Transposable DNA elements as mutagens 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 TE 12 5 of fly genome just two of many different in humans SINEs LINEs 7 of human genome they are ubiquitous mobile genetic parasites selfish DNA stripped down virus or real virus in some cases can insert into DNA causing damage when tamed tamed have been used to generate the most powerful tools in modern molecular genetics control their mobility History of transposable element studies Genetic analysis of unstable genes jumping genes in corn 1948 Rhoades and McClintock unstable fly mutants in 1920s crosses between races unstable bacterial mutants Molecular characterisation ultimately related all three Led by rediscovery of unstable fly mutants in 70 s key breakthrough discovered how to control instability the phenominon of hybrid dysgenesis molecular explanation in early 1980s showed generality and importance of mobile genetic elements let to Nobel prize for McClintock 1983 1 Initial evidence for mobile genetic elements in Drosophila different classes of DNA based on abundance single copy middle repetative 80 different types 5 kb x50 12 5 total first to be cloned highly repetative copia class of middle repetative DNA cloned and characterized in situ DNA hybridization to polytene chromosomes 30 50 sites of hybridization but many differences among wildtype flies One important copia difference between two fly lines white whiteapricot NO copia element at band 3C2 w copia element at band 3C2 Mobile but positions seemed relatively stable in individual lab strains most don t move often and in most cases don t know why when they move white Fig 14 24 once again polytene chromosomes were central to the finding NO copia element at band 3C2 w whiteapricot copia element at band 3C2 w12 was generated by PM hybrid dysgenesis Sved and Kidwell s discovery of strange incompatibility between wildtype races of flies Female parent in cross use the copia DNA to fish out flanking white gene DNA white was one of the first single copy fly genes cloned and transposons had made it possible now in a position to molecularly characterize any mutant allele of white a white mutant generated by hybrid dysgenesis Morgan s mutant that started it all Strain A Male parent in cross Strain A Strain B normal dysgenic 1 hi mutation rate 2 chrom rearrgmts 3 sterility Strain B normal normal degenerate gonads Characterisation of any new strain with respect to P or M identity then Strain X must be M then Strain Y must be P Female parent in cross Male parent in cross M strain maternal for dysgenesis Strain X normal P strain paternal for dysgenesis dysgenic Strain X dysgenic Female parent in cross Male parent in cross M strain maternal for dysgenesis Strain Y dysgenic P strain paternal for dysgenesis dysgenic Strain Y normal 2 Origin of w12 Odd behavior of w hd Female parent in cross Male parent in cross Female parent in cross Male parent in cross M strain M strain dysgenic P strain produced P strain w12 What was wrong with w12 It had a P element transposon in it 2 9 kb DNA if intact M strains have no P elements 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 transposase gene An autonomous element can move by itself has everything it needs transposase gene A nonautonomous element can move only if it gets transposase from somewhere Making a more useful nonautonomous element like white to mark the element your favorite gene s P element ends serve as a vector to move DNA of our choice say from a test tube into a chromosome to make a transgene An autonomous element can provide transposase to move the transgene transposase gene A defective element may be the source of an antitransposase repressor of transposition responsible for the P cytotype w12 stable w12 stable dysgenic w12 stable P element transposon has a very typical simple transposon structure 2 9 kb DNA 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 elements P strain w12 unstable reverts to w wother hypmrph stable generally the P inserts are internally deleted defective elements What is the difference between P and M strains M strain transposase gene Inverted terminal Repeats 31 bp IR ends needed in cis for mobility DNA between them is moved transposase protein can work in trans on IR ends to move DNA Back to the original crosses that define P M strains Female parent in cross Male parent in cross M strain no
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