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How to get fly transgenes from in vitro to in vivo: your favorite gene(s) & w+ P-element (ends) serve as a “vector” to move DNA of our choice Inject DNA into very young embryos ….. aiming for the germ cells. As a GENETIC SOURCE of transposase to get the transgene to insert into chromosomes use a defective (immobile) integrated P-element transposase gene A “stable” source of transposase, since it can’t move modified so that it makes transposase in soma as well as germline Use the engineered mobile genetic element as a mutagen (make it hop randomly into genes): whatever is useful (eg. w+) (a nonautonomous element) starting element: w+ 2 It is a "genetically tagged" mutagen (we can follow it) transposase to mobilize (pseudo-replicative transposition) Tp X starting element: w+ 2 w+ new (random) site of insertion happening In the dysgenic parent's germline 3 collect progeny from the appropriate genetic cross of the dysgenic parent (not carrying the transposase source) X (not carrying the original P) 2 w+ gene 3 Is this a mutant allele of interest? If so, already well marked and easy to clone! P P your favorite gene(s) & w+ (a nonautonomous element) If we are going to want to use as a mutagen (hop into genes)… Lucky thing that M strains exist (strains with no pre-existing source of P transposase or antitransposase to interfere with our controlling non-autonomous element [transgene] mobility) Why do M strains exist? Lab strains taken from the wild before 1950’s don’t have P elements. This DNA parasite invaded D. melanogaster sometime in the 1950s, then rapidly spread nearest P-element relative: in a fly species 50 Myr diverged How? -- an example of horizontal genetic transmission (xfrd in same generation, i.e. gene not introduced from parents (vs. vertical) Are fly-workers to blame for contaminating D. melanogaster? tagged transposon hopped in the dysgenic parent's germline collect progeny from the dysgenic parent w+ gene new (random) site of insertion 3 How do we know that we have even mutated a gene, much less generated a mutant allele of use/interest ? recover the chromosome in a MUTANT SCREEN or SELECTION Steps in forward genetics: decide what to study generate informative mutant alleles (mutagenesis) recover informative mutant alleles study informative mutant allele (do molecular biology) write paper reap rewards NATURE V287:p795 (1980) Nobel Prize 1995 (products of a mutant screen) wildtype gooseberry wildtype gooseberry patch “skins” of dead larvae patch mutant phenotypes informative for understanding pattern formation in metazoans text: 20.3 “The genetic analysis of body-plan development in Drosophila: a comprehensive example. (pp732-745) They regulate… the segmentation regulatory gene hierarchy Ant. Pst. maternal effect Drosophila homeotic gene clusters Mammalian Hox gene clusters gap Mouse embryo pairrule segment polarity Fig. 20.22 p741 Fig. 20.26 p744 Two general categories of mutant allele recovery strategies: (1) genetic screens (“brute force” screens) make mutations randomly, then you sift through chromosomes (often one at a time) looking for mutant alleles of interest/use (2) genetic selections make mutations randomly, then let nature eliminate all undesired mutant alleles so you are only left with the good stuff 2 easier to execute than 1, but often not possible to design & potentially more biased (may get only what you think to look for) By the way: an important but under-appreciated step in genetic analysis: generate mutant allele recover mutant allele study mutant allele maintain mutant allele write paper for each fly line: transfer to 15ml new food every 3 weeks reap rewards Homework problem: How much food (corn meal, molasses, yeast) has T.H.Morgan’s original white1 mutant line consumed since 1910? Strategies & tools that help us recover mutant alleles can also help us maintain them. Maintaining mutant stocks (lines) in model genetic systems: To freeze or not to freeze most microbes (spores are nice) arabidopsis (“the weed”) seeds & corn worm mouse fish fly (embryos) Basic facts to consider in designing screens and selections: (1) Most LOF mutant alleles are recessive (all else being equal) (LOF mutations are the most frequent class) (2) Most null alleles of genes with an obvious LOF phenotype are lethal, or at least sterile. (3) Most “developmentally interesting” genes are essential for viability or fertility Hence: screen/selection schemes must provide for the recovery of recessive lethals and steriles The “diploid advantage” for recessive lethal studies: Diploid: lethal / + alive (fertile) (+ holds the fort) Haploid: often for microbes lethal dead (sterile) (let naked and exposed) rely on conditional lethals in generating mutations: Haploid: lethal dead (sterile) rely on conditional lethals in generating mutations: condition A growth all grow (mutagenize wildtype) vs. condition B no growth only mutants of interest don’t grow mutant genetic screen or screen selection? Two key tricks for microbes: p212: Fig. 7.5 Replica Plating & p558: Fig. 15.15 augmented by: genetic p547: Fig. 15.5 (Penicillin) enrichment selection condition A growth vs. all grow (mutagenized) condition B no growth only mutants of interest don’t grow Two key tricks for microbes: p212: Fig. 7.5 & its use: Fig. 15.15 (p558) Replica Plating genetic screen condition A growth all grow (mutagenized) vs. condition B no growth only mutants of interest don’t grow augmented by: p547: Fig. 15.5 (Penicillin) enrichment genetic selection (diluting out the penicillin) Replica plate on The “diploid advantage” for recessive lethal studies: Diploid: lethal / + alive (fertile) (+ holds the fort) The “diploid handicap” for recessive lethal studies: + masks lethal Haploid: lethal dead (sterile) (let naked and exposed) effects of lethal immediately obvious The problem with diploids in hunting for new recessive mutations: mutagenize Female X Male +/+ +/+ :PARENTS form zygotes + b + + a + + + + + eggs + + + d + + + b + + + c a + + + +/+ sperm + +/b +/+ +/ a F1 PROGENY The problem with diploids in hunting for new recessive mutations: mutagenize Female X Male +/+ +/+ +/+ +/b +/+ +/ a F1 PROGENY given: we are interested in (finding) the a-/a- phenotype How do we know who (if anyone) is carrying a- ? …the individual who can produce a-/a- offspring. The problem with diploids in hunting for new recessive mutations: mutagenize Female X Male +/+ +/+ +/+ +/b +/+ Which is the individual who can produce a-/a- offspring? +/ a F1 PROGENY To whom do we mate to find out? If we can “self” this individual, we are effectively mating to +/a- for sure of course, we had to self everyone: No a-/a- YES! & we know in the F2 a-/a- The problem with diploids in hunting for new recessive mutations: mutagenize Female X Male +/+ +/+ F1 +/+ +/b +/+ +/ a To whom do we mate to find out -- if we can’t self? +/+ +/+ +/+ +/+ +/+ …. we still don’t know in the F2! +/+ +/+ +/+ Meanwhile +/ a +/+ +/ a +/+ The problem with diploids in hunting for new recessive mutations: -- if we can’t self. mutagenize Female X Male +/+ +/+ +/+ +/b X+/+ +/+ (must keep populations separate!) +/+ +/+ +/+ +/+ +/+ Mate inter se at best +/+ +/ a …. we still don’t know in the F2! F1 X+/+ F2 ..but at least now we have potential mates with a- +/+ +/ a +/+ +/ a male? +/+ female? at least some chance: Mate inter se a /a The problem with diploids in hunting for new recessive mutations: -- if we can’t self. mutagenize Female X Male +/+ +/+ +/+ +/b +/+ F1 +/ a X +/+ +/ a +/+ +/+ +/+ +/+ +/+ Mate inter se only +/+ F2 +/+ +/ a +/+ +/+ if we cross them, a-/a- will come: can we do better than mating inter se? a /a It would help if we could keep track of chromosomes: mutagenize a- = mutagenized chromosome with new mut. + = mutagenized but not desired mutant + = non-mutagenized from original Mom + = non-mutagenized from F1 mate Female X Male +/+ +/+ +/+ +/b +/+ F1 +/ a X +/+ +/ a Even nicer if we could eliminate extraneous animals F2 +/+ +/ a +/+ +/+ if we cross them, a-/a- will come: we can do better than mating inter se a /a our friend, Herman Muller had the answer (early ‘30s): (1)used them to determine mutation frequency: …how often a new recessive lethal arose on a given fly chromosome (2) used them to “maintain” deleterious recessive alleles of interest Balancer chromosomes: (a) a chromosome you can distinguish from the others. dominant marker mutant alleles (Bar, Curly, Stubble) (b) a chromosome that will not recombine with others crossover suppressors (multiple inversions) (c) a chromosome that will not “become” homozygous (i.e. that would either be lethal or sterile if homozygous) recessive lethal or sterile alleles