Steps in forward genetics NATURE V287 p795 1980 Nobel Prize 1995 decide what to study generate informative mutant alleles recover informative mutant alleles study informative mutant allele do molecular biology write paper reap rewards text 20 3 The genetic analysis of body plan development in Drosophila a comprehensive example pp732 745 wildtype gooseberry w ildtype gooseberry patch skins of dead larvae patch mutant phenotypes informative for understanding pattern formation in metazoans Two general categories of mutant allele recovery strategies They regulate the segmentation regulatory gene hierarchy Ant Pst maternal effect Drosophila homeotic gene clusters Mammalian Hox gene clusters 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 gap Mouse embryo make mutations randomly then let nature eliminate all undesired mutant alleles so you are only left with the good stuff pairrule segment polarity Fig 20 22 p741 Fig 20 26 p744 2 easier than 1 but often not possible potentially more biased 1 By the way an important but under appreciated step in genetic analysis Maintaining mutant stocks lines in model genetic systems generate mutant allele To freeze or not to freeze recover mutant allele study mutant allele most microbes maintain mutant allele for each fly line transfer to 15ml new food every 3 weeks write paper reap rewards spores are nice arabidopsis the weed seeds corn Homework problem How much food corn meal molasses yeast has T H Morgan s original white1 mutant line consumed since 1910 mouse embryos fish fly worm Strategies tools that help us recover mutant alleles can also help us maintain them 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 The diploid advantage for recessive lethal studies Diploid lethal 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 alive fertile holds the fort Haploid often for microbes lethal dead sterile let naked and exposed Hence screen selection schemes must provide for the recovery of recessive lethals and steriles Haploid lethal dead sterile rely on conditional lethals in generating mutations condition A growth vs all grow mutagenized let naked and exposed condition B no growth only mutants of interest don t grow rely on conditional lethals in generating mutations Two key tricks for microbes condition A growth all grow mutagenize wildtype vs condition B no growth only mutants of interest don t grow mutant genetic screen or selection screen Two key tricks for microbes p212 Fig 7 5 Replica Plating p558 Fig 15 15 p212 Fig 7 5 p558 Fig 15 15 Replica Plating genetic screen augmented by genetic p547 Fig 15 5 Penicillin enrichment selection 2 condition A growth vs all grow mutagenized condition B no growth Two key tricks for microbes only mutants of interest don t grow p212 Fig 7 5 p558 Fig 15 15 augmented by Replica Plating genetic screen Diploid genetic selection p547 Fig 15 5 Penicillin enrichment The diploid handicap for recessive lethal studies The diploid advantage for recessive lethal studies lethal masks lethal alive fertile holds the fort Haploid lethal diluting out the penicillin let naked and exposed Replica plate on The problem with diploids in hunting for new recessive mutations The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic Female X Male a eggs effects of lethal immediately obvious dead sterile mutagenize altv dysgenic PARENTS form zygotes d b b Female X Male a b a F1 PROGENY given we are interested in finding the a a phenotype c sperm b How do we know who if anyone is carrying a the individual who can produce a a offspring a F1 PROGENY The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic given we are interested in the a a phenotype Female X Male The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic Female X Male How do we know who if anyone is carrying a b a F1 PROGENY F1 b a To whom do we mate to find out if we can t self 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 the individual who can produce a a offspring a we still don t know in the F2 Meanwhile a 3 The problem with diploids in hunting for new recessive mutations The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic mutagenize altv dysgenic Female X Male b if we can t self a F1 we still don t know in the F2 X but at least now we have potential mates with a X must keep populations separate F2 a a at best at least some chance Mate inter se a a It would help if we could keep track of chromosomes mutagenize altv dysgenic 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 F2 a if we cross them a a will come p503 Fig 14 17 b F1 a X a F2 Mate inter se a if we cross them a a will come only can we 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 X a a chromosome you can distinguish from the others a Even nicer if we could eliminate the extraneous animals Mate inter se if we can t self Female X Male dominant marker mutant alleles Bar Curly Stubble we can do better than mating inter se a a 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 Balancer chromosomes 1 use them to follow chromosomes in order to create new genotypes such as in mutant screens 2 use them to maintain deleterious recessive alleles of interest 3 essential features a dominant marker mutant alleles b crossover suppressors c recessive lethal or sterile alleles 4 Balancer chromosomes Balancer chromosomes 2 used them to maintain deleterious recessive alleles of interest recall problem
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