Steps in forward genetics decide what to study generate informative mutant alleles recover informative mutant alleles study informative mutant allele do molecular biology write paper reap rewards NATURE V287 p795 1980 Nobel Prize 1995 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 than 1 but often not possible potentially more biased 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 let naked and exposed 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 selection screen 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 p558 Fig 15 15 Replica Plating genetic screen condition A growth all grow mutagenized vs condition B no growth only mutants of interest don t grow augmented by Two key tricks for microbes p212 Fig 7 5 p558 Fig 15 15 p547 Fig 15 5 Penicillin enrichment Replica Plating genetic screen genetic selection diluting out the penicillin Replica plate on The diploid advantage for recessive lethal studies Diploid lethal alive fertile holds the fort Haploid lethal dead sterile let naked and exposed The diploid handicap for recessive lethal studies masks lethal effects of lethal immediately obvious The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic Female X Male a eggs PARENTS d b c form zygotes b sperm a b a F1 PROGENY The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic 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 altv dysgenic given we are interested in the a a phenotype Female X Male How do we know who if anyone is carrying a b 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 the individual who can produce a a offspring The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic Female X Male F1 b a To whom do we mate to find out if we can t self a we still don t know in the F2 Meanwhile a The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic Female X Male b if we can t self a X F1 we still don t know in the F2 X but at least now we have potential mates with a must keep populations separate Mate inter se at best F2 a a at least some chance Mate inter se a a The problem with diploids in hunting for new recessive mutations mutagenize altv dysgenic if we can t self 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 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 X a Even nicer if we could eliminate the 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 p503 Fig 14 17 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 Balancer chromosomes 2 used them to maintain deleterious recessive alleles of interest recall problem without balancer X let let X let X
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