Steps in forward genetics: generate informative mutant allelesrecover informative mutant allelesstudy informative mutant allele (do molecular biology)write paperreap rewardsdecide what to studyNATURE V287:p795 (1980)Nobel Prize 1995wildtypewildtypegooseberrygooseberrypatchpatchmutant phenotypesinformative for understandingpattern formation in metazoans“skins” ofdead larvaetext: 20.3 “The genetic analysis of body-plan developmentin Drosophila: a comprehensive example. (pp732-745)Fig. 20.26 p744Fig. 20.22 p741the segmentation regulatory gene hierarchyAnt.Pst.gappair-rulesegmentpolaritymaternaleffect Mammalian Hox gene clusters Mouse embryoDrosophila homeotic gene clustersThey regulate…Two general categories of mutant allele recovery strategies:(1) genetic screens make mutations randomly, then you sift through chromosomes (often one at a time) looking for mutant alleles of interest/use(2) genetic selectionsmake mutations randomly, then let nature eliminate all undesired mutant alleles so you are only left with the good stuffbut often not possible & potentially more biased2 easier than 1,(“brute force” screens)generate mutant alleleBy the way:an important but under-appreciated step in genetic analysis: recover mutant allelestudy mutant allelewrite paperreap rewardsmaintain mutant allelegenerate mutant allelerecover mutant allelestudy mutant allelewrite paperreap rewardsHomework problem:How much food (corn meal, molasses, yeast) has T.H.Morgan’s original white1 mutant line consumed since 1910? for each fly line: transfer to 15ml new food every 3 weeksStrategies & tools that help us recover mutant alleles can also help us maintain them.Maintaining mutant stocks (lines) in model genetic systems:most microbes(spores are nice)arabidopsis(“the weed”)& cornseedsmousefishflyworm(embryos)To freeze or not to freezeBasic facts to considerin 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 fertilityHence:screen/selection schemes must provide for the recovery of recessive lethals and sterilesThe “diploid advantage” for recessive lethal studies:(+ holds the fort)(let naked and exposed)Diploid:lethal / + alive (fertile)Haploid:lethaldead (sterile)rely on conditional lethals in generating mutations:oftenfor microbesall grow(mutagenizewildtype)only mutants of interest don’t growgrowth vs. no growthcondition A condition BTwo key tricks for microbes:(let naked and exposed)Haploid:lethaldead (sterile)rely on conditional lethals in generating mutations:p212: Fig. 7.5 Replica Plating& p558: Fig. 15.15p547: Fig. 15.5 (Penicillin) enrichmentaugmented by:geneticselectionmutantscreen orselection?geneticscreenall grow(mutagenized)only mutants of interest don’t growgrowth vs. no growthcondition A condition BTwo key tricks for microbes:p212: Fig. 7.5& p558: Fig. 15.15geneticscreenReplica Platingall grow(mutagenized)only mutants of interest don’t growgrowth vs. no growthcondition A condition BTwo key tricks for microbes:p212: Fig. 7.5& p558: Fig. 15.15Replica Plating geneticselectionp547: Fig. 15.5 (Penicillin) enrichmentaugmented by:geneticscreenReplica plate on(diluting outthe penicillin)The “diploid advantage” for recessive lethal studies:(+ holds the fort)(let naked and exposed)Diploid:lethal / + alive (fertile)Haploid:lethaldead (sterile)The “diploid handicap” for recessive lethal studies:+ masks lethaleffects oflethalimmediatelyobviousThe problem with diploids in hunting for new recessive mutations:+++++++eggs+abcd+++++++spermmutagenize(altv.: dysgenic)FemaleMaleX+/++/+:PARENTS+/++/+a+/b+/PROGENYF1ab++++++form zygotesThe problem with diploids in hunting for new recessive mutations:mutagenize(altv.: dysgenic)FemaleMaleX+/++/++/++/+a+/b+/F1 PROGENY…the individual who can produce a-/a- offspring.How do we know who (if anyone) is carrying a- ?given:we are interested in(finding) the a-/a- phenotypeThe problem with diploids in hunting for new recessive mutations:mutagenize(altv.: dysgenic)FemaleMaleX+/++/++/++/+a+/b+/F1 PROGENYHow do we know who (if anyone) is carrying a- ?given:we are interested inthe a-/a- phenotype…the individual who can produce a-/a- offspring.To whom do we mate to find out?If we can “self” this individual,we are effectively mating to +/a- for sureYES! & we know in the F2of course, we hadto self everyone: No a-/a-The problem with diploids in hunting for new recessive mutations:mutagenize(altv.: dysgenic)FemaleMaleX+/++/++/++/+a+/b+/To whom do we mate to find out -- if we can’t self?+/++/++/+a+/+/+a+/…. we still don’tknow in the F2!+/+Meanwhile+/++/++/++/++/+F1The problem with diploids in hunting for new recessive mutations:mutagenize(altv.: dysgenic)FemaleMaleX+/++/++/++/+a+/b+/-- if we can’t self?+/++/+a+/+/+a+/…. we still don’tknow in the F2!+/++/++/++/++/+F1..but at leastnow we havepotential mates with a-F2a /aat least some chance:+/+at bestMateinter seMateinter seX+/+X+/+(must keep populationsseparate!)The problem with diploids in hunting for new recessive mutations:mutagenize(altv.: dysgenic)FemaleMaleX+/++/++/++/+a+/b+/-- if we can’t self?+/++/+a+/+/+a+/+/++/++/++/++/+F1F2a /aif we cross them, a-/a- will come:+/+onlycan wedo betterthanmating inter se?Mateinter se+/+Xmutagenize(altv.: dysgenic)FemaleMaleX+/++/++/++/+a+/b+/+/++/+a+/+/+a+/F1F2a /aif we cross them, a-/a- will come:we cando betterthanmating inter se+/+X+ = mutagenized but not desired mutant+ = non-mutagenized from original Mom+ = non-mutagenized from F1 matea- = mutagenized chromosome with new mut.It would help if we could keep track of chromosomes:Even nicerif we couldeliminate theextraneous animalsour friend, Herman Muller had the answer (early ‘30s):Balancer chromosomes:(a) a chromosome you can distinguish from the others.(b) a chromosome that will not recombine with others(c) a chromosome that will not “become” homozygous (i.e. that would either be lethal or sterile if homozygous)dominant marker mutant alleles (Bar, Curly, Stubble)crossover suppressors (multiple inversions)recessive lethal or sterile alleles(1)used them to determine mutation frequency: …how often a new recessive lethal arose on a given fly chromosome(2) used them to
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