1Reading for Monday’s lecture: (genetic mosaics & chimeras)p518 (“Aneuploid Mosaics…”)pp731-732 (“What cells…”)I will hold office hours during spring break, but at a differenttime than normal:during class time (11a-12N) on March 26 & March 30Using them to follow chromosomes in mutant screensBalancer chromosomes:Aim: find genes that allow cells to know where they are so the cells can know what they should beexpected lof mutant phenotype for “pattern formation” genes:(1) embryonic recessive lethal(2) alterred dentical belt pattern (exoskeleton) in dead embryos vvvvvvvv vvvvvvv vvvvvvvvv vvv vvvvvvv vvvvvvvvv vvv vvvvvvv vvvvvvv vvvvvvv vvvvvvvv vvvvvvvv vvvwildtypevvvvv vvv vvvvvvv vv vvvvvvvvvvvvvvvvvvvvvvv vvvvvvvvvvvvvvvv vvvvvvvvvvvv“bicaudal”Post. Post.(dying fly embryos can still differentiate a lot)(genes generating positional information)Ant. Post.polarity>>>Consider the brute-force screen that led to the last fly Nobel Prize N-V & W: Second chromosome (brute force) screenDTS / CyOfemalescn bwmalesmutagenizeeach son potentiallycarries a new mutantallele of interest…but a different new mutantin eachSecondtake individual malesand mate separately(10,000 crosses)DTS / CyOfemalessingledominanttemperature-sensitivelethalsecond-chromosomebalancersecond-chromosome“markers”(eye color = white)CyO / cn bw & let??sonsX@non-permissiveDTS / CyOfemalessingleCyO / cn bw & let??sonsXCyO / cn bw let-a? CyO / cn bw let-a?sonsdaughtersXeach group of progeny separately (forced incest):CyO / CyO DTS / CyODTS/ cn bw let?unwanted sibs all die@non-permissiveCyO / cn bw let-a?cn bw let-a? / cn bw let-a?CyO / CyOto maintain anynew let mutationdo they all die? (no white eyes?)and if so, when? how?always dieonly after a 2nd generation of 10,000 crossesdid they know which individual sons of mutagenized malescarried a recessive lethal mutation of interest (value)CyO / cn bw let-a? CyO / cn bw let-a?sonsdaughtersXeach group of progeny separately (forced incest):Second chromosome screenDTS / CyOfemalescn bwmalesmutagenizeeach son potentiallycarries a new mutantallele of interestCyO / cn bw mut-a? CyO / cn bw mut-a?sonsdaughterscn bw mut-a? / cn bw mut-a?DTS / CyOfemalessingleXCyO / cn bw & mut??sonsXXonly after a 2nd generation of 10,000 crossesdid they know which original F1 sons carried mutations of valueF1 generationF2 generationBrute forcekeep populationsseparate!…and if looking for maternal-effect mutations, go blindly one generation more!2Second chromosome screenDTS / CyOfemalescn bwmalesmutagenizeCyO / cn bw mut-a? CyO / cn bw mut-a?sonsdaughtersCyO / cn bw mut-a?cn bw mut-a? / cn bw mut-a?CyO / CyODTS / CyOfemalessingleXsonsXXCyO / cn bw & mut??@non-permissive@non-permissiveOR permissiveDTS / cn bw & mut??orTemperature forfirst cross doesn’treally matter:(1) have to hand-pick males anyway(2) males have nomeiotic recombination (so DTS/mut OK)Classic N-V&W screen illustrates two important points:(1) recessiveness (~lof) generally demands multiple generations of blind forced incest crosses (mating siblings) to recover mutant(2) recognizing an informative phenotype is a large part of the genetics gameThe N-V & W advantage: an informative phenotype that could be scored in dead embryos (didn’t demand survival -- or much else!). What if want to study something like eye development instead?ey1 :recessive hypomorph, adults w/ no eyesey-(null) : recessive embryonic lethaley is pleiotropic(multiple “unrelated”phenotypes/functions)…can we overcome the limitations of recessiveness?Attractive features: interesting AND non-essential (and more), but consider:…can we overcome the limitations of pleiotropy?Got lucky with ey1how many other important eye genes missed?&EarlyYES…we shall overcome(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)(2) use targetted genetic mosaics to screen for recessives in the F1 (homozygous clones in heterozygotes …in non-essential tissues only!)but first: already mentioned one way to deal with pleiotropytemperature-conditionalmutant allelests muts. waytoo limitedeveninflies& wormsFARBETTER…can we overcome the limitations of pleiotropy?(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)sevenless/+ (wildtype)vs. sev/sevR7 photoreceptormissing(turned into cone cell)Illustrate with example from fly eye development studies:?conecellphoto-recptrphoto-recptrconecellsignal fromR8 neighborA developmental decision:R7 precursor cellStemmed from original observation:goal: make genes “artificially” haploinsufficientbride-of-sevenless (null eye-specific)seven-in-absentiason-of-sevenlessseven-upOther genes discoveredto be involved in theR7 precursor decision:null sev allele: same thing(hence, eye-specific)null alleles not eye-specific:pleiotropic:How many other pleiotropic genes missed?(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)sev/sevR7 photoreceptormissing(turned into cone cell)?conecellphoto-recptrphoto-recptrconecellsignal fromR8 neighborR7 precursor cellmake genes “artificially” haploinsufficientHow many pleiotropic genes missed?sev- /sev- ; TM3,P{sevB4(ts)}/+designer ts allelesev encodes v-src homolog (human oncogene)screen for dominant mutations that make:24.3oC22.7oCR7 absentR7 presentR7 present (Dominant suppressors)R7 absent (Dominant enhancers)growthtemperaturephenotype(3rd chromosome balancer)(1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function)screen for dominant mutations that make:24.3oC22.7oCR7 absentR7 presentR7 present (Dominant suppressors)R7 absent (Dominant enhancers)growthtemperaturephenotypeFound many pleiotropic lof alleles of both types (incl. recessive lethals).Poising sev+ activity level on a phenotypic threshold made other genes haploinsufficientWildtype fly must normally have an excess of sev+ activity as insurance, so it can tolerate fluctuations in levels of other genes in pathway during development…if take away that cushion, now more sensitive to reductions in other gene levelsbut only with respect to sev function!3sevenless/+
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