Reading 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 different time than normal during class time 11a 12N on March 26 March 30 Balancer chromosomes Using them to follow chromosomes in mutant screens Consider the brute force screen that led to the last fly Nobel Prize N V W Aim find genes that allow cells to know where they are so the cells can know what they should be expected lof mutant phenotype for pattern formation genes genes generating positional information 1 embryonic recessive lethal vvvvvvvvvv vvvvvvvvv vvvvvvvv vvvvvvv vvvvvvvvvv polarity vvvvvvvvv Post vvvvvvvv wildtype vvvvvvv Ant vvvvvvv vvvvvvv vvvvvvvvv vvvvvvvvv vvvvvvvvvv vvvvvvvvvv vvvvvvvvvv vvvvvvvvv vvvvvvvv vvvvvvv 2 alterred dentical belt pattern exoskeleton in dead embryos dying fly embryos can still differentiate a lot Post Post bicaudal Second chromosome brute force screen Second dominant secondtemperature chromosome sensitive balancer lethal DTS CyO females mutagenize X cn bw males non permissive DTS CyO females CyO cn bw single sons second chromosome markers eye color white each son potentially carries a new mutant allele of interest new mutant let but a different in each take individual males and mate separately 10 000 crosses DTS CyO females non permissive CyO cn bw let a daughters X CyO cn bw let single sons X CyO cn bw let a sons each group of progeny separately forced incest unwanted sibs all die CyO CyO DTS CyO DTS cn bw let CyO cn bw let a daughters cn bw let a X CyO sons each group of progeny separately forced incest CyO cn bw let a to maintain any new let mutation cn bw let a cn bw let a CyO CyO do they all die no white eyes and if so when how always die only after a 2nd generation of 10 000 crosses did they know which individual sons of mutagenized males carried a recessive lethal mutation of interest value Second chromosome screen Brute force mutagenize DTS CyO females DTS CyO females cn bw X males each son potentially carries a new mutant allele of interest X CyO cn bw mut CyO cn bw mut a daughters F1 generation single sons X CyO cn bw mut a sons cn bw mut a cn bw mut a F2 generation keep populations separate only after a 2nd generation of 10 000 crosses did they know which original F1 sons carried mutations of value and if looking for maternal effect mutations go blindly one generation more Second chromosome screen Temperature for first cross doesn t really matter mutagenize DTS CyO females cn bw X males non permissive OR permissive DTS CyO females X 1 have to handpick males anyway CyO cn bw mut single sons or 2 males have no meiotic recombination so DTS mut OK DTS cn bw mut non permissive CyO cn bw mut a daughters X CyO cn bw mut a CyO cn bw mut a sons cn bw mut a cn bw mut a CyO CyO 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 can we overcome the limitations of recessiveness 2 recognizing an informative phenotype is a large part of the genetics game The N V W advantage an informative phenotype that could be scored in dead embryos didn t demand survival or much else Early What if want to study something like eye development instead Attractive features interesting AND non essential and more but consider ey1 recessive hypomorph adults w no eyes ey is pleiotropic ey null recessive embryonic lethal multiple unrelated phenotypes functions Got lucky with ey1 how many other important eye genes missed can we overcome the limitations of pleiotropy can we overcome the limitations of pleiotropy YES we shall overcome but first already mentioned one way to deal with pleiotropy FAR BETTER temperature conditional mutant alleles ts muts way too limited even in flies worms 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 1 genetically sensitize the system turn lof recessives into dominants but only with respect to one non essential aspect of the genes function goal make genes artificially haploinsufficient Illustrate with example from fly eye development studies A developmental decision Stemmed from original observation R7 precursor cell signal from R8 neighbor cone photophotorecptr cell recptr sevenless wildtype vs sev sev R7 photoreceptor missing turned into cone cell null sev allele same thing hence eye specific bride of sevenless null eye specific null alleles not eye specific pleiotropic son of sevenless seven in absentia How many other pleiotropic genes missed seven up Other genes discovered to be involved in the R7 precursor decision 1 genetically sensitize the system turn lof recessives into dominants but only with respect to one non essential aspect of the genes function make genes artificially haploinsufficient R7 precursor cell signal from R8 neighbor cone photophotorecptr cell recptr How many pleiotropic genes missed sev sev R7 photoreceptor missing turned into cone cell sev encodes v src homolog human oncogene 3rd chromosome balancer sev sev TM3 P sevB4 ts designer ts allele growth temperature phenotype screen for dominant mutations that make 24 3oC R7 absent R7 present Dominant suppressors 22 7oC R7 present R7 absent Dominant enhancers 1 genetically sensitize the system turn lof recessives into dominants but only with respect to one non essential aspect of the genes function growth temperature phenotype screen for dominant mutations that make 24 3oC R7 absent R7 present Dominant suppressors 22 7oC R7 present R7 absent Dominant enhancers Found many pleiotropic lof alleles of both types incl recessive lethals Poising sev activity level on a phenotypic threshold made other genes haploinsufficient but only with respect to sev function Wildtype 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 levels made genes artificially haploinsufficient R7 precursor cell signal from R8 neighbor cone photocell recptr now other genes in pathway other genes ARE in pathway NOT haploinsufficient haploinsufficient sevenless sevenless wildtype vs sev sev R7 photoreceptor missing turned into cone
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