Balancer chromosomes Reading for Monday s lecture genetic mosaics chimeras 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 p518 Aneuploid Mosaics expected lof mutant phenotype for pattern formation genes genes generating positional information 1 embryonic recessive lethal vvvvvvvvv vvvvvvvv vvvvvvv vvvvvvvvv vvvvvvvv vvvvvvv vvvvvvvvvv vvvvvvv Post Post polarity wildtype vvvvvvvv Ant vvvvvvv vvvvvvv vvvvvvvvv vvvvvvvvv vvvvvvvvvv vvvvvvvvvv vvvvvvvvvv 2 alterred dentical belt pattern exoskeleton in dead embryos dying fly embryos can still differentiate a lot vvvvvvvvv 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 vvvvvvvvvv pp731 732 What cells Post bicaudal Second Second chromosome brute force screen dominant secondtemperature chromosome sensitive balancer lethal DTS CyO females mutagenize X males non permissive DTS CyO females second chromosome markers eye color white cn bw CyO cn bw each son potentially carries a new mutant allele of interest but a different new mutant in each let single sons take individual males and mate separately 10 000 crosses DTS CyO females non permissive X CyO cn bw let single sons CyO cn bw let a daughters unwanted sibs all die cn bw let a X CyO sons CyO CyO each group of progeny separately forced incest Second chromosome screen CyO cn bw let a daughters cn bw let a X CyO sons DTS CyO females DTS CyO females CyO cn bw let a to maintain any new let mutation cn bw let a cn bw let a do they all die no white eyes and if so when how Brute force mutagenize each group of progeny separately forced incest cn bw X males each son potentially carries a new mutant allele of interest X CyO cn bw mut CyO CyO 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 DTS CyO DTS cn bw let 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 1 Second chromosome screen Temperature for first cross doesn t really matter mutagenize DTS CyO cn bw X females males non permissive OR permissive DTS CyO X females 1 have to handpick males anyway CyO cn bw mut single sons 2 males have no meiotic recombination so DTS mut OK or DTS cn bw mut 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 non permissive CyO cn bw mut a daughters X CyO cn bw mut a ey1 recessive hypomorph adults w no eyes ey null recessive embryonic lethal CyO cn bw mut a sons Got lucky with ey1 cn bw mut a cn bw mut a CyO CyO can we overcome the limitations of pleiotropy ey is pleiotropic multiple unrelated phenotypes functions how many other important eye genes missed 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 YES we shall overcome but first already mentioned one way to deal with pleiotropy temperature conditional mutant alleles FAR BETTER ts muts way too limited even in flies worms 1 genetically sensitize the system turn lof recessives into dominants but only with respect to goal make genes artificially haploinsufficient Illustrate with example from fly eye development studies A developmental decision R7 precursor cell signal from photo R8 neighbor cone photorecptr cell recptr 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 Stemmed from original observation 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 one non essential aspect of the genes function make genes artificially haploinsufficient R7 precursor cell signal from photo R8 neighbor cone photorecptr cell recptr How many pleiotropic genes missed sev sev R7 photoreceptor missing turned into cone cell sev encodes v src homolog human oncogene 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 3rd chromosome balancer sev sev TM3 P sevB4 ts designer ts allele Found many pleiotropic lof alleles of both types incl recessive lethals growth temperature phenotype screen for dominant mutations that make Poising sev activity level on a phenotypic threshold made other genes haploinsufficient but only with respect to sev function 24 3oC R7 absent R7 present Dominant suppressors 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 22 7oC R7 present R7 absent if take away that cushion now more sensitive to reductions in other gene levels Dominant enhancers 2 made genes artificially haploinsufficient R7 precursor cell signal from cone photo R8 neighbor cell recptr sevenless wildtype vs sev sev R7 photoreceptor missing turned into cone cell Point to keep in mind will not necessarily identify every relevant gene in pathway this way
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