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Reading on inversions and their genetic consequences pp501-503 genetic mosaics: pp152-4 (Mitotic recombination…and cancer) p518 (“Aneuploid Mosaics…”) pp731-2 (“What cells…”) I'll be holding office hours (once) during spring break and at a special time: Wed, March 26, 11a-12N I’ve asked to reserve a room from 7-9pm Wednesday, April 9, for a review session in connection with the midterm on Monday, April 14. I’ll announce where the session will be held as soon as I know myself. In connection with genetic screens & selections: it would help if we could keep track of chromosomes and eliminate extraneous animals so that we don't have to rely on random inter se matings for the F2 cross: mutagenize Female X Male +/+ +/+ +/+ +/b +/+ F1 +/ a X +/+ +/ a F2 +/+ +/ a +/+ +/+ a /a Herman Muller gave fly workers Balancer chromosomes for this purpose (early ‘30s - '40s): …not only to facilitate mutant screens (and selections) but also to faciliate the maintenance of the deleterious alleles recovered in such screens and selections. Three key features: (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 cannot become homozygous recessive lethal or sterile alleles p503: Fig. 14.17 (read about inversions and their genetic consequences on pp501-503) Balancer chromosomes: (2) use them to “maintain” deleterious recessive alleles of interest …hence parents must have carried let and some siblings do Problem without balancer: let/+ X let/+ +/+ & let/+ & let/let Must rely on matings with the let carriers to maintain the let allele. Balancer chromosomes: (2) use them to “maintain” deleterious recessive alleles of interest Problem without balancer: let/+ X let/+ +/+ & let/+ & let/let let/+ X let/+ +/+ & let/+ & let/let Three possible let/+ X random sib matings: +/+ +/+ X +/+ +/+ & let/+ +/+ …but with a balancer chromosome: let/Bal = let -A- let-B+ Dom+ /Bal, let-A+ Dom let -B- Inv let-A-/Bal X let-A-/Bal progeny just like parents by default let-A-/let-A- lethal let-A-/Bal O.K. Bal/Bal Balanced lethal condition lethal Balancer chromosomes: (2) used them to “maintain” deleterious recessive alleles of interest What about an X-linked recessive lethal? female let-A-/Bal male recessive female-specific sterile X let-A-/Y or Bal /Y lethal Balanced condition lethal or sterile let-A-/Bal O.K. female Bal/Bal sterile female let-A-/Y lethal male Bal /Y O.K. male Balancer chromosomes: (1) use 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 how they should differentiate expected lof mutant phenotype for “pattern formation” genes: (genes generating positional information) (1) embryonic recessive lethal vvvvvvvvvv vvvvvvvvv vvvvvvvv vvvvvvv vvvvvvvvvv wildtype polarity>>> vvvvvvvvv Post. vvvvvvvv Ant. vvvvvvv 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” 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 wildtype polarity>>> vvvvvvvvv Post. vvvvvvvv Ant. vvvvvvv 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 Second chromosome (brute force) screen dominant secondtemperature- chromosome sensitive balancer lethal DTS / CyO mutagenize X females cn bw males @non-permissive temp. for progeny DTS / CyO females X CyO / cn bw single sons second-chromosome “markers” (eye color = white) each son potentially carries a new recessive mutant allele of interest …but a different new mutant in each & let?? 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 from a particular male are kept separate (forces 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 The progeny from forced incest: CyO / cn bw let-a? cn bw let-a? / cn bw let-a? to maintain any do they all die? (no white eyes?) new let mutation in a and if so, when? how? balanced lethal condition 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) Second chromosome screen mutagenize DTS / CyO females Brute force cn bw X males each son potentially carries a new mutant allele of interest DTS / CyO females X CyO / cn bw & mut?? F1 generation single sons CyO / cn bw mut-a? daughters cn bw mut-a? X CyO / 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! Temperature for first cross doesn’t really matter: mutagenize DTS / CyO females X @non-permissive OR permissive DTS / CyO females X cn bw males (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 Either way, this is all we get. CyO / cn bw mut-a? daughters cn bw mut-a? X CyO / sons CyO / cn bw mut-a? 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? 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-(null) : recessive embryonic lethal Got lucky with ey1 ey is pleiotropic (multiple “unrelated” phenotypes/functions) 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 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 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: One of many cell fate decisions made during eye development: R7 precursor cell signal (from ? photo- R8 neighbor) cone photorecptr cell recptr R7 precursor cell signal from ? photo- R8 neighbor cone photorecptr cell recptr Other genes discovered to be involved in the R7 precursor decision: The observation that started it all: sevenless/+ (wildtype) vs. sev/sev R7 photoreceptor missing (turned into cone cell) sev mutant allele was a null (hence, eye-specific) bride-of-sevenless (null eye-specific) null alleles not eye-specific: pleiotropic: son-of-sevenless seven-in-absentia seven-up 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) make genes “artificially” haploinsufficient Isolate mutant alleles that interfere with eye development but do not disrupt other (perhaps essential) functions that some genes may have. R7 photoreceptor missing sev/sev (turned into cone cell) sev encodes v-src homolog (human oncogene) (3rd chromosome balancer) sev- /sev- ; TM3, P{sevB4(ts)} /+ designer ts allele modeled after ts human 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) Clearly at either of these two temperatures, the system governing the R7 decision is poised on a phenotypic threshold 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 IN AN F1 GENETIC SCREEN: dominant enhancers or suppressors of the R7 phenotype. But many of these DOMINANT "modifiers" were also recessive lethal (pleiotropic -- had other essential functions). 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 R7 precursor cell signal from cone photo- R8 neighbor cell recptr Asked: what genes work with sevenless to control this developmental decision? Found those other genes by making them “artificially” haploinsufficient with respect to the sev function, but NOT haploinsufficient with respect to other functions that they might have. now other genes in pathway other genes in ARE pathway NOT haploinsufficient haploinsufficient sevenless/”+“ adjust level to poise system on phenotypic threshold ….then can look for newly induced dominant enhancer or suppressor alleles Point to keep in mind: …will not necessarily identify every relevant gene in pathway this way sevenless: receptor in R7 cell that responds to signal from R8 bride-of-sevenless: ligand (signal molecule) generated in R8 no new mutant alleles found in sev sensitized screen!