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Reading for Monday’s lecture: ( genetic mosaics & chimeras )

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.

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Reading for Monday’s lecture: ( genetic mosaics & chimeras )

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  1. 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

  2. Consider the brute-force screen that led to the last fly Nobel Prize N-V & W: v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v Ant. Post. v v v v v v Post. Post. v v v v v v v v v v v v v v v v polarity>>> wildtype “bicaudal” Balancer chromosomes: Using them to follow chromosomes in mutant screens 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 (2) alterred dentical belt pattern (exoskeleton) in dead embryos (dying fly embryos can still differentiate a lot)

  3. Second dominant temperature- sensitive lethal mutagenize second- chromosome balancer second-chromosome “markers” (eye color = white) X cn bw DTS / CyO males females @non-permissive CyO / cn bw & let?? DTS / CyO females sons single Second chromosome (brute force) screen each son potentially carries a new mutant allele of interest …but a different new mutant in each take individual males and mate separately (10,000 crosses)

  4. @non-permissive X unwanted sibs all die CyO / cn bw let-a? CyO / cn bw let-a? sons daughters each group of progeny separately (forced incest): CyO / CyO DTS / CyO DTS/ cn bw let? X CyO / cn bw & let?? DTS / CyO females sons single

  5. CyO / CyO CyO / cn bw let-a? cn bw let-a? / cn bw let-a? X CyO / cn bw let-a? CyO / cn bw let-a? sons daughters each group of progeny separately (forced incest): to maintain any new let mutation 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)

  6. Brute force mutagenize F1 generation F2 generation …and if looking for maternal-effect mutations, go blindly one generation more! Second chromosome screen cn bw DTS / CyO X males females each son potentially carries a new mutant allele of interest DTS / CyO X CyO / cn bw & mut?? females sons single X CyO / cn bw mut-a? CyO / cn bw mut-a? sons daughters keep populations separate! cn bw mut-a? / cn bw mut-a? only after a 2nd generation of 10,000 crosses did they know which original F1 sons carried mutations of value

  7. mutagenize @non-permissive OR permissive or DTS / cn bw & mut?? Second chromosome screen Temperature for first cross doesn’t really matter: cn bw DTS / CyO X males females (1) have to hand- pick males anyway (2) males have no meiotic recombination (so DTS/mut OK) CyO / cn bw & mut?? X single DTS / CyO sons females @non-permissive X CyO / cn bw mut-a? CyO / cn bw mut-a? sons daughters CyO / CyO CyO / cn bw mut-a? cn bw mut-a? / cn bw mut-a?

  8. 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 (multiple “unrelated” phenotypes/functions) ey-(null) : recessive embryoniclethal Got lucky with ey1 how many other important eye genes missed? …can we overcome the limitations of pleiotropy?

  9. FAR BETTER …can we overcome the limitations of pleiotropy? YES…we shall overcome but first: already mentioned one way to deal with pleiotropy ts muts. way too limited even in flies & worms temperature-conditional mutant alleles (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!)

  10. signal from R8 neighbor cone cell ? R7 precursor cell sevenless/+ (wildtype) vs. sev/sev R7 photoreceptor missing (turned into cone cell) photo- recptr photo- recptr cone cell son-of-sevenless seven-in-absentia seven-up (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: null sev allele: same thing (hence, eye-specific) bride-of-sevenless (null eye-specific) Other genes discovered to be involved in the R7 precursor decision: null alleles noteye-specific: pleiotropic: How many other pleiotropic genes missed?

  11. signal from R8 neighbor cone cell ? R7 precursor cell photo- recptr photo- recptr (3rd chromosome balancer) sev-/sev- ; TM3,P{sevB4(ts)}/+ designer ts allele cone cell growth temperature phenotype (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 photoreceptor missing (turned into cone cell) sev/sev sev encodes v-src homolog (human oncogene) How many pleiotropic genes missed? screen for dominant mutations that make: 24.3oC R7 absent R7 present(Dominant suppressors) 22.7oC R7 present R7 absent(Dominant enhancers)

  12. growth temperature phenotype (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: 24.3oC 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

  13. signal from R8 neighbor R7 precursor cell sevenless/+ (wildtype) vs. sev/sev R7 photoreceptor missing (turned into cone cell) photo- recptr cone cell adjust level to poise system on phenotypic threshold sevenless/”+“ made genes “artificially” haploinsufficient now other genes in pathway ARE haploinsufficient other genes in pathway NOT haploinsufficient ….then look for newly induced dominant enhancer or suppressor alleles

  14. 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! Point to keep in mind: …will not necessarily identify every relevant gene in pathway this way

  15. Another way around the limitations of pleiotropy in genetic screens: (2) use targetted genetic mosaics to screen for recessives in the F1 (homozygous clones in heterozygotes …in non-essential tissues only!) …recover new recessives in the F1??? Based on a phenominon discovered (‘30s) by former chair of U.C. Zoology Dept: mitotic recombination but improved upon enormously in modern times …only possible because of a very strange aspect of fly chromosome behavior: homologous chromosomes pair during mitotic interphase

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