Reading for Mondays lecture genetic mosaics chimeras p

Reading for Monday’s lecture: (genetic mosaics & chimeras) p 518 (“Aneuploid Mosaics…”) pp 731 -732 (“What cells…”) I will hold office hours during spring break, but at a different time than normal: during class time (11 a-12 N) 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 vvvvv vvvvvvv polarity>>> vvvvv Post. vvvv wildtype vvvvvvv Ant. vvvvvvvvv vvvvvvvvvv vvvvvvv (2) alterred dentical belt pattern (exoskeleton) in dead embryos (dying fly embryos can still differentiate a lot) Post. “bicaudal”

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

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

Cy. O / cn bw let-a? daughters cn bw let-a? X Cy. O /sons each group of progeny separately (forced incest): Cy. O / cn bw let-a? to maintain any new let mutation cn bw let-a? / cn bw let-a? Cy. O / Cy. O do they all die? (no white eyes? ) and if so, when? how? always die only after a 2 nd 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 / Cy. O females Brute force cn bw X males each son potentially carries a new mutant allele of interest X Cy. O / cn bw & mut? ? Cy. O / cn bw mut-a? daughters F 1 generation single sons cn bw mut-a? X Cy. O / sons cn bw mut-a? / cn bw mut-a? F 2 generation keep populations separate! only after a 2 nd generation of 10, 000 crosses did they know which original F 1 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 / Cy. O females cn bw X males @non-permissive OR permissive DTS / Cy. O females X (1) have to handpick males anyway Cy. O / cn bw & mut? ? single sons or (2) males have no meiotic recombination (so DTS/mut OK) DTS / cn bw & mut? ? @non-permissive Cy. O / cn bw mut-a? daughters cn bw mut-a? X Cy. O / sons Cy. O / cn bw mut-a? Cy. O / Cy. O

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: ey 1 : recessive hypomorph, adults w/ no eyes ey is pleiotropic -(null) ey : recessive embryonic lethal (multiple “unrelated” phenotypes/functions) Got lucky with ey 1 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 F 1 (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: R 7 precursor cell signal from ? photo- R 8 neighbor cone photorecptr cell recptr sevenless/+ (wildtype) vs. sev/sev R 7 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 R 7 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 R 7 precursor cell signal from ? photo- R 8 neighbor cone photorecptr cell recptr How many pleiotropic genes missed? R 7 photoreceptor sev/sev missing (turned into cone cell) sev encodes v-src homolog (human oncogene) (3 rd chromosome balancer) sev- /sev- ; TM 3, P{sev. B 4(ts)}/+ designer ts allele growth temperature phenotype screen for dominant mutations that make: 24. 3 o. C R 7 absent R 7 present (Dominant suppressors) 22. 7 o. C R 7 present R 7 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. 3 o. C R 7 absent R 7 present (Dominant suppressors) 22. 7 o. C R 7 present R 7 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 R 7 precursor cell signal from cone photo- R 8 neighbor cell recptr now other genes in pathway other genes in ARE pathway NOT haploinsufficient sevenless/”+“ sevenless/+ (wildtype) vs. sev/sev R 7 photoreceptor missing (turned into cone cell) adjust level to poise system on phenotypic threshold …. then 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 R 7 cell that responds to signal from R 8 bride-of-sevenless: ligand (signal molecule) generated in R 8 no new mutant alleles found in sev sensitized screen!

Another way around the limitations of pleiotropy in genetic screens: (2) use targetted genetic mosaics to screen for recessives in the F 1 (homozygous clones in heterozygotes …in non-essential tissues only!) …recover new recessives in the F 1? ? ? Based on a phenominon discovered (‘ 30 s) 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