1. Reading on inversions and their genetic consequences pp501-503 genetic mosaics: pp152-4 (Mitotic recombination…and cancer) 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. 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

2. Female +/+ Male mutagenize X +/+ +/++/+ +/++/+ a +/+/ b +/+/ F1 +/++/+ +/++/+ a +/+/ +/++/+ a +/+/ F2 a /a +/+ X 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: In connection with genetic screens & selections:

3. Herman Muller gave fly workers Balancer chromosomes for this purpose (early ‘30s - '40s): (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 …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:

4. p503: Fig. 14.17 (read about inversions and their genetic consequences on pp501-503)

5. Balancer chromosomes: (2) use them to “maintain” deleterious recessive alleles of interest +/++/+let/+let/let && let/+ X Problem without balancer: …hence parents must have carried let and some siblings do Must rely on matings with the let carriers to maintain the let allele.

6. Balancer chromosomes: (2) use them to “maintain” deleterious recessive alleles of interest Problem without balancer: +/++/+let/+let/let && +/++/+ let/+ & X +/++/+ X +/++/+ +/++/+ +/++/+ X +/++/+ let/let && let/+ X Three possible random sib matings:

7. …but with a balancer chromosome: let/Bal -A - let -A + Dom Dom + let Inv-B - let-B + let-A - /Bal X let-A - /let-A - lethal let-A - /Bal O.K. Bal/Bal lethal Balanced lethal condition progeny just like parents by default let/Bal, =

8. Balancer chromosomes: (2) used them to “maintain” deleterious recessive alleles of interest What about an X-linked recessive lethal? lethal let-A - /Bal O.K. female Bal/Bal sterile female Balanced condition lethal or sterile recessive female-specific sterile /Y/Y let-A - /Y lethal male Bal /Y O.K. male let-A - /Bal let-A - /Y X or Bal /Y female male

9. (1) use them to follow chromosomes in mutant screens Balancer chromosomes: 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: (1) embryonic recessive lethal (2) alterred dentical belt pattern (exoskeleton) in dead embryos 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 wildtype 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 “bicaudal” Post. (dying fly embryos can still differentiate a lot) (genes generating positional information) Ant. Post. polarity>>> Consider the brute-force screen that led to the last fly Nobel Prize N-V & W:

10. Using them to follow chromosomes in mutant screens Balancer chromosomes: 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: (1) embryonic recessive lethal (2) alterred dentical belt pattern (exoskeleton) in dead embryos 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 wildtype 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 “bicaudal” Post. (dying fly embryos can still differentiate a lot) (genes generating positional information) Ant. Post. polarity>>> Consider the brute-force screen that led to the last fly Nobel Prize N-V & W:

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

12. DTS / CyO females single CyO / cn bw & let?? sons X X each group of progeny from a particular male are kept separate (forces incest) CyO / cn bw let-a? sons daughters CyO / CyODTS / CyO DTS/ cn bw let? unwanted sibs all die @non-permissive

13. CyO / cn bw let-a? cn bw let-a? / cn bw let-a? CyO / CyO to maintain any new let mutation in a balanced lethal condition 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) CyO / cn bw let-a? sons daughters X The progeny from forced incest:

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

15. DTS / CyO females cn bw males mutagenize CyO / cn bw mut-a? cn bw mut-a? / cn bw mut-a? CyO / CyO X X DTS / CyO females single sons X CyO / cn bw & mut?? @non-permissive OR permissive DTS / cn bw & mut?? or Temperature for first cross doesn’t really matter: (1) have to hand- pick males anyway (2) males have no meiotic recombination (so DTS/mut OK) CyO / cn bw mut-a? sons daughters @non-permissive Either way, this is all we get.

16. 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 (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!). …can we overcome the limitations of recessiveness? &Early What if want to study something like eye development instead?

17. ey -(null) : recessive embryonic lethal ey is pleiotropic (multiple “unrelated” phenotypes/functions) Attractive features: interesting AND non-essential (and more), …can we overcome the limitations of pleiotropy? Got lucky with ey 1 how many other important eye genes missed? ey 1 :recessive hypomorph, adults w/ no eyes but consider:

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

19. (1) genetically sensitize the system: turn lof recessives into dominants (but only with respect to one non-essential aspect of the genes’ function) Illustrate with example from fly eye development studies: ? cone cell photo- recptr photo- recptr cone cell signal (from R8 neighbor) One of many cell fate decisions made during eye development: R7 precursor cell goal: make genes “artificially” haploinsufficient

20. sevenless/+ (wildtype) vs. sev/sev R7 photoreceptor missing (turned into cone cell) ? cone cell photo- recptr photo- recptr cone cell signal from R8 neighbor R7 precursor cell The observation that started it all: bride-of-sevenless (null eye-specific) seven-in-absentia son-of-sevenless seven-up Other genes discovered to be involved in the R7 precursor decision: sev mutant allele was a null (hence, eye-specific) null alleles not eye-specific: pleiotropic: How many other pleiotropic genes missed?

21. (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.

22. sev - /sev - ; / + sev/sev R7 photoreceptor missing (turned into cone cell) sev encodes v-src homolog (human oncogene) screen for dominant mutations that make: 24.3 o C 22.7 o C R7 absent R7 present R7 present (Dominant suppressors) R7 absent (Dominant enhancers) growth temperature phenotype designer ts allele modeled after ts human allele P{sev B4(ts) } (3rd chromosome balancer) TM3, Clearly at either of these two temperatures, the system governing the R7 decision is poised on a phenotypic threshold

23. screen for dominant mutations that make: 24.3 o C 22.7 o C R7 absent R7 present R7 present (Dominant suppressors) R7 absent (Dominant enhancers) growth temperature phenotype Found many pleiotropic lof alleles of both types IN AN F1 GENETIC SCREEN: dominant enhancers or suppressors of the R7 phenotype. Poising sev + activity level on a phenotypic threshold made other genes haploinsufficient 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 but only with respect to sev function! But many of these DOMINANT "modifiers" were also recessive lethal (pleiotropic -- had other essential functions).

24. 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. R7 precursor cell photo- recptr cone cell signal from R8 neighbor ….then can look for newly induced dominant enhancer or suppressor alleles adjust level to poise system on phenotypic threshold sevenless/”+“ other genes in pathway NOT haploinsufficient now other genes in pathway ARE haploinsufficient Asked: what genes work with sevenless to control this developmental decision?

25. 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!