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The date for the review for the midterm has been changed to Thursday eve. (7-9p) April 10 since there is a conflict with a night exam on the 9th. I’ll announce where the session will be held as soon as I know myself. Reading on sex: pp85-88 (In many species…) pp109-110 review (Analysis of rare mistakes…) pp516-518 (Changes in chrom. # --to--Some euploid…) Table 14.2 (p526) pp664-665 (RNA splicing helps regulate gene expression) pp669-676 except 670-71 (Sex determination in Drosophila…) On Monday: (1) genetically sensitize the system: “turn” lof recessives into dominants (but only with respect to one non-essential aspect of their function) Poising the activity level of “your favorite gene” on a phenotypic threshold to make other genes that work with it in a regulatory pathway haploinsufficient …but only with respect to the functioning of “your favorite gene.” So that: (1) we can identify new mutations of interest in the F1 generation (first generation after mutagenizing the parents) AND (2) can overcome some complications of pleiotropy …so that we can more easily study the non-vital aspects of the functioning of genes that ALSO have vital functions (1) genetically sensitize the system: “turn” lof recessives into dominants (but only with respect to one non-essential aspect of their function) Based on the rationale that: Wildtype organism must normally have an excess of most genes’ activity as insurance against fluctuations in the levels of activity of various genes in a pathway during development …if take away that cushion for any one gene in a pathway, now make the normal operation of the pathway …with respect to that one gene… more vulnerable to reductions in the levels of other gene products with which it works 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 without making them dominant !!! In your text's glossary: genetic mosaics see: mosaics “an organism containing tissues of different genotypes” cells …all derived from a single initial cell (zygote) genetic mosaic vs: organism (genetic) chimera: an embryo or animal composed of cells from two or more different organisms individuals genetic mosaics are extremely useful for determining where a particular gene’s function is needed: Your text short-changes the key concept of cell autonomy in gene function Fig 20.14: + + - + + genetic mosaics are extremely useful for determining where a particular gene’s function is needed: nonmosaic wildtype L2 L1 L2 L1 phenotypic effect of ag- vs. ag+ differentiation non-mosaic ag- mutant L2 L1 L2 L1 In what cell type (L2 vs. L1) is ag+ function needed to elicit normal differentiation ( ) of L1? Where is ag+ needed? mosaic white marks ag- nonmosaic wildtype ag- ag+ mosaic L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 ag+ non-mosaic ag- mutant L2 L1 ag- L1 differentiates abnormally despite being ag+ L1 differentiates normally despite being ag- ag seems to work in L2 to signal L1 (like boss) For ag, the cell whose phenotype is mutant is not the cell whose genotype is mutant. Hence ag is not cell autonomous with respect to the L1 phenotype The “genetic marker” is cell autonomous (that’s why it was chosen),since the cell whose marker phenotype is mutant is the cell whose marker genotype is mutant. …if a cell’s genotype in the mosaic dictates that cell’s phenotype nonmosaic wildtype ag- L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 L2 L1 ag+ non-mosaic ag- mutant ag is nonautonomous ag+ ag+ ag- ag- ag seems to work in L2 to signal L1 When discussing autonomy/nonautonomy, need to specify the phenotype in question: …ag is non-autonomous with respect to the L1 differentiation phenotype …perhaps ag is autonomous with respect to generating the signal in L2. (if we had an assay for the signal, we might see that phenotype directly) T.H.Morgan published on genetic mosaics in 1914: do males normally have white eyes? X X X X first nuclear division of an XX zygote Hence, ~50% w+ vs. w…mosaic patches too large for our purposes X X w-/w+ w-/-- XX XO XO XX otherwise diploid (AA) stable thereafter Fig. 14.33 Another way around the limitations of pleiotropy in genetic screens: (2) use genetic mosaics to screen for recessives in the F1 …look for homozygous mutant clones in otherwise heterozygous animals …identify (and recover) new recessives in the F1 even works for new mutants that are recessive lethal or sterile provided -- one generates clones in only a small fraction of all cells or -- one generates clones only in non-essential tissues Another way around the limitations of pleiotropy in genetic screens: (2) use genetic mosaics to screen for recessives in the F1 …look for homozygous mutant clones in otherwise heterozygous animals 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 Stern’s observation: …for fly heterozygous for recessive cell-autonomous l.o.f. alleles of two genes: yellow(body)- singed(bristles)+ yellow(body)+ singed(bristles)- If irradiated (ionizing) during development occasionally saw odd ADULT fly: deduced: mistake in mitosis, not new mutant alleles most flies wildtype patch of y/y ? patch of sn/sn ? “twin spot” Fig. 5.23 p152 Consider what it is about mitosis that insures daughter cells will have the same genotype as their mother cell: XmXp y- sn+ y+ sn- y- sn+ m y- p y+ sny+ sn- then: if instead: y- sn+ y+ sn- y- sn+ y+ sn- sn+ m m y- sn+ y- sn+ p p y+ sny+ sn- m p m p = m m p p y- sn+ y+ sn- m y- sn+ y+ sn- m p p What if DNA breaks and improper repair after S phase change relationship between genes and centromeres: y- sn+ y+ sn- y- sn+ m y- p y+ sny+ sn- sn+ m m mistake p p NO CHANGE if instead: yellow y- sn+ y+ sny- sn+ y+ sn- m m m m p p y- sn+ y- sn+ m y+ sny+ sn- m p twin-spot singed p DNA breaks and improper repair after S phase generate the “twin-spot” of cells homozygous for y and sn y- sn+ m y+ sn- p y- sn+ y- sn+ y+ sny+ sn- twin-spot: because progeny of the y/y & sn/sn original cells tend to stay together ADULT EXTERIOR size of patches depends on when abberrant mitosis occurs m m mistake p p y- sn+ y+ sn- y- sn+ y+ snGrowth m m m m p p m y/y (yellow) y- sn+ y- sn+ patch (clone) of sn/sn (singed) y+ sny+ sn- m patch (clone) of p p Fig. 5.24 (p152) What if we had induced a NEW recessive MUTATION on a mutagenized y- sn+ chromosome (in the father’s sperm) that affected cell growth parameters? y- m- sn+ y+ m+sn- y- m- sn+ p y- m- sn+ m y+ m+ sny+ m+sn- p p mistake m m y- m- sn+ p m + y m+ sn- m p y- m- sn+ m y+ m+ sn- pp m zygote m-/mm-/m+ odd patch of y/y yellow patch of sn/sn singed y- m- sn+ y- m- sn+ p y+ m+sny+ m+ sn- p m m ..appearance of an ABNORMAL homozygous yellow patch next to homozygous singed patch would SIGNAL that the female carried an interesting new mutant allele y- m- sn+ y+ m+sn- y- m- sn+ p y- m- sn+ m y+ m+ sny+ m+sn- p p mistake m m y- m- sn+ p m + y m+ sn- m p y- m- sn+ m y+ m+ sn- pp m zygote m-/mm-/m+ odd patch of y/y yellow patch of sn/sn singed y- m- sn+ y- m- sn+ p y+ m+sny+ m+ sn- p m m ..and such a female would be fully viable even if the new recessive mutant allele would have been lethal (perhaps even embryonic lethal -- killing long before adult stage) if a significant fraction of her cells had been homozygous for it. odd patch of y/y yellow m-/mm-/m+ patch of sn/sn singed ADULT EXTERIOR hence avoids complications of pleiotropy …lethal either because of a defect in the same function affecting adult cuticle, or because of a defect in some other process Problems that had limited the use of radiation-induced mitotic recombination for genetic screens: -- mitotic recombination infrequent -- position of exchange not controlled -- radiation used to induce is damaging -- tissues in which occurs are not controlled Solution: induce using site-specific yeast recombination system FLP: recombinase (protein that catalyzes recombination at FRTs) FRT: DNA target (34 bp) site for recombinase FRT site y- sn+ y+ sn- y- sn+ m y- p y+ sny+ sn- source of FLPase …but only induced when & where FLPase induced sn+ m m p p targeted “mistake” yellow y- sn+ y+ sny- sn+ y+ sn- m m m m p p y- sn+ y- sn+ m y+ sny+ sn- m p twin-spot e.g. eye precursor cells singed p lats/lats clone in eye l(3)93B/l(3)93B clone in eye of lats/+ adult of l(3)93B/+ adult (“large tumor suppressor”) 3rd instar larvae left: wildtype right: lats/lats (doomed) (Xu et al, Develop.121:1053 1995) homozygous lats clone on thorax of lats/+ adult fly (Xu et al, Develop.121:1053 1995) Genetic mosaics have zillions of uses besides just facilitating mutant isolation …and geneticists have ways of controlling exactly when and where FLPase is generated …and hence exactly when and where mitotic recombination is induced