This proposal whose broad goal is to understand the molecular basis of terminal sexual differentiation. In Drosophila, somatic sex determination is controlled by a relatively well characterized genetic hierarchy whose terminal members include the intersex (ix) and doublesex (dsx) genes. A fundamental hypothesis of the application is that this hierarchy controls somatic sex via the differential regulation of target genes at the mRNA level. The proposed research aims to characterize such targets by identifying genes whose level of expression varies depending on the sex of the organism and/or the genetic state of the sex-determination genes. A distinction is made between genes that are sex-specific by virtue of expression in sex- specific tissues, and genes that are differentially expressed in tissues common to both sexes. To date, potential target genes identified by subtractive hybridization or other methods are limited to sex specific tissues such as the gonads. Although the initial specification of the tissue is dependent upon the sex hierarchy, the subsequent expression of the """"""""target"""""""" genes is controlled independently by tissue specific factors. The proposed two specific aims. For the first aim, differentially expressed genes will be detected using differential display reverse transcription PCR (DDRT-PCR or """"""""differential display""""""""). This is a recently developed method in which RNA samples are reverse transcribed and PCR amplified using specific arbitrary small primers in combination with a set of anchored oligo(dT) primers; a given primer pair will amplify a distinct set of approximately 150-250 small 3' cDNA fragments. These are resolved on polyacrylamide gels and the results from two different RNA samples compared for differences. By repeating the procedure with multiple pairs of primers, thousands of mRNAs can potentially be analyzed. In the initial analysis, a temperature-sensitive mutation at the intersex locus will be used in an attempt to induce a male-like pattern of expression in flies which are chromosomally and morphologically female. Newly eclosed adult females which are hemizygous for the temperature- sensitive allele and which have been raised at the permissive (18 degrees) temperature will be shifted to the restrictive temperature (29 degrees) for periods ranging from one day to three weeks. Genotypically identical siblings will be maintained at the permissive temperature to serve as the """"""""female"""""""" control. mRNA from both samples will be isolated, and differential display will be performed to identify cDNA fragments which are quantitatively or qualitatively different between the two samples. Finally, the differential display strategy will be used for three additional comparisons: diplo-X pupae which are hemizygous for the intersex allele will be shifted to 29 degrees at mid-pupation and compared to unshifted female siblings at the late pupal stage; adult males and females that are derived from vasa tudor mutant mothers, and therefore lack a germ line, will be compared; and a temperature- sensitive mutation in the tra2 locus will be used to determine the effects of shifting the hierarchy into the male mode at a position upstream of the doublesex and intersex genes following eclosion. cDNAs which show reproducible differences by differential display will be cloned, and sex-specific or sex-differential expression will be confirmed by probing and quantitating slot blots of mRNA isolated from males and females, male and female gonads, and males and females which lack a germline. Similar methods will be used to address the influence of the sex determination hierarchy on the differential expression, using mRNA from appropriate adults which carry either the temperature-sensitive intersex or tra2 mutations and which have been either shifted or maintained post-eclosion. For the second specific aim, Dr. Chase proposes to identify dominant, second-site suppressors of the temperature-sensitive intersex mutation. The screen to be employed is based upon the restoration of fertility to hemizygous females, which are normally sterile when raised at the restrictive temperature. Male flies which carry a deficiency for intersex will be mutatgenized with EMS and mated to females heterozygous for the temperature-sensitive ix mutation. Progeny will be raised at 29 degrees, and hemizygous females will be mated en masse to marked males. Any embryos laid by fertile females will be collected, reared to adulthood, and retested for the presence of the suppressor. In subsequent generations, suppressor mutations will be mapped by conventional methods, tested for possible interactions with other intersex alleles or mutations in other sex-determination genes, and analyzed for intrinsic phenotypes.
