Bruce S. Baker - US grants

During the coming year we will continue to focus our major efforts on the analysis of sex determination in Drosophila. Our work on sex determination is divided between the molecular genetic of new functions involved in the control of sex. Our work on the first of these genes that we cloned (doublesex, dsx) will, in the immediate future, be focused on understanding the complex pattern of transcripts that are produced by this gene. We will sequence appropriate portions of cDNAs and the genome in order to define the exact differences between the different transcripts. To date we have identified several different internal splice options that are used as well as two different 3' ends. It seems likely that at least one transcript has a 5' end different from that of the other transcripts. When portions of the genome that are unique to each transcript are identified we will make probes (both labeled nucleic acid and antibodies) to examine the pattern of usages of the different gene products temporally and spatially. We have sequenced one cDNA that appears to have a complete open reading frame and are in the process of expressing it in E. coli in order to begin a set of experiments aimed at inquiring whether it is a DNA binding protein and whether in interacts with sequences in or near sex specific differentiation functions known to be under dsx control. The tra-2 gene has also been recently cloned in our laboratory and substantial efforts during the coming year will be directed at it basic characterization. Our initial analyses of mutants in and around tra-2 and its transcription pattern suggest that it is at least 10 kb in size and transcriptionally complex. We will also be undertaking molecular screens aimed at isolating both new sex determination regulatory genes and new sexual differentiation functions in the next couple of months. Genetic analyses are being carried out on new female lethal locus on the X chromosome and on the intersex gene. In addition several potential new sex determination regulatory genes have been cloned based on their extensive homology with dsx sequences (referred to as dsx cognates, dsc s). Mutants in the gene that we believe corresponds to one dsc have been isolated and shown to interact with mutants at other genes known to be in sex determination.

DESCRIPTION: Dosage compensation of X-chromosome gene expression has been an intriguing regulatory problem for several decades but not until the past few years has much progress been made to understand the underlying genetic system controlling it. It is now clear that the mechanisms of dosage compensation are quite distinct in the three most studied eukaryotic system, Drosophila C. elegans, and man. Drosophila dosage compensates by hyperactivation of the single male X chromosome, C. elegans reduces the expression of each of the two females X chromosomes, and human dosage compensation is achieved via inactivation of one of the female X-chromosomes. The genetic analysis of dosage compensation in Drosophila has lead to the discovery of four male specific lethal genes and which appear to be the primary mediators of dosage compensation while the sex-lethal gene, also a determinant of sex-determination, lies upstream in the regulatory hierarchy. The four male-lethal genes have been cloned and characterized in recent years with Dr. Baker's lab playing a prominent role in this work. In one of the more dramatic visual demonstrations , it was shown that these proteins bind at numerous site over the X chromosome but only a few site on the autosomes. The male-lethal proteins appear to bind to the same site and their binding is inter-dependent. Attempts by the Baker lab and other labs to identify the specific binding site sequences have been less successful, but recently Dr. Baker has developed FISH based method to finally provide the technical means of deletion mapping the binding sites. The first specific aim is to determine how the MSL-2 gene is regulated. Preliminary studies indicate that MSL-2 is negatively regulated by Sxl to prevent dosage compensation in females. Sxl's regulation of MSL-2 appears to be mediated via the 5' and 3' untranslated regions of MSL-2. A male-specific intron is retained in females and this retention requires Sxl. Potential Sxl binding sites are present within this intron as well as the 3'UTR. The MSL-2 gene will be dissected to map sequence element that mediated post-transcriptional regulation. The second specific aim is to identify the X-chromosome sequences that mediate dosage compensation. Two approaches will be used: search for sequences that bind to the putative MSL-2 DNA binding domain and use chromosomal rearrangements to delimit a cosmid size segment of DNA that contains a strong MSL band at its normal position on the X chromosome and then introduce a cLone of this DNA into autosomal sites. In addition a mixture of MSL-1 and MSL-2 will be used in selection experiments on pools of random 25-mer oligonucleotides to identify specific sequence elements. The third specific aim is to identify other components of the dosage compensation pathway by using genetic screens. Dr. Baker argues that the other components may have be involved in general aspects of transcription and chromatin structure. Consequently, mutations in such genes would be expected to be lethal in both sexes, unlike the four previously identified msls. The genetic screen will be for mutations that dominantly suppress or enhance ectopic expression of MSL-2 in females. Replacement of either the 5' or 3' UTR of MSL-2 results in ectopic expression in females and, if expressed at high enough levels, reduces female viability. Lower ectopic expression leads to some MSL complex formation but does not affect female viability. Loss of function mutations in genes that function positively with MSL-2 should be detected as dominant suppressors of impaired female viability whereas loss of function mutations in the genes that function negatively should act as dominant enhancers and lead to impaired female viability. The fourth specific aim is related to several issues related to dosage compensation during embryogenesis and in non-polytene cells. Apparently dosage compensation at the earliest stages of embryogenesis is controlled by Sxl but is independent of the msl genes and the developmental stage at which msl mediate dosage compensation begins has not been determined. Experiments are proposed to determine whether or not the MSL proteins are localized to the male X-chromosomes in non-polytene chromosomes during embryogenesis. The fifth specific aim is to investigate the evolution of dosage compensation in the genus Drosophila and in other closely related genera. Immuno detection of MSL-2 proteins on salivary gland proteins of other species will be performed and msl genes from other species will be cloned. Of particularly interest is the notion that dosage compensation may have evolved independently several times since the X-chromosomes of Drosophila have undergone dramatic episodes of evolution necessitating subsequent addition of dosage compensation to chromosome arms that were previously autosomal.

DESCRIPTION: Fruitless (fru) is the first gene in a branch of the Drosophila sex determination hierarchy. Fru appears to govern all, or nearly all, aspects of male species-specific sexual behavior. This proposal focuses on several aspects of (1) how fru acts to lay down the potential for a complex behavior in the CNS, and (2) how that behavior is controlled by the CNS.

DESCRIPTION (provided by applicant): Our previous studies led us to propose that a sex determination regulatory gene, fruitless (fru), is responsible for building the potential for male sexual behavior into the CNS during Drosophila development. The transcripts of the distal (P1) fru promoter are sex-specifically spliced to produce female- and male-specific mRNAs. It is the P1 derived male-specific FRU proteins (FRU/M) that are needed for male sexual behavior. FRU/M proteins are required for all, or nearly all, aspects of male sexual behavior, from the initial recognition of a potential mate, through to the transfer of bodily fluids and the duration of copulation. That fru functions during developmental to establish the potential for male sexual behavior is suggested by the temporal and spatial patterns of expression of the FRU/M proteins. These proteins are expressed almost exclusively in the CNS. Expression is maximal in the mid-pupal period when they are expressed in ca. 1700 cells (2% of the CNS); this time coincides with the period of major morphogenetic events that shape the adult CNS. It is on the developmental origins of these neurons, the characteristics that distinguish them, and the roles they play in male sexual behavior that this grant is focused. We address the following topics. (1) The behavioral roles of individual groups of FRU/M-expressing neurons. (2) To being to understand these neurons functions we are (A) identifying the types of neurons they are, (B) addressing whether homologously positioned groups of fru P1-expressing neurons in males and females differ in their projection patterns, branching patterns, or synapses, and (C) for selected groups of neurons, determine which cells they are connected to so as to begin to elucidate the neuronal circuitry underlying male courtship behavior. (3) We will determine when the FRUM cells are born during development, whether FRUM cells found in clusters are clonally related to one another, and the reasons why some groups of cells expressing the P1 promoter are found in only one sex. (4) The FRU proteins are putative BTB Zn-finger transcription factors, and thus likely regulate the expression of other genes; therefore we will (A) determine whether FRU proteins function as homo- or heterodimers with other BTB domain proteins, (B) investigate the role of the unique 101 amino acid N-terminus of the male-specific FRU proteins, (C) determine the consensus DNA binding sites of the different FRU Zn-finger pairs, and (D) identify genes that are regulated by FRU proteins. (5) We will determine whether fru is the "master" gene for male sexual behavior: is FRUM both necessary and sufficient for male courtship behavior.