The long term goal of the work proposed is to characterize the molecular events that regulate early germ cell differentiation during oogenesis as a model for how specialized cell types arise from stem cell populations. The focus of these investigations is a small class of genes that mutate to produce tumorous egg chambers (tec genes), a phenotype that identifies the timing of the defect as close to the time that stem cell daughters are born. From the point of view of relatedness to health issues, the potential impact of insights derived from this work is three-fold: (1.) to understand how defects in germline stem cells, and their meiotic daughter cells, contribute to infertility, (2.) to understand how disruptions in the processes represented by tec genes might contribute to germline tumors in humans (i.e. dysgerminomas, Gondos), (3.) to understand how the products of genes like tec genes of Drosophila participate in cell cycle regulation. A new tec gene, bag-of-marbles (bam), has been identified and characterized. Important features of the gene are; (1.) it is required for germ cell differentiation in both males and females, (2.) it shares partial sequence similarity with another tec gene, ovarian tumors.
The specific aims of this proposal can be stated as the following: (1.) to determine what cells in ovary and testis express bam , (2.) to characterize physical properties of bam protein using anti-bam antibodies, (3.) to characterize more fully the genetic function(s) of bam by isolating new alleles, (4.) to test for evidence of genetic interactions between bam and other tec genes, (5.) to isolate genomic and cDNA clones and determine nucleotide sequence and pattern of transcript expression for a second new tec gene, stonewall. The distribution of bam antigen in egg chambers, testes and embryos will be determined using anti-bam antibodies. Remobilization of the mutagenic transposon will be used to make new alleles of bam in order to determine the full range of its' mutability and thereby identify the extent of its' biological role(s). To determine the specificity of bam+ activity in germline and/or somatic cells, a new approach using a high efficiency method for inducing mitotic recombination will be exploited (Golic & Lindquist). Genetic tests for intergenic interactions to reveal possible biochemical interactions will be performed using flies heterozygous for bam and previously identified tec genes as well as newly identified tec genes (McKearin & Spradling). From the collection of single P-element induced female sterile mutations, six alleles of a new tec gene, stonewall, have been recovered and genomic DNA containing the gene cloned. Molecular investigations similar to those carried for bam will be initiated.
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