This project concerns the mechanism by which the C. elegans vitellogenin genes are controlled during development. The vitellogenins are specified by a small family of genes, the transcription of which begins during the larval-to-adult molt in the intestine of the hermaphrodite. The genes are totally inactive in males and in hermaphrodite larvae. Thus these genes are presumably regulated by two groups of genes that have been heavily studied in C. elegans: the sex determination genes and the heterochronic genes. The object of this project is to determine how these regulatory loci control the activity of the vit genes. Two repeated heptameric sequences, VPE1 and VPE2, have been identified in the promoter regions of all of the vit genes, and their functions are currently being tested in a transgenic system. It is now clear that no more than 174 upstream base pairs are required for correctly regulated, high level expression. Analysis of strains with point mutations in some of the VPE's has demonstrated that some are required for expression, while others are individually dispensable. In this application, experiments are proposed to answer specific questions about how the VPE's function, e.g. are the two types of VPE element interchangeable? Are their locations of critical importance? The possibility of sequences downstream of the cap site being required will also be investigated, using a lacZ fusion construction. A C. elegans transcription factor gene (elt-1) related to the erythrocyte- specific transcription factor, GATA-1, has recently been cloned. It has been demonstrated using a yeast in vivo system that the elt-1 product recognizes VPE2 or a very close relative of this sequence. This system will be exploited to determine the exact requirements for the elt-1 binding site. The elt-1 mRNA and protein products will be localized to determine whether the elt-1 product could be a regulator of the vit genes. Mutations in the elt-1 gene will be obtained in order to allow investigation of what roles its product may play in the worm. In addition, a gene that encodes the transcription factor that binds to VPE1 will be cloned by screening a lambdagtII cDNA expression library with a DNA fragment composed of multiple VPE1 sequences. In order to determine how the timing and sex determination pathways interact with the vit promoters, genetic interactions between heterochronic lin mutations that result in delayed or premature vit synthesis, and mutations in VPE1 or VPE2 will be sought. Disruption of the effect of sex determination mutants on fusion gene expression by mutations in the VPE's will also be searched for. In order to identify additional regulators of vit gene expression, mutations that express inappropriately (i.e. in males, larvae or non-intestinal tissues) or those that can suppress the reduced expression due to mutations in the VPE's will be screened for. Finally the function of the vitellogenins will be investigated by screening for mutations that result in complete loss of expression.
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