We propose a genetic and molecular biological approach to the study of the Drosophila gene zeste, its interactions with the unlinked loci white, bithorax, and decapentaplegic, and its involvement in synapsis-dependent gene expression (transvection). These experiments will utilize cloned DNA sequences isolated in laboratory and shown to contain all information required for normal zeste expression. Attention will first be given to the completion of a detailed analysis of the structure and nucleotide sequence of the wild-type gene. Next, nucleotide sequence changes and alterations in zeste transcripts differentiating mutant from wild-type zeste loci will be determined. The resolution of these studies will be increased by employing mutations generated by in vitro mutagenesis of cloned DNA subsequently introduced into the Drosophila genome by germ-line transformation. These experiments should aid in the understanding of the manner in which mutations at zeste can lead to a myriad of phenotypic consequences, and perhaps define regions of the gene involved in particular aspects of zeste function. To address the molecular roles played by putative protein products of the zeste locus, two major approaches will be employed. First, careful measurements will be taken of the amount and nature of white transcripts and protein products in the presence of zeste mutations as a function of Drosophila development. These experiments will help establish at which stage of the expression of the target genes zeste functions. Next, antibodies will be generated against antigenic determinants specified by zeste. These antibodies will be utilized to describe the tissue-specific and intracellular distributions of zeste locus products, and to explore the possibility that zeste locus products interact specifically with sequences in the vicinity of the white locus known to be required for the zeste phenotype. Additional studies will attempt to determine whether overexpression of zeste results in defined phenotypic consequences, to study the action of unlinked genes which modify the zeste phenotype, and to explore the nature of DNA sequences in yeast homologous to zeste. It is expected that these experiments will have an important bearing on the study of gene regulation in metozoan organisms, and of the means by which the somatic pairing of chromosomes may influence gene expression.
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