The long-term objective of this research is to identify and analyze some of the key genes and gen products that mediate sexual differentiation and zygote development in the unicellular eukaryotic flagellate, Chlamydomonas reinhardtii. The organism has two haploid mating types, mt and m, and mt/mt diploid zygotes, created by the fusion of complementary gametes, are uniquely capable of sporulation and meiosis. The project will employ two approaches. In the first, sequences identified as lying within the mt locus, which we have recently cloned from both mating types, will be used in transformation experiments to rescue mutants defective in mating/development. In the second, mutants defective in mating/development will be generated by insertional mutagenesis, which tags the mutated homologue. Three genes are directly targeted for analysis. 1) The imp-1 gene has been localize to within a 6 kb restriction fragment of the mt locus; its product is anticipated to participate in gametic adhesion, and its identification may lead us to its mt homologue and hence an understanding of how mating type-specific cell-cell recognition is achieved. 2) the iso-1 gene, tagged by an insertion event, functions only in mt gametes; its product is anticipated to activate the expression of mt-specific genes, and repress the expression of mt-specific genes, that are dispersed throughout the genome. 3) The imp-11 gene, which resides within the cloned mt- locus but has yet been localized to a specific domain, is anticipated to activate the expression of iso-1 and also to act with the postulated mt- specific protein P to switch on the zygote differentiation program. Gel- retardation assays will supplement our standard molecular-biology approaches to imp-11 (and iso-1) analysis, the expectation being that both imp-11 and P can be identified by their ability to bind to cloned sequences upstream of zygote-specific genes. The identification and analysis of these genes may illuminate the evaluation and the operation of sexual differentiation.
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