The primary long-term goal of this research is to elucidate how differentiation of two distinct cell types may be programmed in the genome of a multicellular organism, using as a model Volvox carteri, which exhibits complete division of labor between mortal somatic cells and immortal germ cells. The secondary long-term goal is to use Volvox and its close unicellular and colonial relatives to study the molecular genetic basis for the evolution of such a germ/soma dichotomy. The following specific studies are proposed: 1. Detailed molecular analysis of the regA gene, to gain insight into how this central important gene acts to preclude somatic cells from redifferentiating as reproductive cells. Steps include cloning of the gene, analysis of the sequence organization of the gene in somatic and reproductive cells (in order to test the hypothesis that expression of the gene is regulated by DNA rearrangement), and analysis of patterns of regA transcription, and translation and cellular distribution of its protein product. 2. Development of a transformation system that could be used initially to test the function of putative regA clones, and later to study other genes of developmental interest. Steps include testing of four different DNA-delivery methods with a variety of vectors bearing prokaryotic selectable markers, and development of vectors with homologous selectable markers. 3. Analysis of genes regulating other steps in development of the germ/soma dichotomy, including saturation mapping of such genes, screens for revertants and/or second-site suppressors nd physical mapping of the genes by use of restriction fragment length polymorphisms 4. Study of the phylogeny and stage-specific and/or cell-type-specific patterns of expression of several families of """"""""V/C"""""""" genes which are transcribed in Volvox, but have few if any homologues in the genome of the related unicellular organism, Chlamydomonas. The health-relatedness of this research derives from the analogy that it is possible to draw between somatic ell redifferentiation in regA mutants of Volvox and neoplasia in humans. In regA mutants, somatic cells (which are normally terminally differentiated and post-mitotic) redifferentiate and proliferate without restraint. It is possible that understanding how regA functions in wild type Volvox to prevent such restrained proliferation might provide novel insights that could be drawn upon in exploring growth control in higher eukaryotes.
