STUDIES ON THE GENETIC ORGANIZATION OF TETRAHYMENA

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The long term objectives include (1) a mapping of genes in the germinal and highly differentiated somatic nuclei of Tetrahymena thermophila so that mechanisms in differentiation can be studied, (2) a detailed understanding of meiosis, including environmental and genetic factors that influence chromosome pairing and recombination, and (3) the genetic factors involved in ribosomes biosynthesis. All of the proposed studies exploit the separation of germinal and somatic functions into two different nuclei in Tetrahymena. Specific aims include: (1) The isolation of strains missing arms of chromosomes, and containing novel associations of chromosome arms. This will be done by crosses using complex, multiple monosomics. The crosses will be done in circumstances that prevent the production of new somatic nuclei, and therefore allow the viable recovery of cells with nullisomic germinal nuclei. (2) The creation of a genetic map based on mitotic recombination. This will be done by searching for induced homozygosity of specific markers. It is necessary since meiotic recombination is too active to allow mapping of genes on chromosomes. Measurements comparing the effect of ultra violet radiation on mutant formation and mitotic recombination will be performed. (3) A study of meiotic recombination in the gene coding for the ribosomal RNA (the rDNA). These studies exploit the single copy organization of the rDNA in the germinal nucleus. Drug resistant mutants, and mutations effecting ribosome confirmation will be isolated. Mutations in the rDNA will be identified by crosses with strains carrying a gene that distorts its amplicication in the somatic nucleus, and will be localized to specific ribosomal subunits by in vitro testing. They will be pinpointed by DNA sequence analysis. Specific combinations of mutations in this gene will be used to measure the exact amount of meiotic recombination in control cells, and then in cells in unusual environments and carrying unusual karyotypes (monosomics and nullisomics). This study should provide detailed information on how environment and genotype can influence aspects of meiosis. (4) Improved methods of freezing and storage of these complex cells will be developed based on the effects of prolonged starvation.