The mechanism of coordinate expression of the ribosomal protein genes in Saccharomyces cerevisiae will be investigated, utilizing recombinant DNA molecules that contain ribosomal protein genes whose expression can be readily assayed in vivo. Fusions between the yeast rp39(1078) or rp52 genes and the E. coli lacIZ gene will be constructed and transformed into yeast. The expression of Beta-galactosidase from these genes will be measured in vivo to ascertain which sequences contiguous to or within these genes are necessary for their expression. The in vivo expression of cloned wild type (CRYS1) and resistance (cryR1) alleles of the cryptopleurine resistance gene coding for rp59 will also be measured. Site directed and random mutations generated in vitro within and adjacent to these rp genes and gene fusions will be screened for those that prevent or alter the expression of the genes in vivo, by transforming the mutated genes into yeast, and assaying the lac or Cry phenotypes. The expression of rp genes during shifts in cellular growth rates and during the stringent response will be probed using the cloned rp genes. Altered expression of the mutated rp genes under these same circumstances will be screened. Gene dosage experiments with these cryR1, rp 39-lacIZ and rp52-lacIZ genes will be performed to determine whether the balance or rp synthesis in yeast is maintained by autogenous regulation. Null mutations in each of the duplicated rp39 genes as well as the single copy rp52 and rp59 genes will be constructed by integrative disruption, to determine the functionality and essentiality of these genes. Cloned RNA2, RNA3, RNA11 and SRN2 genes will be used to determine the nature, location, interaction, and function of the RNA gene products in order to determine the role of these gene products in mRNA processing in yeast. The cloned rp59 and rp52 genes will be utilized as templates for studying mRNA splicing in vivo.
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