Characterization and functional analyses of the CRT1-CBP1-NUC1 gene cluster in the yeast Saccharomyces cerevisiae.
AdvisorDieckmann, Carol L.
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PublisherThe University of Arizona.
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AbstractTwo yeast nuclear genes (NUC1 and CRT1) that flank CBP1 were studied. NUC1 is located 230 bp downstream of CBP1 and encodes a major mitochondrial nuclease. In strains which carry double stranded RNA (dsRNA) virus L-A, disruption of NUC1 causes a dramatic increase in the level of L-A dsRNA genome and its product, the viruslike particle coat protein. This phenotype is similar to that observed in a strain with a mutation in the nuclear gene encoding the most abundant outer mitochondrial membrane protein, porin. The disruption of NUC1 does not affect the level of porin. Mutations in NUC1 are recessive with respect to virus overexpression and the overexpression phenotype can be suppressed by the presence of M type dsRNA. In both NUC1 and nuc1 strains, virus abundance is increased by growing cells in a medium containing a nonfermentable carbon source, an effect independent of the increase in coat protein and viral genome due to nuc1 mutations or to the absence of M. Since mutations in NUC1 affect the abundance of L-A but not M, NUC1 defines a new family of nuclear genes involved in dsRNA metabolism. CRT1 is located 270 bp upstream of CBP1 and encodes a function related to yeast respiration. CRT1 produces two RNA species which are 0.8 and 0.9 kb. The longer transcripts are initiated upstream of the open reading frame and are expressed constitutively, while the shorter transcripts are initiated within the open reading frame and are induced 8 to 10-fold upon carbon source derepression. Sequence analysis revealed that the open reading frame could encode a basic protein with a molecular weight of 30.7 kDa. The deduced amino acid sequence indicates that the CRT1 protein has putative zinc finger, leucine zipper, and acidic blob domains, which are typical of transcriptional regulatory proteins. A complete deletion of CRT1 from the genome has no apparent phenotype. Thus, CRT1 is not an essential yeast gene. A gene (CRT2) that duplicates the function of CRT1 was identified in a synthetic lethal/respiratory deficiency screen. The function of either CRT1 or CRT2 is sufficient to support respiration in yeast. Therefore, CRT1 may encode a regulatory factor involved in the expression of genes encoding respiratory functions.