The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UMES. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL] promoter.In the budding yeast Saccharomyces cerevisiae, expression of glucose-repressed genes in response to glucose starvation requires the SNF1 protein-serine/threonine kinase (1). The SNF1 kinase is widely conserved, and its mammalian counterpart, AMP-activated protein kinase, is involved in regulation of lipid metabolism and cellular stress responses (2). In yeast, genetic evidence indicates that one of the functions of SNF1 is to relieve transcriptional repression mediated by the SSN6-TUP1 repressor complex. This complex is tethered to glucose-repressed promoters, including that of SUC2 (encoding invertase), by the DNA-binding protein MIG1 (3-5). Mutations in SSN6 or TUPI completely relieve glucose repression of SUC2 and bypass the requirement for the SNF1 kinase. A migl mutation partially relieves repression, suggesting that other DNA-binding proteins are also involved (6).Mutations in the SSN3 and SSN8 genes were isolated as suppressors of the snfl mutant defect in SUC2 derepression (7). This selection also yielded mutations in six other SSN genes, including MIG1 (= SSNJ) and SSN6. The ssn3 and ssn8 mutations are weak suppressors alone but show strong synergy with migl. Moreover, in strains wild type for SNF1, both ssn3 and ssn8 act synergistically with migl to relieve glucose repression of SUC2 (6). These findings implicate SSN3 and SSN8 in negative regulation of SUC2 expression. Both mutations also cause flocculence.To explore the regulatory roles of SSN3 and SSN8, we have cloned the genes by complementation. Sequence analysis showed homology to the cyclin-dependent protein kinase (cdk) family and cyclin C, respectively. Many protein kinases involved in cell cycle control are composed of a catalytic subunit, the cdk, and an activating/targeting subunit, the cyclin. We present genetic and biochemical evidence that SSN3 and SSN8 constitute a cdk-cyclin pair. Genetic evidence suggests a general role for SSN3-SSN8 in transcriptional control.*
Mutations in the SNF8 gene impair derepression of the SUC2 gene, encoding invertase, in response to glucose limitation of Saccharomyces cerevisiae. We report here the cloning of the SNF8 gene by complementation. Sequence analysis predicts a 26,936-dalton product. Disruption of the chromosomal locus caused a five-fold decrease in invertase derepression, defective growth on raffinose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf8 null mutation with spt6/ssn20 and ssn6 suppressors distinguished SNF8 from the groups, SNF1, SNF4 and SNF2, SNF5, SNF6. Notably, the snf8 ssn6 double mutants were extremely sick. Mutations of SNF8 and SNF7 showed similar phenotypes and genetic interactions, and the double mutant combination caused no additional phenotypic impairment. These findings suggest that SNF7 and SNF8 are functionally related.
The molecular mechanisms underlying axonal pathfinding are not well understood. In a genetic screen for mutations affecting the projection of the larval optic nerve we isolated the abstrakt locus. abstrakt is required for pathfinding of the larval optic nerve, and it also affects development in both the adult visual system and the embryonic CNS. Here we report the molecular characterization of abstrakt. It encodes a putative ATP-dependent RNA helicase of the DEAD box protein family, with two rare substitutions in the PTRELA and the RG-D motifs, thought to be involved in oligonucleotide binding: serine for threonine, and lysine for arginine, respectively. Two mutant alleles of abstrakt show amino acid exchanges in highly conserved positions. A glycine to serine exchange in the HRIGR motif, which is involved in RNA binding and ATP hydrolysis, results in a complete loss of protein function; and a proline to leucine exchange located between the highly conserved ATPase A and PTRELA motifs results in temperature-sensitive protein function. Both the broad requirement for abstrakt gene function and its ubiquitous expression are consistent with a molecular function of the abstrakt protein in mRNA splicing or translational control.
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