In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS] and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not rasi, ras2, or In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase encoded by the CYR] gene (allelic to the CDC35 gene) through RAS] and RAS2 gene products (3, 5, 37). As a result of this activation, cyclic AMP (cAMP) stimulates the cAMP-dependent protein kinase (A kinase), whose regulatory subunit is encoded by the BCYJ gene and whose catalytic subunit is encoded by the three interchangeable genes TPKJ, TPK2, and TPK3 (47,48). This cAMP-dependent protein kinase pathway plays a key role in the nutritional control of the G1/G0 switch (2). The RAS] and RAS2 genes of S. cerevisiae encode GDPand GTP-binding proteins with an intrinsic GTPase activity and are closely related to the ras genes of higher eucaryotic organisms. By analogy to transducins G. and G1, RAS proteins are believed to be transducers which activate their effectors when bound to GTP but not when bound to GDP (43). In S. cerevisiae, biochemical evidence of the activity of the GTP-bound form as a positive effector of adenylate cyclase has been presented (15). The product of the CDC25 gene is required for cAMP production (5). This effect is mediated by RAS proteins, as deduced from the existence of different mutations in the RAS2 gene that suppress the cdc25 mutations. One is a Gly -* Val-19 substitution which mimics oncogenic variants and leads to a lower GTPase activity (25).The second is a spontaneous mutation selected as a suppressor of a cdc25 mutation which corresponds to a Thr -k Ile-152 substitution (4). This mutation leads to spontaneous GDP-GTP exchange by increasing the guanyl nucleotide exchange rate on RAS proteins (9). This finding suggests that the CDC25 gene product is a positive regulator which acts upstream of RAS proteins, most likely as a GDP-GTP exchange factor.We have recently described the cloning of a DNA fragment that suppresses the cdc25 mutation but not the rasi, truncated open reading frame (ORF) that shares 47% identity with the C-terminal part of the CDC25 gene at the amino acid level. The product of this fragment has been expressed in Escherichia coli. Partially purified protein strongly enhances the release of GDP from the S. cerevisiae RAS2-GDP or c-Ha-ras p21-GDP complex and then promotes faster GDP-GTP exchange (10). We had previously named the corresponding gene SCD25 (1), but since this name was already used, we changed SCD25 to SDC25. In this report, we describe the cloning of the N-terminal part of the SDC25 gene and further characterize the complete gene. Homology with the CDC25 gene is also present in the N-terminal part, although this homology is weaker than that found in the C-terminal part. In contrast with the C-terminal part, the complete gene on a multicopy plasmid did not suppress the CDC25 gene defect, although it was transcribed and translat...
A Dictyostelium discoideum DNA fragment that complements the ura3 and the ura5 mutants of Saccharomyces cerevisiae has been sequenced. It contains an open reading frame of 478 codons capable of encoding a polypeptide of molecular weight 52475. This gene, named DdPYR5-6, encodes a bifunctional protein composed of the orotate phosphoribosyl transferase (OPRTase) and the orotidine-5'-phosphate decarboxylase (OMPdecase) domains described for UMP synthase in mammals. The existence of separate domains for the two activities was suspected because deletion of the N-terminal coding segment of the gene eliminated the ura5 but not the ura3 complementing activity. We have now confirmed that the two parts of the open reading frame share homology with known OPRTase and OMPdecase sequences. Several blocks of sequence are conserved among OPRTase from bacteria, fungi and slime mold and one of them corresponds to the consensus sequence for phosphoribosylbinding sites. The OMPdecase domain shows extensive similarity with the yeast and Neurospora crassa enzymes, suggesting that they have evolved from an ancestral gene which was fused to the OPRTase gene in D. discoideum. It is less related to the bacterial enzyme but all these sequences present conserved blocks of homology which could identify the active site. The codon usage is strongly biased in a manner similar to that found for other D. discoideum genes. The flanking DNA contains homopolymers of A and T and alternating sequences that are characteristic of the gene organization in D. discoideum.
In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.
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