The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools.

The CDC25 gene is transcribed at a very low level in S. cerevisiae cells. We have studied the effects of an overexpression of this regulatory gene by cloning either the whole CDC25 open reading frame (pIND25-2 plasmid) or its 3' terminal portion (pIND25-1 plasmid) under the control of the inducible strong GAL promoter. The strain transformed with pIND25-2 produced high levels of CDC25 specific mRNA, induced by galactose. This strain does not show any apparent alteration of growth, both in glucose and in galactose. Instead the yeast cells transformed with pIND25-1, that overexpress the 3' terminal part of CDC25 gene, grow very slowly in galactose medium, while they grow normally in glucose medium. The nucleotides were extracted from transformed cells, separated by HPLC and quantitated. The ATP/ADP and GTP/GDP ratios were almost identical in control and in pIND25-2 transformed strains growing in glucose and in galactose, while the strain that overexpresses the 3' terminal portion of CDC25 gene showed a decrease of ATP/ADP ratio and a partial depletion of the GTP pool. The disruption of RAS genes was only partially able to 'cure' this phenotype. A ras2-ts1, ras1::URA3 strain, transformed with pIND25-1 plasmid, was able to grow in galactose at 36 degrees C. These results suggest that the carboxy-terminal domain of the CDC25 protein could stimulate an highly unregulated GTPase activity in yeast cells by interacting not only with RAS gene products but also with some other yeast G-proteins.     

Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis

Summary The cell division cycle gene CDC25 was replaced by various disrupted and deleted mutant copies. Mutants disrupted at a central position of the gene, or lacking 532 residues within the amono-terminal half of the gene product grow normally in glucose, but not in acetate media, and they fail to sporulate as homozygous diploids. Disruptions or deletions within the carboxy-terminal half are lethal, except for the deletion of the 38 carboxy-terminal residues, which are required for sporulation but not for growth in glucose or acetate media. It is concluded that distinct domains of the CDC25 gene product are involved in the control of mitosis and/or meiosis.     

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.     

Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene.

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.     

SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae.

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.     

Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae

The nucleotide sequence of the CLS4 gene controlling Ca2+ regulatory process of bud emergence, which was cloned previously [Ohya et al., J. Bacteriol. 165 (1986) 28-33], was determined. The CLS4 (CDC24) locus encodes a protein consisting of 736 amino acid (aa) residues with an Mr of 83,970. By primer extension mapping, the mRNA start point was located 139 bp upstream from the translation start codon. The predicted CLS4 protein was hydrophilic with two serine + threonine-rich domains in the middle and C-terminal regions. It has two putative Ca2+-binding regions, one being partly homologous to the Ca2+-binding domain of the S-100a protein and the other that of alpha-lactalbumin.     

Cloning and characterization of the Saccharomyces cerevisiae CDC6 gene.

The yeast cell division cycle gene CDC6 was isolated by complementation of a temperature-sensitive cdc6 mutant with a genomic library. The amino acid sequence of the 48 kDalton CDC6 gene product, as deduced from DNA sequence data, includes the three consensus peptide motifs involved in guanine nucleotide binding and GTPase activity, a target site for cAMP-dependent protein kinase and a carboxy-terminal domain related to metallothionein sequences. A plasmid-encoded CDC6-beta-galactosidase hybrid protein was located at the plasma membrane by indirect immunofluorescence. Disruption experiments indicate that the CDC6 gene product is essential for mitotic growth.     

Iron requirement for GAL gene induction in the yeast Saccharomyces cerevisiae

Iron is an essential nutrient. Its deficiency hinders the synthesis of ATP and DNA. We report that galactose metabolism is defective when iron availability is restricted. Our data support this connection because 1) galactose-mediated induction of GAL promoter-dependent gene expression was diminished by iron limitation, and 2) iron-deficient mutants grew slowly on galactose-containing medium. These two defects were immediately corrected by iron replacement. Inherited defects in human galactose metabolism are characteristic of the disease called galactosemia. Our findings suggest that iron-deficient galactosemic individuals might be more severely compromised than iron-replete individuals. This work shows that iron homeostasis and galactose metabolism are linked with one another.     

Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae

The GAL1 promoter is one of the strongest inducible promoters in the yeast Saccharomyces cerevisiae. In order to improve recombinant protein production we have developed a fluorescence based method for screening and evaluating the contribution of various gene deletions to protein expression from the GAL1 promoter. The level of protein synthesis was determined in 28 selected mutant strains simultaneously, by direct measurement of fluorescence in living cells using a microplate reader. The highest, 2.4-fold increase in GFP production was observed in a gal1 mutant strain. Deletion of GAL80 caused a 1.3-fold increase in fluorescence relative to the isogenic strain. GAL3, GAL4 and MTH1 gene deletion completely abrogated GFP synthesis. Growth of gal7, gal10 and gal3 also exhibited reduced fitness in galactose medium. Other genetic perturbations affected the GFP expression level only moderately. The fluorescence based method proved to be useful for screening genes involved in GAL1 promoter regulation and provides insight into more efficient manipulation of the GAL system.