Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae.

We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a 2.0-kilobase heat-inducible mRNA. This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into stationary phase. The STI1 gene encodes a 66-kilodalton protein, as determined from the sequence of the longest open reading frame. The putative STI1 protein, as identified by two-dimensional gel electrophoresis, migrated in the region of 73 to 75 kilodaltons as a series of four isoforms with different isoelectric points. STI1 is not homologous to the other conserved HSP70 family members in yeasts, despite similarities in size and regulation. Cells carrying a disruption mutation of the STI1 gene grew normally at 30 degrees C but showed impaired growth at higher and lower temperatures. Overexpression of the STI1 gene resulted in substantial trans-activation of SSA4 promoter-reporter gene fusions, indicating that STI1 may play a role in mediating the heat shock response of some HSP70 genes.     

Functional analysis of mutations in the PIS gene, which encodes Saccharomyces cerevisiae phosphatidylinositol synthase

Abstract The PIS gene for an enzyme phosphatidylinositol synthase having an increased K _m for myo-inositol, was isolated from Saccharomyces cerevisiae . The mutant PIS gene contained a CAA codon at position 114 instead of the CAC codon observed in the wild-type gene, resulting in alteration of the amino acid from His to Gln. Oligonucleotide mediated site-directed mutagenesis of PIS at codon 114 revealed that mutant genes with codons for Ala, Thr and Leu could support yeast cell growth in vivo, but those for Asp, Lys and Tyr could not. All mutant enzymes when expressed in Escherichia coli showed greatly reduced in vitro activity.     

Isolation and characterization of WHI3, a size-control gene of Saccharomyces cerevisiae

WHI3 is a gene affecting size control and cell cycle in the yeast Saccharomyces cerevisiae. The whi3 mutant has small cells, while extra doses of WHI3 produce large cells, and a large excess of WHI3 produces a lethal arrest in G1 phase. WHI3 seems to be a dose-dependent inhibitor of Start. Whi3 and its partially redundant homolog Whi4 have an RNA-binding domain, and mutagenesis experiments indicate that this RNA-binding domain is essential for Whi3 function. CLN3-1 whi3 cells are extremely small, nearly sterile, and largely nonresponsive to mating factor. Fertility is restored by deletion of CLN2, suggesting that whi3 cells may have abnormally high levels of CLN2 function.     

Isolation and characterization of OLE1, a gene affecting fatty acid desaturation from Saccharomyces cerevisiae

The unsaturated fatty acid (ufa) requiring ole1 mutant of Saccharomyces cerevisiae appears to produce a defective delta-9 fatty acid desaturase. This enzyme catalyzes double bond formation between carbons 9 and 10 of palmitoyl and stearoyl coenzyme A. A DNA fragment isolated by complementation of an ole1 strain repairs the ufa requirement in mutant cells. Genetic analysis of the cloned DNA fragment indicates that it is allelic to the OLE1 gene. Disruption of a single copy of the wild type gene in a diploid strain produces both wild type and nonreverting ufa-requiring haploid progeny upon sporulation. Membrane lipids of the disrupted haploid strains contain only ufas supplied in the growth medium. The recovery of activity in both wild type and disrupted segregants was examined after removal of ufas from the growth medium. Following ufa deprivation disruptant cells grew normally for about three generations and then at a slower rate for at least 0.6 generations. During that time cellular ufas dropped from 63 to 7.3 mol % of the total fatty acids. No production of the 16:1 and 18:1 products of the desaturase was observed in disruptant cells, whereas desaturation in wild type control cells was evident 2 h after deprivation. These results indicate that 1) the OLE1 gene is essential for production of monounsaturated fatty acids and is probably the structural gene for the delta-9 desaturase enzyme. 2) A large part of membrane ufas present under normal culture conditions are not essential for growth and cell division.     

Proline biosynthesis in Saccharomyces cerevisiae: molecular analysis of the PRO1 gene, which encodes gamma-glutamyl kinase.

The PRO1 gene of Saccharomyces cerevisiae encodes the 428-amino-acid protein gamma-glutamyl kinase (ATP:L-glutamate 5-phosphotransferase, EC 2.7.2.11), which catalyzes the first step in proline biosynthesis. Amino acid sequence comparison revealed significant homology between the yeast and Escherichia coli gamma-glutamyl kinases throughout their lengths. Four close matches to the consensus sequence for GCN4 protein binding and one close match to the RAP1 protein-binding site were found in the PRO1 upstream region. The response of the PRO1 gene to changes in the growth medium was analyzed by measurement of steady-state mRNA levels and of beta-galactosidase activity encoded by a PRO1-lacZ gene fusion. PRO1 expression was not repressed by exogenous proline and was not induced by the presence of glutamate in the growth medium. Although expression of the PRO1 gene did not change in response to histidine starvation, both steady-state PRO1 mRNA levels and beta-galactosidase activities were elevated in a gcd1 strain and reduced in a gcn4 strain. In addition, a pro1 bradytrophic strain became completely auxotrophic for proline in a gcn4 strain background. These results indicate that PRO1 is regulated by the general amino acid control system.     

Primary structure and product characterization of the Saccharomyces cerevisiae CHO1 gene that encodes phosphatidylserine synthase

An open reading frame of 828 base pairs was found in the CHO1 gene region of Saccharomyces cerevisiae by nucleotide sequencing analysis. Its enhanced expression with the aid of the PHO5 regulatory sequence resulted in an overproduction of a protein with a molecular weight of approximately 30,000, which in turn was converted by proteolysis to active phosphatidylserine synthase, whose molecular weight was approximately 23,000. The larger protein was concluded to be the primary product of the CHO1 gene, since its amino-terminal sequence was identical to that deduced from the nucleotide sequence of the above open reading frame, except for the terminal methionine residue. A partial homology in primary structures was noticed between this yeast enzyme and phosphatidylglycerophosphate synthase of Escherichia coli which also uses CDP-diacylglycerol as a substrate. The overproduced phosphatidylserine synthase in both microsomal and extensively purified fractions displayed two different Km values for L-serine, i.e., 0.14 mM at low L-serine concentrations and 9.5 mM at high L-serine concentrations. This may indicate a negatively cooperative regulation of this enzyme activity or the presence of two active components with different affinities for L-serine.     

Isolation and characterization of the Saccharomyces cerevisiae EKI1 gene encoding ethanolamine kinase.

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.     

Isolation and characterization of the positive regulatory gene ADR1 from Saccharomyces cerevisiae.

The DNA segments containing the ADR1 gene and a mutant allele, ADR1-5c, have been isolated by complementation of function in Saccharomyces cerevisiae. The ADR1 gene is required for synthesis of the glucose-repressible alcohol dehydrogenase (ADHII) when S. cerevisiae cells are grown on a nonfermentable carbon source, whereas the ADR1-5c allele allows ADHII synthesis even during glucose repression. A plasmid pool consisting of yeast DNA fragments isolated from a strain carrying the ADR1-5c allele was used to transform a strain containing the adr1-1 allele, which prevents ADHII depression. Transformants were isolated which expressed ADHII during glucose repression. A plasmid isolated from one of these transformants was shown to carry the ADR1-5c allele by its ability to integrate at the chromosomal adr1-1 locus. The wild-type ADR1 gene was isolated by colony hybridization, using the cloned ADR1-5c gene as a probe. The ADR1-5c and ADR1 DNA segments were indistinguishable by restriction site mapping. A partial ADR1 phenotype could be conferred by a 1.9-kilobase region, but DNA outside of this region appeared to be necessary for normal activation of ADHII by the ADR1 gene.     

Isolation and characterization of the Saccharomyces cerevisiae XPT1 gene encoding xanthine phosphoribosyl transferase

A new Saccharomyces cerevisiae gene, XPT1, was isolated as a multicopy suppressor of a hypoxanthine phosphoribosyl transferase (HPRT) defect. Disruption of XPT1 affects xanthine utilization in vivo and results in a severe reduction of xanthine phosphoribosyl transferase (XPRT) activity while HPRT is unaffected. We conclude that XPT1 encodes XPRT in yeast.     

YRA1, an essential Saccharomyces cerevisiae gene, encodes a novel nuclear protein with RNA annealing activity.

The complexity of eukaryotic mRNA processing suggests a need for certain factors, called RNA chaperones, that can modulate RNA secondary structure as well as the interactions between pre-mRNA and trans-acting components. To identify factors that may fulfill this role in the yeast Saccharomyces cerevisiae, we fractionated whole-cell extracts and assayed for activity that could facilitate a specific RNA-RNA annealing reaction. We detected one strong RNA annealing activity and purified it to homogeneity. This previously undescribed factor, Yra1p, is localized to the nucleus; its sequence contains one RNP-motif RNA-binding domain. The YRA1 gene contains a 766-nt intron, the second-largest identified in this organism, and Yra1p serves an essential, nonredundant function. Taken together, our findings indicate that Yra1p is likely to have an important role in S. cerevisiae nuclear pre-mRNA metabolism.     

The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific exo-1,3-beta-glucanase which contributes to ascospore thermoresistance.

A number of genes have been shown to be transcribed specifically during sporulation in Saccharomyces cerevisiae, yet their developmental function is unknown. The SPR1 gene is transcribed during only the late stages of sporulation. We have sequenced the SPR1 gene and found that it has extensive DNA and protein sequence homology to the S. cerevisiae EXG1 gene which encodes an exo-1,3-beta-glucanase expressed during vegetative growth (C. R. Vasquez de Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebrada, E. Mendez, and F. del Ray, Gene 97:173-182, 1991). We show that spr1 mutant cells do not hydrolyze p-nitrophenyl-beta-D-glucoside or laminarin in a whole-cell assay for exo-1,3-beta-glucanases. In addition to the absence of this enzymatic activity, spr1 mutant spores exhibit reduced thermoresistance relative to isogenic wild-type spores. These observations are consistent with the notion that SPR1 encodes a sporulation-specific exo-1,3-beta-glucanase.     

Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae.

The srb1-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on an osmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions. A DNA fragment which complements both of these phenotypic effects has been cloned. This clone contains a functional gene which is transcribed into a 2.3-kb polyadenylated mRNA molecule. Transformation of yeast strains carrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsense suppressor. Integrative transformation and gene disruption experiments, when combined with classical genetic analysis, confirmed that the cloned fragment contained the wild-type SRB1 gene. The integrated marker was used to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis. A disruption mutant created by the insertion of a TRP1 marker into SRB1 displayed only the lysis ability phenotype and was not dependent on an osmotic stabilizer for growth. Lysis ability was acquired by growth in (or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding. It is inferred that budding cells lyse with a higher probability and that weak points in the wall at the site of budding are involved in the process. The biotechnological potential of the cloned gene and the disruption mutant is discussed.     

Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.