Showing papers in "Yeast in 1985"

Primary structure of the Saccharomyces cerevisiae GAL7 gene.

Abstract: We present the nucleotide sequence of a 1599-base pair (bp) DNA fragment containing the entire GAL7 gene that encodes galactose-1-phosphate uridyltransferase of Saccharomyces cerevisiae. The deduced peptide was composed of 364 amino acid residues. The expected molecular weight was 42,005 daltons, which agreed with the observed value for the purified enzyme. The 3'-end of the GAL7 transcript mapped at a position 82 bp downstream from the UAA termination codon by the S1 nuclease protection experiment. We constructed a GAL7'-lac'Z fusion on various types of yeast plasmid vectors. The fused gene on any type of vector was induced by galactose and repressed by glucose as for the GAL7 gene on the chromosome. The response of GAL7'-lac'Z fusion to gal4 delta and gal80 delta regulatory mutations was also similar to the response of the chromosomal GAL7 gene. By using various deletions in the 5'-flanking region of the gene fusion, we delimited the sequence essential for galactose controlled expression with a 180 bp-fragment of DNA lying 92 bp upstream of the transcription initiation site.

Isolation and characterization of a phosphoprotein phosphatase-deficient mutant in yeast.

Abstract: The ppd1 mutant of yeast, Saccharomyces cerevisiae, was isolated as a suppressor of the cyr2 mutation which caused alteration of the catalytic subunit of cAMP-dependent protein kinase. Three peaks of phosphoprotein phosphatase activity (peak I, II and III) were identified by DEAE-Sephacel chromatography of crude extracts of the wild-type strain. The ppd1 mutant was deficient in peak III phosphoprotein phosphatase activity. The peak III enzyme efficiently utilized the phosphorylated forms of NAD-dependent glutamate dehydrogenase and trehalase as substrate. The ppd1 mutation did not suppress the cyr1, CYR3 or ras1 ras2 mutations. The ppd1 locus was located on chromosome II and had identical characteristcs with glc1. The ppd1 mutation suppressed the G1 arrest caused by nutritional limitation, but maintained sensitivity to mating pheromone. In diploids homozygous for the ppd1 mutation, no premeiotic DNA replication and commitment to intragenic recombination occurred and no spores were formed, suggesting that the accumulation of phosphorylated proteins in the absence of one of the phosphoprotein phosphatases is required for mitosis but not for the initiation of meiosis.

The PET18 locus of Saccharomyces cerevisiae: a complex locus containing multiple genes.

Abstract: The basis of pleiotropy shown by the pet18 mutants of Saccharomyces cerevisiae (rho-0,KIL-0 and temperature sensitive growth) was examined by cloning the fragment which complements the defect in growth at 37 degrees C of the pet18 mutants. The cloned DNA could complement the defect in the maintenance of the killer plasmid but did not give the cell the ability to maintain mitochondrial DNA. Sequence analysis of the cloned DNA revealed the presence of four open reading frames, at least two of which are necessary for the complementation activity. By using the cloned DNA as a probe, we found that two independent pet18 mutants have a deletion covering the entire sequence contained in the probe. From these results we predict that the traits of the pet18 mutants that concern temperature sensitivity and killer of the pet18 mutants are controlled by a separate gene(s) from that which participates in the maintenance of mitochondrial DNA.

On the mechanism of exclusion of M2 double-stranded RNA by L-A-E double-stranded RNA in Saccharomyces cerevisiae.

Abstract: L-A-E double-stranded RNA (dsRNA), when introduced into cells carrying L-A-H and M2 dsRNAs, does not eliminate the L-A-H dsRNA, but (i) L-A-E does lower the copy number of L-A-H dramatically and (ii) L-A-E eliminates M2 dsRNA from the cell. That these two effects of L-A-E are related is shown by the fact that mutants of a strain carrying L-A-H and M2 selected for their resistance to exclusion of M2 by L-A-E [effect (ii)] have an altered L-A-H whose copy number is not lowered by L-A-E [effect (i)]. Although the L-A in K1 strains (L-A-HN in all cases examined) differs significantly both genetically and physically from the L-A in the K2 strain studied (L-A-H), the L-A-HN from the K1 strains can maintain M2 dsRNA, and the L-A-H from the K2 strains can maintain M1 dsRNA.

Partial restoration of meiosis in an apomictic strain of Saccharomyces cerevisiae: A model system for investigation of nucleomitochondrial interactions during sporulation

Abstract: In an apomictic strain of Saccharomyces cerevisiae (ATCC 4117-H2) which undergoes a single nuclear division during sporulation and consequently forms asci containing two uninucleate diploid spores, a study was undertaken to investigate the effects of cultivation in three presporulation media (YPA; YNB; SMM) on nuclear division and ascosporogenesis in sporulation medium. Comparison of effects of presporulation culture in these media on the number of spores formed per ascus showed that a marked induction (30 +/- 4.3 per cent) of three- and four-spored asci could occur in sporulation medium following cultivation in a defined YNB medium supplemented with a 1 per cent solution of vitamins and containing decreased ammonium sulphate and increased glucose levels. Experiments in which the concentrations of glucose and of ammonium sulphate were varied simultaneously indicated that the initial presporulation carbon to nitrogen source ratio is an important factor in determining tetrad formation in sporulation medium. Nuclear staining demonstrated two classes of asci: binucleate (one- and two-spored) and tetranucleate (three- and four-spored). Genetic evidence and data concerning effects of inclusion in sporulation medium of a meiotic inhibitor (glucose) indicated spores in tetrads were haploid rather than diploid. This ability to condition a significant number of cells for meiotic rather than apomictic differentiation made possible investigation of effects of mitochondrial inhibitors on both developmental processes simultaneously. It was found possible to selectively inhibit meiotic development by inclusion in sporulation medium of appropriate concentrations of specific inhibitors. Moreover, the data suggest meiotic sporulation is more strictly dependent than apomictic sporulation on mitochondrial function.

Expression of the Drosophila 70,000 Dalton heat shock protein is translationally controlled in yeast.

Abstract: Plasmid pPW229, containing the 2.25 kilobase transcribed sequence for the 70,000 Dalton heat shock protein of Drosophila, was integrated into plasmid CV13 and used to transform Saccharomyces cerevisiae. Upon a heat shock, at 41 degrees C for 20 min, a new 70,000 Dalton protein appeared in the transformants. This protein was not detected in transformants grown at 23 degrees C, nor in transformants carrying the hybrid plasmid from which the structural gene for the 70,000 Dalton protein had been deleted. RNA was isolated from transformants grown at 23 degrees C and from transformants heat shocked at 41 degrees C. RNA complementary to the Drosophila heat shock gene was present in the transformants, grown either at 23 degrees C or heat shocked. No complementary RNA was detected in yeast cells transformed with the hybrid plasmid from which the structural gene had been deleted. The Drosophila heat shock gene in yeast appears to be transcribed constitutively but translated only under heat shock conditions.