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R Kunisawa

Publications and source records attributed to R Kunisawa.

15 recordsLinked to original sources
PubMed1993

Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability.

Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first step in the biosynthesis of PtdIns-4,5-bisphosphate (PtdIns[4,5]P2). Hydrolysis of PtdIns[4,5]P2 in response to extracellular stimuli is thought to initiate intracellular signaling cascades that modulate cell proliferation and differentiation. The PIK1 gene encoding a PtdIns 4-kinase from the yeast Saccharomyces cerevisiae was isolated by polymerase chain reaction (PCR) with oligonucleotides based on the sequence of peptides derived from the purified enzyme. The sequence of the PIK1 gene product bears similarities to that of PtdIns 3-kinases from mammals (p110) and yeast (Vps34p). Expression of PIK1 from a multicopy plasmid elevated PtdIns 4-kinase activity and enhanced the response to mating pheromone. A pik1 null mutant was inviable, indicating that PtdIns4P and presumably PtdIns[4,5]P2 are indispensable phospholipids.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae.

Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes. Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes. Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner. To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12. In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes. The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway. Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C. The MOT1 gene was cloned by complementation of this temperature-sensitive lethality. Construction of a null allele confirmed that MOT1 is an essential gene. MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases. The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.

Adenosine Triphosphatases↗
PubMed1991

Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase.

Calcineurin, or phosphoprotein phosphatase type 2B (PP2B), is a calmodulin-regulated phosphoprotein phosphatase. We isolated a gene encoding a yeast PP2B homolog (CNA1) by screening a yeast genomic DNA library in the expression vector lambda gt11, first with 125I-labeled yeast calmodulin and then with a human cDNA encoding the catalytic (or A) subunit of calcineurin. The predicted CNA1 gene product is 54% identical to its mammalian counterpart. Using the polymerase chain reaction (PCR) with oligonucleotide primers based on sequences conserved between CNA1 and mammalian PP2B genes, we isolated a second gene, CNA2. CNA2 is identical to PP2Bw, a partial cDNA clone previously described by others as originating from rabbit brain tissue. Our findings demonstrate that a unicellular eukaryote contains phosphoprotein phosphatases of the 2B class. Haploid cells containing a single cna1 or cna2 null mutation, or both mutations, were viable. MATa cna1 cna2 double mutants were more sensitive than wild-type cells or either single mutant to growth arrest induced by the mating pheromone alpha factor and failed to resume growth during continuous exposure to alpha factor. Thus, calcineurin action antagonizes the mating-pheromone response pathway.

Amino Acid Sequence↗
PubMed1991

Multiple Ca2+/calmodulin-dependent protein kinase genes in a unicellular eukaryote.

We purified a Ca2+/calmodulin (CaM)-dependent protein kinase (CaM kinase) from the yeast Saccharomyces cerevisiae with properties similar to mammalian type II CaM kinases. Degenerate oligonucleotides designed on the basis of the amino acid sequence of tryptic peptides from the 55 kd subunit of the yeast CaM kinase were used to isolate its gene from a set of lambda gt11-yeast genomic DNA phage clones initially selected by the ability to bind 125I-labelled yeast CaM. The cloned gene (CMK1) encodes an open reading frame that is homologous to the sequences of vertebrate type II CaM kinases. Several criteria demonstrated that the CMK1 gene product is the 55 kd polypeptide. Neither over-production (11-fold) nor complete elimination of the CMK1 gene product had any detectably deleterious effect on yeast cell growth. Extracts from cmk1 delta cells, which lacked detectable p55 using an antiserum raised against a Staphylococcus aureus protein A-CMK1 fusion protein, possessed significant residual Ca2+/CAM-dependent protein kinase activity. Using the CMK1 gene as a probe at low stringency, a second gene (CMK2) encoding another CaM-dependent protein kinase with striking sequence similarity to CMK1 was cloned. Deletion of CMK2, or both CMK1 and CMK2, was not lethal, although loss of CMK2 caused a slow rate of spore germination.

Amino Acid Sequence↗
PubMed1990

A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle.

Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast S. cerevisiae. A single gene (PKC1) was isolated that encodes a putative protein kinase closely related to the alpha, beta, and gamma subspecies of mammalian PKC. Deletion of PKC1 resulted in recessive lethality. Cells depleted of the PKC1 gene product displayed a uniform phenotype, a characteristic of cell division cycle (cdc) mutants, and arrested cell division at a point subsequent to DNA replication, but prior to mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle.

Amino Acid Sequence↗
PubMed1989

A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae.

MATa cells carrying an sst2 mutation are unable to recover from the G1-specific cell cycle arrest induced by the mating pheromone alpha factor. The KSS1 gene, when overexpressed, suppresses this adaptation defect. KSS1 overexpression also suppresses the recovery defect manifested by cells expressing an alpha factor receptor lacking its 136 amino acid cytoplasmic tail. Because SST2 product and the receptor tail contribute independently to events that allow recovery from pheromone-induced growth arrest, KSS1 function defines a third independent process that promotes desensitization. The KSS1 gene encodes an apparent protein kinase homologous to the CDC28 (S. cerevisiae) and cdc2+ (S. pombe) gene products. The recovery-promoting activity of the KSS1 gene requires a functional WHI1 gene, which encodes a yeast homolog to animal cyclins, suggesting that the KSS1 and WHI1 proteins act in the same growth control pathway.

Amino Acid Sequence↗
PubMed1984

Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor.

S. cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor. Both molecules are excised from larger precursor polypeptides. In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly. In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form. All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues. Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses. The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation. The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo. Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase.

Arginine↗
PubMed1980

Evolution in Pseudomonas fluorescens.

The relationships among 93 strains of Pseudomonas fluorescens were investigated by (1) a numerical taxonomic analysis on the results of 150 phenotypic tests, (2) DNA hybridization studies using 16 reference strains, (3) quantitative microcomplement fixation studies using six reference strains with antibodies directed against the protein azurin. In general, the strains fell into distinct clusters. Assignment to these clusters on the basis of azurin immunological similarity showed 98% agreement with assignment based on DNA homology, suggesting that many genes will follow the same pattern. Of the strains that clustered on the basis of genotype (DNA, azurin) 88% also clustered on the basis of phenotype. The occasional noncongruency observed between the genotypic and phenotypic data may be due to the variable rates of phenotypic evolution. These results provide a perspective on the roles of horizontal and vertical transfer of genes in the evolution of this bacterial group.

Azurin↗
PubMed1969

Comparative study of the structure of gas vacuoles.

The fine structure of gas vacuoles was examined in two blue-green algae, two green bacteria, three purple sulfur bacteria, and two halobacteria. The gas vacuole is a compound organelle, composed of a variable number of gas vesicles. These are closed, cylindrical, gas-containing structures with conical ends, about 80 to 100 nm in width and of variable length, ranging from 0.2 to over 1.0 mum. The wall of the gas vesicle is a non-unit membrane 2 to 3 nm in thickness, bearing very regular striations with a periodicity of 4 nm, oriented more or less at right angles to the long axis of the cylinder. This fine structure could be clearly resolved in isolated gas vesicles prepared from a blue-green alga and from Halobacterium halobium, and its presence in the gas vesicles of the green bacterium Pelodictyon clathratiforme was inferred from thin sections. The gas vacuole thus appears to be a homologous organelle in all of these procaryotic groups. Minor differences with respect to the length and arrangement of the gas vesicles were observed. In blue-green algae and green bacteria, the vesicles are relatively long and tend to be arrayed in parallel bundles; in purple sulfur bacteria and Halobacterium, they are shorter and more irregularly distributed in the cell.

Bacteria↗