Biomedical subjects

F Hishinuma

Publications and source records attributed to F Hishinuma.

At least 37 records · Page 2Linked to original sources

PubMed1991

Estrogen delays entry of the yeast Saccharomyces cerevisiae into meiosis.

Estrogen has been suggested to influence the cell cycle of haploid yeast cells in the early G1 phase of mitosis, its effect possibly being mediated by control of the level of cAMP (TANAKA, S. et al. (1989). Cell, 57: 675-681). Therefore, we were interested in whether estrogen also affects the meiotic phase of diploid yeast cells. Accordingly, we measured the amounts of adenylate cyclase mRNA and intracellular cAMP, the proportions of dividing cells and 4n cells and the doubling times of diploid yeast cells during the presporulation stage in the presence and absence of estrogen. The amount of adenylate cyclase mRNA was found to decrease rapidly within 24 hours after inoculation of cells onto sporulation-promoting plates (YPA plates). The cAMP level of these cells also decreased rapidly. Mitotic cell division continued for 18 hours after cell inoculation, but about 24 hours after inoculation, the amount of cAMP per cell had decreased to a minimum and the cells began to enter meiosis. By contrast, when the cells were inoculated onto YPA plates in the presence of estrogen, their intracellular cAMP and adenylate cyclase mRNA levels became higher than those in control cultures without estrogen and cell division continued for 24 hours. But after 30 hours their intracellular cAMP level decreased to a minimum and they began to enter meiosis. These results show that estrogen delayed the entry of diploid yeast cells into meiosis on sporulation-promoting plates and suggest that its effect may be mediated by control of the level of cAMP.

Adenylyl Cyclases↗

PubMed1990

Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae.

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.

Animals↗

PubMed1990

Integration of heterologous genes into the chromosome of Saccharomyces cerevisiae using a delta sequence of yeast retrotransposon Ty.

Distribution of a delta (delta) sequence of the Ty element on a chromosome of the yeast Saccharomyces cerevisiae was analysed by pulsed-field gel electrophoresis. More than 100 copies of the delta sequence were nonrandomly distributed on the chromosome. Using the delta sequence as a recombination site, mouse alpha-amylase and human beta-endorphin genes were introduced into the chromosomal DNA. The integration occurred on a particular chromosome in each case and the copy number was estimated as three to five. It was also found that single- or multi-copy integration occurred at a single or multiple sites on the particular chromosome. The integrants secreted alpha-amylase and beta-endorphin by three-to fivefold compared with single-copy integrants. This type of integration was mitotically stable over a period of 50 generations under non-selective conditions.

Chromosomes↗

PubMed1990

Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.

Alleles↗

PubMed1989

Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae.

The DNA coding for human nerve growth factor (hNGF) was chemically synthesized and introduced into Saccharomyces cerevisiae. Expression and secretion of hNGF was obtained by use of the yeast phosphoglycerate kinase-encoding gene promoter and the pre-pro sequence of the yeast alpha-mating factor. Immunoblotting with antiserum raised against a protein A-hNGF fusion protein, allowed the detection of an immunoreactive material secreted into the culture medium. A preparation from the culture medium, partially purified by ion-exchange column chromatography, stimulated neurite outgrowth from rat pheochromocytoma PC12h cells.

Amino Acid Sequence↗

PubMed1989

Estrogen can regulate the cell cycle in the early G1 phase of yeast by increasing the amount of adenylate cyclase mRNA.

The effects of beta-estradiol (estrogen; a minor component of yeast cells) on S. cerevisiae cells in the G0 and G1 phases were examined. Results showed that estrogen stimulated the recovery of growth from G0 arrest induced by nutrient limitation or ts mutation of cdc35 (adenylate cyclase) in the early G1 phase, and inhibited entry into the resting G0 phase by increasing the intracellular cAMP level. However, estrogen had no effect on late G1 arrest induced by the alpha factor or ts mutation of cdc36. Estrogen was found to lead to higher steady-state levels of adenylate cyclase mRNA but not to affect the expression of the RAS1 and RAS2 genes, although these can also alter the intracellular cAMP level. These results suggest that estrogen influences the cell cycle of yeast in the early G1 phase by controlling the level of cAMP through the increase of adenylate cyclase mRNA.

Adenylyl Cyclases↗

PubMed1989

Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein.

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987]. The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.

Fungal Proteins↗

PubMed1989

Pseudomonas aeruginosa cytotoxin: the nucleotide sequence of the gene and the mechanism of activation of the protoxin.

The gene encoding cytotoxin (ctx) was cloned from Pseudomonas aeruginosa 158 and the nucleotide sequence was determined. The structural gene of ctx encodes the procytotoxin of 286 amino acid residues with a molecular mass of 31,681 Daltons. Procytotoxin was activated by removal of 20 amino acid residues from the C terminus with trypsin. The cloned ctx gene was not expressed in either an Escherichia coli strain or a cytotoxin non-producing strain of P. aeruginosa. An expression system for the ctx gene was constructed by placing the structural gene of ctx downstream of tac promoter on a broad host-range vector plasmid.

Amino Acid Sequence↗

PubMed1988

A novel yeast secretion signal isolated from 28K killer precursor protein encoded on the linear DNA plasmid pGKL1.

Saccharomyces cerevisiae harboring linear dsDNA plasmids, pGKL1 and pGKL2, secretes a killer toxin consisting of 97, 31 and 28 kilodalton subunits (Nucleic Acids Res., 15, 1031-1046, 1987). We isolated the DNA encoding the N-terminal pre-sequence of the 28K precursor protein and constructed a new secretion vector in S. cerevisiae. Mouse alpha-amylase fused to the 28K signal sequence was secreted into the culture medium with a high efficiency similar to those fused to the mating factor alpha and 97K-31K killer signal sequences. This data clearly indicates that 28K presequence functions as a secretion signal. Glycosylated and nonglycosylated alpha-amylase molecules were detected in the culture medium. The secretion of alpha-amylase was blocked by sec18-1 mutation. The secreted alpha-amylase recovered from the medium was found to migrate faster in SDS-polyacrylamide gel than the precursor form of alpha-amylase synthesized in vitro. These lines of evidence suggest that mouse alpha-amylase fused to 28K killer signal sequence was processed, glycosylated and secreted through the normal secretion pathway of the yeast.

Amino Acid Sequence↗

PubMed1988

Sequence-directed bends of DNA helix axis at the upstream activation sites of alpha-cell-specific genes in yeast.

The MF alpha 1 gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone alpha factor, is an alpha-cell-specific gene whose expression is regulated by the mating-type locus, MAT. Two upstream activation sites (UASMF alpha 1s), which are binding sites for an activator protein, MAT alpha 1, mediate alpha-cell-specific expression of this gene. We show here that DNA fragments containing the UASMF alpha 1 region exhibited anomalous slow electrophoretic mobilities on gels at lower temperature, but not at higher temperature, that is characteristic of bent DNA. We confirmed the sequence-directed bend at the UASMF alpha 1 region by employing a circular permutation analysis and a DNA cyclization assay. Deletion analyses revealed the existence of two bends in this region, each of which overlaps each UASMF alpha 1 element. The two bends were almost in phase; they lie in a nearly same plane with a same direction. We also show the existence of sequence-directed bend at the UAS region of the STE3 gene, another alpha-specific gene in S. cerevisiae. These bent DNAs may be involved in transcriptional regulation of this set of genes.

Base Sequence↗

PubMed1988

Evidence for preferential multiplication of the internal unit in tandem repeats of the mating factor alpha genes in Saccharomyces yeasts.

We have determined DNA sequences of the mating factor alpha genes of Saccharomyces uvarum and Saccharomyces italicus and compared them to that of the MF alpha 1 gene of S. cerevisiae. The DNA sequences of the mating factor genes in both species were almost completely identical to that of the MF alpha 1 gene of S. cerevisiae except for the number of tandem repeated units; these latter consisted of a spacer peptide and a mature mating factor and there were three units in S. uvarum and five units in S. italicus compared with four units in the MF alpha 1 of S. cerevisiae. From the detailed comparison of DNA sequences of the spacer peptide-mating factor units from these three species, the high sequence homology can be recognized in the internal units of the tandem repeats. This suggests that the internal units might be multiplied preferentially in the tandem repeated units of mating factor genes.

Amino Acid Sequence↗

PubMed1988

Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae.

We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.

Genes, Fungal↗

PubMed1987

Expression of beta-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain.

A 50-base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.

Animals↗

PubMed1987

A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1.

The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae. The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity. The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha. The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S. cerevisiae.

Amino Acid Sequence↗

PubMed1987

Expression and identification of immunity determinants on linear DNA killer plasmids pGKL1 and pGKL2 in Kluyveromyces lactis.

The linear dsDNA plasmids, pGKL1 (8.9 kb) and pGKL2 (13.4 kb) discovered in Kluyveromyces lactis, confer killer and immunity characteristics upon various yeast strains. We have devised an immunity assay and have been able to show the expression of an immunity phenotype in the K. lactis transformants harbouring conventional circular plasmids which contain DNA fragments of pGKL1. Using this expression system, the immunity determinant on pGKL1 was identified as ORF5. In addition, the presence of pGKL2 was proved to be essential for the expression of the immunity phenotype. This is the first demonstration of this new pGKL2 function, as distinct from its known functions for the replication and maintenance of pGKL1 in yeast cells.

DNA Restriction Enzymes↗

PubMed1987

Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomous replication of hybrid plasmids in Saccharomyces cerevisiae.

By use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker. The replication properties of hybrid plasmids in yeasts were investigated. We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids. Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.

Base Sequence↗

PubMed1987

Molecular cloning of an amylase gene of Bacillus circulans.

An amylase gene of Bacillus circulans was cloned in B. subtilis and its nucleotide sequence was determined. The putative proamylase consists of 528 amino acids, which correspond to a molecular weight of 58,776. Homologous regions with other amylases of Bacillus species were found. A sigma 55-type promoter is located at about 250 bp upstream from the starting codon. This promoter was also functional in Escherichia coli, and able to express beta-galactosidase activity.

Amino Acid Sequence↗

PubMed1987

Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae.

The MF alpha 1 gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone alpha factor, is an alpha-specific gene whose expression is regulated by the mating-type locus. To study the role of sequences upstream of MF alpha 1 in its expression and regulation, we generated two sets of promoter deletions: upstream deletions and internal deletions. By analyzing these deletions, we have identified a TATA box and two closely related, tandemly arranged upstream activation sites as necessary elements for MF alpha 1 expression. Two upstream activation sites were located ca. 300 and 250 base pairs upstream of the MF alpha 1 transcription start points, which were also determined in this study. Each site contained a homologous 22-base-pair sequence, and both sites were required for maximum transcription level. The distance between the upstream activation sites and the transcription start points could be altered without causing loss of transcription efficiency, and the sites were active in either orientation with respect to the coding region. These elements conferred cell type-specific expression on a heterologous promoter. Analysis with host mating-type locus mutants indicates that these sequences are the sites through which the MAT alpha 1 product exerts its action to activate the MF alpha 1 gene. Homologous sequences with these elements were found in other alpha-specific genes, MF alpha 2 and STE3, and may mediate activation of this set of genes by MAT alpha 1.

Base Sequence↗