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I Uno

Publications and source records attributed to I Uno.

At least 37 records · Page 2Linked to original sources
PubMed1993

Characterization of the cyr1-2 UGA mutation in Saccharomyces cerevisiae.

cyr1-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyr1-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyr1-2 mutation. Construction of chimeric genes restricted the region containing the cyr1-2 mutation, and the cyr1-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyr1-2 mutation site result in null mutations. The complete open reading frame of the cyr1-2 gene expressed under the control of the GAL1 promoter complemented cyr1-d1 in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyr1-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.

Adenylyl Cyclases↗
PubMed1993

Direct sequencing of superoxide dismutase genes from two bacterial strains amplified by polymerase chain reaction.

The nucleotides of the Mn-superoxide dismutase (SOD) gene of Bacillus circulans and the Fe-SOD gene of Aerobacter aerogenes were sequenced by PCR. These SOD genes were specifically amplified by using oligonucleotide primers corresponding to the amino-terminal amino acid sequences and the antisense strand primer corresponding to the common amino acid sequence near the carboxyl-terminus among various Mn- and Fe-SODs thus far sequenced. The PCR products amplified from B. circulans and A. aerogenes genes contained a 486-nucleotide sequence encoding 162 amino acids and a 507-nucleotide sequence encoding 169 amino acids, respectively. Each sequence seemed to contain most of the open reading frame encoding the SOD protein when compared with other sequenced SODs. The two amino acid sequences deduced from the nucleotide sequences of PCR products had an identity of 66.1%. However, the SODs from B. circulans and A. aerogenes were immunologically distinct from each other judging from an immunoprecipitation test. The two SODs had high homologies with other bacterial Mn-SODs, especially the highest homology of 75.4% and 66.7%, respectively, with the B. stearothermophilus Mn-SOD. Genomic Southern hybridization suggested that each PCR product of the bacterial genes that were synthesized and sequenced was the product of the sole SOD gene in each bacterium.

Amino Acid Sequence↗
PubMed1992

Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1.

Staurosporine is an antibiotic that specifically inhibits protein kinase C. Fourteen staurosporine- and temperature-sensitive (stt) mutants of Saccharomyces cerevisiae were isolated and characterized. These mutants were divided into ten complementation groups, and characterized for their cross-sensitivity to K-252a, neomycin, or CaCl2. The STT1 gene was cloned and sequenced. The nucleotide sequence of the STT1 gene revealed that STT1 is the same gene as PKC1. The STT1 gene conferred resistance to staurosporine on wild-type cells, when present on a high copy number plasmid. STT1/stt1::HIS3 diploid cells were more sensitive to staurosporine than STT1/STT1 diploid cells. Analysis of temperature-sensitive stt1 mutants showed that the STT1 gene product functioned in S or G2/M phase. These results suggest that a protein kinase (the STT1 gene product) is one of the essential targets of staurosporine in yeast cells.

Alkaloids↗
PubMed1991

A dominant interfering mutation (CYR3) of the Saccharomyces cerevisiae RAS2 gene.

The dominant cyclic AMP-requiring mutation CYR3 had been previously reported as a mutation in the regulatory subunit of cyclic AMP-dependent protein kinase. However, recharacterization revealed that the CYR3 mutation was a nonconditional dominant lethal mutation and was a missense allele of RAS2 which results from the substitution of aspartic acid for glycine at amino acid 22.

Fungal Proteins↗
PubMed1991

Genetic and molecular analyses of the SUP201 gene: a tRNA(3Arg) nonsense suppressor of yeast cyrl-2.

The temperature-sensitive cyr1-2 mutant in Saccharomyces cerevisiae produces low levels of adenylate cyclase and cyclic AMP at 25 degrees C and is unable to synthesize repressible acid phosphatase at 25 degrees C. Suppressor mutants of cyr1-2 were isolated by detecting acid phosphatase activity. One of the dominant suppressor mutations isolated was designated SUP201 and characterized. The SUP201 mutant gene was isolated from a gene library made from cyr1-2 SUP201 mutant DNA. Nucleotide sequence analysis of the cloned SUP201 gene revealed that the SUP201 gene was a mutated tRNA gene flanking GCN4, which worked as a UGA suppressor.

Base Sequence↗
PubMed1991

Isolation and characterization of the adenylate cyclase structural gene of Neurospora crassa.

A single gene (nac) encoding an adenylate cyclase was cloned from the genomic DNA library of Neurospora crassa, using the DNA fragment encoding the catalytic domain of adenylate cyclase of Saccharomyces cerevisiae as a probe. The open reading frame of this gene (6900 base pairs) was interrupted three time by introns. The protein encoded consists of 2300 amino acids and has adenylate cyclase activity. N. crassa adenylate cyclase has a high degree of homology with the catalytic domains of yeast and bovine brain adenylate cyclases.

Adenylyl Cyclases↗
PubMed1991

Suppression of the cr-1 mutation in Neurospora crassa.

We have cloned a DNA fragment, which hybridized with the adenylate cyclase gene (CYR1) of Saccharomyces cerevisiae, from genomic DNA libraries of Neurospora crassa. The cr-1 mutation was able to be suppressed by introducing this DNA fragment on a cosmid vector, judging from recovery of the adenylate cyclase activity and the abnormal morphology.

Adenylyl Cyclases↗
PubMed1990

The adenylate cyclase/protein kinase cascade regulates entry into meiosis in Saccharomyces cerevisiae through the gene IME1.

Entry into meiosis in Saccharomyces cerevisiae cells is regulated by starvation through the adenylate cyclase/cAMP-dependent protein kinase (AC/PK) pathway. The gene IME1 is also involved in starvation control of meiosis. Multicopy IME1 plasmids overcome the meiotic deficiency of bcy1 and of RASval19 diploids. Double mutants ime1 cdc25 and ime1 ras2 are sporulation deficient. These results suggest that IME1 comes after the AC/PK cascade. Furthermore, the level of IME1 transcripts is affected by mutations in the AC/PK genes CDC25, CYR1 and BCY1. Moreover, the addition of cAMP to a cyr1-2 diploid suppresses IME1 transcription. The presence in a bcy1 diploid of IME1 multicopy plasmids does not cure the failure of bcy1 cells to arrest as unbudded cells following starvation and to enter the G0 state (thermotolerance, synthesis of unique G0 proteins). This indicates that the pathway downstream of the AC/PK cascade branches to control meiosis through IME1, and to control entry into G0 and cell cycle initiation, independently of IME1.

Adenylyl Cyclases↗
PubMed1990

The functional domain of adenylate cyclase associated with entry into meiosis in Saccharomyces cerevisiae.

Diploid yeast cells that carry a part of the CYR1 gene deficient in a region coding for the N-terminal domain of adenylate cyclase were growth arrested and accumulated unbudded cells after inoculation into complete medium or nitrogen-free medium, but produced many cells which had one or more buds after incubation in sporulation medium. The cells incubated in sporulation medium had abnormal spindles which were free from the spindle pole bodies, larger in size, or frequently distributed in cytoplasm. The levels of cyclic AMP in these cells did not decrease to the wild-type level after transfer to the sporulation medium and remained at a constant level. The results suggest that the N-terminal domain of adenylate cyclase is associated with the regulatory function for sporulation. The environmental signals for sporulation may be transferred to the adenylate cyclase system through a factor that negatively interacts with the N-terminal domain of this enzyme.

Adenylyl Cyclases↗
PubMed1989

Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature 333, 188-190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined. The incorporation of [32P]Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase. The incorporation of [32P]Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains. The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in [32P]Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis. We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP. The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells. The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases. Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells. The effect was most prominent in membranes from cyr1-2 and ras2 cells. These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.

1-Phosphatidylinositol 4-Kinase↗
PubMed1989

Isolation and characterization of temperature-sensitive mutations in the RAS2 and CYR1 genes of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.

Adenylyl Cyclases↗
PubMed1988

Essential role for phosphatidylinositol 4,5-bisphosphate in yeast cell proliferation.

The responses of mammalian cells to external signals are commonly mediated by intracellular secondary messengers, among which are the breakdown products of phosphatidylinositol 4,5-bisphosphate (PIP2): 1,2-diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3) (refs 1-7). Although phosphoinositide turnover in the yeast Saccharomyces cerevisiae has been shown to be regulated by glucose and sterol, as yet no definitive function has been ascribed to yeast phosphoinositides. We have recently developed a monoclonal antibody specific for PIP2 and reported that it inhibits mitogenesis of mammalian cells stimulated by platelet-derived growth factor and bombesin. We now report that when introduced into yeast cells by electroporation this antibody inhibits their growth. Furthermore, several yeast mutants with temperature-dependent growth defects are altered in their sensitivity to our antibody and are found to have specific alterations in their phosphoinositide metabolism.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

Localization of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae.

The subcellular distribution of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae cells was determined by subcellular fractionation and indirect immunofluorescence microscopy using the bcy1 mutant deficient in the regulatory subunit as control. The regulatory subunit of cAMP-dependent protein kinase showing cAMP-binding activity was identified as a single protein of 50 kDa by photoaffinity labeling and immunoblotting. The regulatory subunit was concentrated in a nuclear fraction in addition to a cytoplasmic fraction. By comparison of the regulatory subunit distribution with the DNA localization, the area detected by the indirect immunofluorescence was identified as the nucleus.

Carrier Proteins↗