Biomedical subjects

K Shirahige

Publications and source records attributed to K Shirahige.

At least 19 recordsLinked to original sources

PubMed2001

The cloning and characterization of the CDC50 gene family in Saccharomyces cerevisiae.

We have cloned a gene that complements the cold-sensitive growth of cdc50-1 mutant strain of Saccharomyces cerevisiae at 14 degrees C. The CDC50 gene was found to be identical to YCR094w on chromosome III and contains 1173 nucleotides encoding 391 amino acids. We found a missense mutation at the first initiation codon of cdc50-1. The disruption of the CDC50 gene revealed that it is not essential for growth, but the disruptant caused the same cold-sensitive phenotype as cdc50-1, suggesting that the cdc50-1 is a null mutation resulted from the mutation in the first codon. The cdc50-1 mutant arrests at START in G1 phase at the non-permissive temperature. The CDC50 gene product has strong structural similarity to two other proteins in Saccharomyces cerevisiae encoded by YNR048w and YNL323w. The over-expression of either YNR048w or YNL323w suppressed the cdc50-1 mutant and the double disruption of either CDC50 and YNR048w or CDC50 and YNL323w resulted in a severe slow-growth phenotype. We conclude that these three genes constitute a family with redundant function. We also found that the CDC39 gene was a multicopy suppressor of cdc50-1 mutation, suggesting that the CDC50 family is involved in regulation of transcription via CDC39.

Amino Acid Sequence↗

PubMed2001

A member of the YER057c/yjgf/Uk114 family links isoleucine biosynthesis and intact mitochondria maintenance in Saccharomyces cerevisiae.

BACKGROUND: Two paralogs, YIL051c and YER057c, in the Saccharomyces cerevisiae genome are members of the YER057c/Yigf/Uk114 family, which is highly conserved among Eubacteria, Archaea and Eukarya. Although the molecular function of this protein family is not clear, previous studies suggest that it plays a role in the regulation of metabolic pathways and cell differentiation. RESULTS: Yil051cp is 70% identical in amino acid sequence to Yer057cp, and differs in that the former is longer by 16 amino acids containing, in part, the mitochondrial targeting signal at the N-terminus of the protein. An HA-tagged protein of Yil051cp is localized strictly in mitochondria, while that of Yer057cp is found in both cytoplasm and nucleus. Disruption of YIL051c (yil051cDelta) resulted in severe growth retardation in glucose medium due to isoleucine auxotroph, and no growth in glycerol medium due to the loss of mitochondria. An extract prepared from yil051cDelta cells showed no transaminase activity for isoleucine, while that for valine or leucine was intact. Haploid yil051cDelta cells newly isolated from the YIL051c/yil051cDelta hetero-diploids gradually lost mitochondrial DNA within 24 h in the absence of, but not in the presence of, an isoleucine. Mutants either requiring leucine (leu2-112) or isoleucine-valine (bat1Delta, bat2Delta) in a YIL051c background showed no changes in mitochondrial DNA maintenance in the absence of requirements. CONCLUSIONS: Based on these results, we named Yil051c as Ibm1 (Isoleucine Biosynthesis and Mitochondria maintenance1) and concluded that: (i) Ibm1p determines the specificity of isoleucine biosynthesis, probably at the transamination step, (ii) Ibm1p is required for the maintenance of mitochondrial DNA when isoleucine is deficient, and (iii) Isoleucine compensates for the lack of Ibm1p. Taken together, Ibm1p may act as a sensor for isoleucine deficiency as well as a regulator determining the specificity for branched amino acid transaminase.

Amino Acid Sequence↗

PubMed2000

Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures.

An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.

Antibodies, Monoclonal↗

PubMed2000

Progression of cell cycle monitored by dielectric spectroscopy and flow-cytometric analysis of DNA content.

A dielectric method has already been developed for the real-time monitoring of cell cycle progression in synchronized cell culture (Asami et al., 1999). This method, in combination with DNA content analysis by fluorescence-activated cell sorting (FACS), was applied to the synchronized cell culture of a CDC28-13th mutant (Saccharomyces cerevisiae). In synchronous cell growth, relative permittivity epsilon (or dielectric constant) for the culture broth showed cyclic changes at low frequencies below 0.5 MHz, being correlated to phases in the cell cycle that were simultaneously determined by FACS. The epsilon increased in the period from S phase to G(2) phase and decreased between M and G(1) phases. Peaks in these cyclic changes of epsilon indicated the time when daughter cells segregated from mother cells.

Cell Cycle↗

PubMed1998

Regulation of DNA-replication origins during cell-cycle progression.

We have shown previously that chromosome VI of Saccharomyces cerevisiae contains nine origins of DNA replication that differ in initiation frequency and replicate sequentially during the S phase of the cell cycle. Here we show that there are links between activation of these multiple origins and regulation of S-phase progression. We study the effects of a DNA-damaging agent, methyl methane sulphonate (MMS), and of mutations in checkpoint genes such as rad53 on the activity of origins, measured by two-dimensional gel analysis, and on cell-cycle progression, measured by fluorescence-activated cell sorting. We find that when MMS slows down S-phase progression it also selectively blocks initiation from late origins. A rad53 mutation enhances late and/or inefficient origins and releases the initiation block by MMS. Mutation of rad53 also results in a late origin becoming early replicating. We conclude that rad53 regulates the timing of initiation of replication from late origins during normal cell growth and blocks initiation from late origins in MMS-treated cells. rad53 is, therefore, involved in the cell's surveillance of S-phase progression. We also find that orc2, which encodes subunit 2 of the origin-recognition complex, is involved in suppression of late origins.

Cell Cycle↗

PubMed1998

Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae.

BACKGROUND: The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis-element, ACS (ARS Consensus Sequence), essential for DNA replication. The protein-DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression. Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S. cerevisiae. RESULTS: Topological changes in the replication origins of the S. cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding. The footprinting method detected the inhibition and enhancement of UV-induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits. Furthermore, footprint patterns were reproduced in vitro with purified ORC. The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase. Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1-arrested cells. CONCLUSION: The application of in vivo UV photofootprinting in studies of topological changes of S. cerevisiae replication origins revealed the presence of two modes of topological ORC-ACS interaction. The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC-ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication.

Base Sequence↗

PubMed1997

The efficiency and timing of initiation of replication of multiple replicons of Saccharomyces cerevisiae chromosome VI.

BACKGROUND: A complete set of nine ARSs was identified (the tenth ARS in this paper), mapped on chromosome VI of Saccharomyces cerevisiae, and characterized for functional elements. RESULTS: The level of activity of all ARSs as chromosomal replication origins was determined by neutral/neutral 2D gel-electrophoresis. These origins were classified into three groups: (i) three high frequency origins used once nearly every cell cycle, (ii) four intermediate frequency origins used once in two to three cycles and (iii) two low frequency origins used in fewer than 5% of cell cycles. These variations in initiation frequency among origins of chromosome VI are present in three common laboratory wild-type strains. Each origin is initiated at a fixed time and passively replicated by incoming replication forks at a fixed time during a synchronous S phase. Replication of each arm of the chromosome starts from one major origin located one-fifth (left arm) and one-third (right arm) of the distance from the centromere, and expands sequentially in both directions. Two telomere vicinity origins are replicated last. Time of initiation and replication of the last replicating origin, Ori609, was remarkably variable from cell to cell. CONCLUSIONS: Chromosome VI of S. cerevisiae contains nine replication origins that comprise five active replicons under normal cell growth conditions. A clear correlation was found between the efficiency of initiation and the order of replication. The timing of initiation of most origins, except for the first and last, is coincident with the time of passive replication by incoming forks from neighbouring origins.

Cell Cycle↗

PubMed1996

Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae.

A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC.

Cell Division↗

PubMed1994

Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae.

We have analyzed the relationship between autonomously replicating sequence (ARS) structure and function for three ARS (ARS605, ARS607 and ARS609) from chromosome VI of Saccharomyces cerevisiae by systematic XhoI-linker mutation in the ARS consensus sequence (ACS) and flanking sequences. All mutations that encroached upon the ACS destroyed ARS activity. DNA sequences stimulative for ARS function were identified on either side of the ACS of ARS605 and only on the 3'-side of the ACS of ARS607. In ARS609, however, no such stimulative sequences were observed. Base substitutions complementary to the wild-type sequence of those stimulative regions, in ARS605 and ARS607, that did not change the delta G of unwinding nor affected ARS activity suggests that these regions have, at least, a function as DNA-unwinding elements (DUE). ARS605, ARS607 and ARS609 DNA are of low delta G value and showed hypersensitivity to single-strand-specific nuclease when inserted in negatively supercoiled plasmid. Linker mutations inhibitory for ARS activity (5L11 and 7L14) also caused significant changes in local nucleotide (nt) sensitivity within the ACS and its adjoining regions. Complementary base substitutions, however, did not affect these changes in local nt sensitivity. These results imply that the stimulative regions flanking the ACS are necessary to produce an optimum conformation around the ACS which may be important for full ARS activity.

Base Composition↗

PubMed1994

A protein which binds preferentially to single-stranded core sequence of autonomously replicating sequence is essential for respiratory function in mitochondrial of Saccharomyces cerevisiae.

From yeast nuclear extract, we have identified several DNA-protein complexes using the T-rich strand of core consensus sequence of autonomously replicating sequence by gel shift assay. One of them showed preferential binding to the T-rich sequence of the DNA. We have partially purified a protein constituent of this complex and cloned its gene. The gene has an open reading frame encoding a protein of 380 amino acids (M(r) = 42,100) which is processed to a mature protein of 371 amino acids (M(r) = 40,900). The protein has neither significant amino acid homology with any previously reported proteins nor characteristic motifs. A putative HAP2/HAP3/HAP4 binding sequence was found at about 1 kilobase upstream of the gene. Disruption of the chromosomal gene revealed that the gene was neither essential for cell viability nor involved in DNA replication, but was essential for mitochondrial respiratory function. We therefore named the gene MRF1 for mitochondrial respiratory function 1. In a mrf1 null mutant the absorption spectra of cytochromes b, a, and a3 were undetectable, although mitochondrial DNA and protein synthesis in mitochondria were intact. Antibodies against MRF1 detected the antigen localized predominantly in the nucleus in vivo. These results suggest that MRF1 is a transcriptional regulatory protein of some genes whose products are necessary for the functional assembly of mitochondrial respiratory proteins.

Amino Acid Sequence↗

PubMed1993

Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae.

We have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI. The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4. Nine ARS fragments were identified, and their positions were mapped on the chromosome. Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations. The organization of functional elements composed of core and stimulative sequences was found to be variable. Single core sequences were identified in eight of nine ARSs. The remaining ARS (ARS603) essential element is composed of two core-like sequences. The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS. Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs. In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence. In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids.

Base Sequence↗

PubMed1991

The direct cloning of the yeast genome using the gap-filling method and the complete physical mapping of Saccharomyces cerevisiae chromosome VI.

The ordered clone library of chromosome VI of Saccharomyces cerevisiae has been constructed by Olson et al. [Proc. Natl. Acad. Sci. USA 83 (1986) 7826-7830, and personal communication]. It is composed of four contiguous stretches from the chromosome, each of 40-70 kb. There remained three gaps of unknown length between these four contigs. We applied the 'gap-repair' method to clone these three gap regions directly from the yeast chromosome. All three gap regions, ranging from 7 to 22 kb, were successfully cloned without any structural changes. Together with these gap regions, a precise physical map of EcoRI and HindIII sites was constructed over the 230-kb fragment which covers most of chromosome VI except for two telomeres.

Chromosome Mapping↗

PubMed1990

Molecular cloning, genetic characterization and DNA sequence analysis of the recM region of Bacillus subtilis.

In Bacillus subtilis the recM gene, whose product is associated with DNA repair and recombination, has been located between the dnaX and rrnA genes. The recM gene has been cloned and analyzed. Analysis of the nucleotide sequence (3.741-kilobase) around recM revealed five open reading frames (orf). We have assigned recM and dnaX to two of this orf, given the gene order dnaX-orf107-recM-orf74-orf87. The organization of genes of the dnaX-orf107-recM region resembles the organization of genes in the dnaX-orf12-recR region of the Escherichia coli chromosome. Proteins of 24.2 and 17.0 kDa would result from translation of the wild type and in vitro truncated recM genes, and radioactive bands of proteins of molecular weights of 24.5 and 17.0 kDa were detected by the use of the T7promoter-expression system. The RecM protein contains a potential zinc finger domain for nucleic acid binding and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP. Strains, in which the recM gene has been insertionally inactivated, were generated and show a phenotype essentially the same as previously described recM mutants.

Adenosine Triphosphate↗

PubMed1978