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M Harata

Publications and source records attributed to M Harata.

14 recordsLinked to original sources
PubMed1999

The nuclear actin-related protein of Saccharomyces cerevisiae, Act3p/Arp4, interacts with core histones.

Act3p/Arp4, an essential actin-related protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.

Actins↗
PubMed1999

Two isoforms of a human actin-related protein show nuclear localization and mutually selective expression between brain and other tissues.

Actin-related proteins (Arps), which are divergent, but apparently homologues to actin, are categorized into 10 classes. While Arps belonging to classes 1-3 were found to be localized in the cytoplasm across eukaryotic phyla, other classes of Arps were found mostly in invertebrates and suggested to contribute to structural modulation of chromatin. Here we report the identification and the characterization of two human isoforms of an Arp not belonging to classes 1-3, which we designated hArpN alpha and hArpN beta. Both proteins were expressed in HeLa cells and they were found localized within the nucleus. Most interestingly, in different human tissues, hArpN alpha and beta were found to be expressed mutually exclusively, and the expression of hArpN alpha was absolutely restricted to the brain. These findings suggest that, in vertebrates, members of distantly related Arps might have tissue-specific functions in the nucleus, possibly through structural modulation of chromatin.

Actins↗
PubMed1998

Triplet repeat-containing ribosomal protein L14 gene in immortalized human endothelial cell line (t-HUE4).

A cDNA encoding human 60S ribosomal subunit protein L14 (hRL14) was isolated from a human immortal endothelial cell line, t-HUE4. This cell line was established via a series of cell lines cultured in a serum-free and a protein-free medium, and a directional cDNA library has been constructed and screened in search for the genes modulating protein synthesis machinery in cell proliferation. A putative full-length clone with an open reading frame of 220 amino acids; predicted molecular weight of 23.6 kDa. A significant identity for hRL14 was observed with rat RL14 (85% identity), with exception of COOH-terminal region, but not with any eukaryote amino acid sequences so far deposited to database. The typical features of ribosomal proteins were observed in hRL14, as seen in nuclear targeting sequences necessary for the transport from cytoplasm to nucleolus, a bZIP like (basic region-leucine zipper) element for the binding to rRNA, and the internal repeat sequences; the pentapeptide QKA(A/S)X. The COOH-terminal region of the transcripts contained fifteen triplet repeats (GCT; alanine) at nucleotide 465 to 509, which is significantly expanded compared to the rat RL14. However, the repeat number was all the same among the normal human endothelial cell line and the cell lines established in the course of t-HUE4 establishment. A single band with about 800 bases was identified by Northern blot analysis without tissue specificity. This GCT repeat was found to be one of the longest uninterrupted repeats in a coding sequence, which were associated with the highest degree of polymorphism.

Amino Acid Sequence↗
PubMed1997

Purification and nucleic-acid-binding properties of a Saccharomyces cerevisiae protein involved in the control of ploidy.

Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum. The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kühne, C. & Karwan, A. (1995) Yeast 11, 929-944]. Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins. In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scp160p. Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellulose and anti-Scp160p affinity chromatography. In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA. Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes. Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm.

Amino Acid Sequence↗
PubMed1997

A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of chicken and marked decrease of its immunoreactivity during the progression of S phase.

A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunoreactivity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.

Amino Acid Sequence↗
PubMed1995

The actin-related protein Act3p of Saccharomyces cerevisiae is located in the nucleus.

Actin-related proteins, a group of protein families that exhibit about 50% sequence identity among each other and to conventional actin, have been found in a variety of eukaryotic organisms. In the budding yeast Saccharomyces cerevisiae, genes for one conventional actin (ACT1) and for three actin-related proteins (ACT2, ACT3, and ACT5) are known. ACT3, which we recently discovered, is an essential gene coding for a polypeptide of 489 amino acids (Act3p), with a calculated molecular mass of 54.8 kDa. Besides its homology to conventional actin, Act3p possesses a domain exhibiting weak similarity to the chromosomal protein HMG-14 as well as a potential nuclear localization signal. An antiserum prepared against a specific segment of the ACT3 gene product recognizes a polypeptide band of approximately 55 kDa in yeast extract. Indirect immunofluorescence experiments with this antiserum revealed that Act3p is located in the nucleus. Nuclear staining was observed in all cells regardless of the stage of the cell cycle. Independently, immunoblotting experiments with subcellular fractions showed that Act3p is indeed highly enriched in the nuclear fraction. We suggest that Act3p is an essential constituent of yeast chromatin.

Actins↗
PubMed1994

An essential gene of Saccharomyces cerevisiae coding for an actin-related protein.

Actin filaments provide the internal scaffold of eukaryotic cells; they are involved in maintenance of cell shape, cytokinesis, organelle movement, and cell motility. The major component of these filaments, actin, is one of the most well-conserved eukaryotic proteins. Recently genes more distantly related to the conventional actins were cloned from several organisms. In the budding yeast, Saccharomyces cerevisiae, one conventional actin gene, ACT1 (coding for the filament actin), and a so-called actin-like gene, ACT2 (of unknown function), have so far been identified. We report here the discovery of a third member of the actin gene family from this organism, which we named ACT3. The latter gene is essential for viability and codes for a putative polypeptide, Act3, of 489 amino acids (M(r) = 54,831). The deduced amino acid sequence of Act3 is less related to conventional actins than is the deduced amino acid sequence of Act2, mainly because of three unique hydrophilic [corrected] segments. These segments are found inserted into a part of the sequence corresponding to a surface loop of the known three-dimensional structure of the actin molecule. According to sequence comparison, the basal core structure of conventional actin may well be conserved in Act3. Our findings demonstrate that, unexpectedly, there exist three members of the diverse actin protein family in budding yeast that obviously provide different essential functions for survival.

Actins↗
PubMed1993

W-heterochromatin of chicken; its unusual DNA components, late replication, and chromatin structure.

About 65% of DNA in the chicken W chromosome has been shown to consist of XhoI and EcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kb XhoI and the 1.2-kb EcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescence in situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed along XhoI and EcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the beta-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of the HaeIII-digested MSB-1 nuclei yielded a chromatin fraction in which XhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for the XhoI family sequence were present in this fraction.

Actins↗
PubMed1989

Presence of female-specific bent-repetitive DNA sequences in the genomes of turkey and pheasant and their interactions with W-protein of chicken.

Two female-specific repetitive DNA units, the 0.4 kb PstI and 0.5 kb TaqI sequences, were detected in the genomic DNA of turkey and pheasant, respectively, by Southern blot hybridization under non-stringent conditions with the W chromosome-specific 0.7 kb XhoI repetitive unit of chicken as a probe. Cloning and sequencing of these two repetitive units revealed that they shared features with the XhoI family repetitive unit of chicken although the overall similarities of the nucleotide sequences were less than 60%. In common with the chicken XhoI family they consisted of tandem repeats of about 21 bp, the majority of which contained (A)3-5 and (T)3-5 clusters separated by six or seven relatively G + C-rich sequences, and they behaved as bent DNA molecules on polyacrylamide gel electrophoresis at room temperature. W-protein, purified from chicken liver nuclei and shown to bind with high affinity to the XhoI family repetitive unit, also bound with the cloned repetitive units from turkey and pheasant DNase I footprint analysis suggested that the mode of interaction of W-protein with these units was similar to that with the 0.7 kb XhoI sequence. On the other hand, W-protein did not bind to the female-specific 0.4 kb BamHI repetitive unit from the Bobwhite quail. The 0.4 kb BamHI sequence contained some A and T clusters but these clusters did not appear in phase with the pitch of DNA helix and the repetitive unit did not show DNA bending.

Animals↗
PubMed1988

Purification and characterization of W-protein. A DNA-binding protein showing high affinity for the W chromosome-specific repetitive DNA sequences of chicken.

A protein component, which binds with high affinity to the W chromosome-specific XhoI family repetitive DNA of chicken (Tone, M., Sakaki, Y., Hashiguchi, T., and Mizuno, S. (1984) Chromosoma (Berl.) 89, 228-237), was detected in the 0.35 M NaCl extract of the female chicken liver nuclei. This protein, designated as W-protein, was substantially purified by phosphocellulose, hydroxyapatite, and DEAE-Toyopearl column chromatography. Molecular weight of W-protein was estimated to be about 72,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but it seems to form multimeric structure having apparent molecular weight of about 2.3 X 10(6) under nondenaturing conditions. W-Protein binds strongly to both 0.7- and 1.1-kb repeating units of the XhoI family, both of which show curved DNA characteristics, and weakly to the AATAT-satellite sequence of Drosophila melanogaster. Stable binding of W-protein requires greater than or equal to 300 base pairs of the 0.7-kilobases sequence, or more than 14 tandem repeats of the 21-base pair internal repeating unit of the 0.7-kilobase sequence. DNA footprint analysis and effects of some DNA-binding compounds suggest that the DNA double helix wraps around W-protein or its multimeric form contacting through A-T-rich minor grooves. A possible role of W-protein in the formation of W heterochromatic body is discussed.

Animals↗
PubMed1977

Abnormal urogenital organs occurring spontaneously in inbred ACI and Kyoto-notched rats.

Spontaneous malformations of urogenital organs found in inbred rat foetuses of ACI/NHok strains were further investigated with a postnatal survey involving other inbred strains. A new type of malformation occurred in Kyoto-notched rats. The present findings suggested that the malformations have different hereditary characteristics caused by different maldevelopmental mechanisms and are probably due to recessive polygenes.

Animals↗