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M Yanagida

Publications and source records attributed to M Yanagida.

At least 163 records · Page 9Linked to original sources
PubMed1992

A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere.

Fission yeast centromeres vary in size but are organized in a similar fashion. Each consists of two distinct domains, namely, the approximately 15-kilobase (kb) central region (cnt+imr), containing chromosome-specific low copy number sequences, and 20- to 100-kb outer surrounding sequences (otr) with highly repetitive motifs common to all centromeres. The central region consists of an inner asymmetric sequence flanked by inverted repeats that exhibit strict identity with each other. Nucleotide changes in the left repeat are always accompanied with the same changes in the right. The chromatin structure of the central region is unusual. A nucleosomal nuclease digestion pattern formed on unstable plasmids but not on stable chromosome. DNase I hypersensitive sites correlate with the location of tRNA genes in the central region. Autonomously replicating sequences are also present in the central region. The behavior of truncated minichromosomes suggested that the central region is essential, but not sufficient, to confer transmission stability. A portion of the outer repetitive region is also required. A larger outer region is necessary to ensure correct meiotic behavior. Fluorescence in situ hybridization identified individual cens. In the interphase, they cluster near the nuclear periphery. The central sequence (cnt+imr) may play a role in positioning individual chromosomes within the nucleus, whereas the outer regions (otr) may interact with each other to form the higher-order complex structure.

Base Sequence↗
PubMed1992

Fission yeast pap1-dependent transcription is negatively regulated by an essential nuclear protein, crm1.

The fission yeast pap1+ gene encodes an AP-1-like transcription factor that contains a leucine zipper motif. We identified a target gene of pap1, the p25 gene. The 5' upstream region of the p25 gene contains an AP-1 site, and by DNase I footprint analysis, we showed that the pap1 protein binds to the AP-1 site as well as to a 14-bp palindrome sequence. p25 is overproduced when the pap1+ gene is overexpressed, whereas p25 is not produced at all in the pap1 deletion mutant. p25 was previously found to be overproduced in strains carrying cold-sensitive crm1 mutations whose gene product is essential for viability and is thought to play an important role in maintenance of a proper chromosomal architecture. Deletion and site-directed mutagenesis of sequences upstream of the p25 gene demonstrated that the AP-1 site as well as the palindrome sequence are crucial for transcriptional activation either by pap1 overproduction or by the cold-sensitive crm1 mutation; pap1+ is apparently negatively regulated by crm1+. Moreover, we found that cold-sensitive crm1 mutations are suppressed by the deletion of pap1+, further indicating a close relationship between crm1+ and pap1+. The crm1 protein is highly conserved; the budding yeast homolog, CRM1, which complements the fission yeast cold-sensitive crm1 mutation, was isolated and found to also be essential for viability. These results suggest the functional importance of chromosome structure on the regulation of gene expression through the pap1 transcription factor.

Amino Acid Sequence↗
PubMed1992

NG-methyl-L-arginine, an inhibitor of L-arginine-derived nitric oxide synthesis, stimulates renal sympathetic nerve activity in vivo. A role for nitric oxide in the central regulation of sympathetic tone?

Continuous production of endothelium-derived nitric oxide (NO) in peripheral vessels has been shown to modulate vascular resistance and blood pressure. NO is also formed in the brain upon activation of glutamate receptors, which are thought to mediate central autonomic reflexes. In the present study we assessed whether NO plays a role in central autonomic regulation. For this, we have investigated the effects of NG-methyl-L-arginine (NMA), a selective inhibitor of NO synthesis from L-arginine, on sympathetic renal nerve activity (RNA), blood pressure, and heart rate in the anesthetized rat. NMA elicited a dose-dependent sustained increase in blood pressure (approximately 20 and 30 mm Hg, 5 minutes after 10 and 50 mumol/kg i.v., respectively). Heart rate and RNA decreased transiently (15 beats per minute and 40%, respectively); RNA subsequently increased (100%) while blood pressure remained elevated. Baroreceptor deafferentation markedly altered these responses to NMA; the transient decreases in heart rate and RNA were abolished, whereas the increases in RNA and blood pressure were significantly potentiated. After spinal C-1-C-2 transection, there was no increase in RNA, and blood pressure increased to a smaller extent. L-Arginine blocked the NMA-induced increases in blood pressure and RNA. Thus, in addition to modulating vascular resistance by a peripheral action, NO may also play a role in the central regulation of sympathetic tone.

Animals↗
PubMed1992

Visualization of centromeric and nucleolar DNA in fission yeast by fluorescence in situ hybridization.

The nucleolar and centromeric DNAs of the fission yeast Schizosaccharomyces pombe were visualized in the nucleus by fluorescence in situ hybridization using repetitive ribosomal and centromeric DNAs as the probes. The rDNAs were seen in the nuclear domain previously assigned as nucleolar, that is, the region into which the rod-like chromatin protrudes from the hemispherical chromosomal domain. Using mitotically-arrested cells containing condensed chromosomes, it was demonstrated that the rDNAs were present on the smallest chromosome III, consistent with genetic data. Using a centromeric repetitive element as the hybridization probe, the centromere of chromosome III, cen3, which contains the largest number of the repetitive elements, was visualized. The centromere in interphase cells is located near the periphery of the nucleus as a single dot. Early in mitosis, however, it divides into two and is situated in the middle of the short mitotic spindle. After spindle extension in anaphase, the centromeric DNA is present at both ends of the spindle, that is, near the spindle pole bodies. The movement of cen3 during mitosis (anaphase A and B) is discussed in relation to spindle dynamics and chromosome separation.

Cell Cycle↗
PubMed1992

Purification of rat megakaryocyte colony-forming cells using a monoclonal antibody against rat platelet glycoprotein IIb/IIIa.

We recently reported the production and characterization of four monoclonal antibodies (MoAbs) against rat platelet glycoprotein IIb/IIIa (GPIIb/IIIa). In this study we developed a simple and efficient three-step procedure, based on positive selection by immunoadsorption (panning) using one MoAb, P55, to purify rat megakaryocyte colony-forming cells (megakaryocyte colony-forming units, CFU-MK) from normal bone marrow. Cells obtained after each step were assayed for their ability to form megakaryocyte colonies in the presence of Concanavalin A (Con A)-stimulated rat spleen cell-conditioned medium in soft agar cultures. Marrow cells were first separated on discontinuous Percoll gradients. Cells sedimented at densities between 1.063 and 1.082 g/ml were depleted of cells adherent to plastic tissue culture dishes. The nonadherent cells were further incubated on dishes coated with P55 MoAb. CFU-MK were enriched about 50-fold in the adsorbed cell fraction. This sequential fractionation procedure resulted in a 345-fold (range 276 to 412-fold) enrichment of rat CFU-MK over whole bone marrow cells. The average cloning efficiency of CFU-MK in the final fraction was about 7% (range 5%-9.2%) of the nucleated cells. The overall recovery of CFU-MK averaged 20% (range 9%-29%). The panning step provided a 46-fold enrichment of megakaryocyte burst-forming cells (megakaryocyte burst-forming units, BFU-MK), whose average cloning efficiency in the post-panning fraction was 0.14% (range 0.07%-0.2%). In addition, erythroid burst-forming cells (erythroid burst-forming units, BFU-E) were also significantly enriched by panning, but to a lesser degree than BFU-MK and CFU-MK. By contrast, granulocyte-macrophage colony-forming cells (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid colony-forming cells (erythroid colony-forming units, CFU-E) were not enriched by panning. CFU-MK obtained after panning formed megakaryocyte colonies in the presence of recombinant rat interleukin 3 (rIL-3), mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), or human erythropoietin (hEPO), as has been reported for murine CFU-MK in whole marrow cells. The highly enriched populations of rat CFU-MK should thus provide a basis for the further study of the regulation of megakaryocytopoiesis.

Animals↗
PubMed1991

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver.

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37 degrees C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.

Amino Acid Sequence↗
PubMed1991

A large number of tRNA genes are symmetrically located in fission yeast centromeres.

We report here that the fission yeast centromere regions in the three chromosomes contain no less than 36 symmetrically arranged tRNA-coding sequences, and many of them are located within the inner inverted regions that are thought to be essential for the centromere function. There are 11 different species of tRNA-coding sequences, and four of them are identical to those previously known in this organism. This high-density distribution of tRNA genes in the centromere regions is surprising, as the fission yeast centromeres were thought to form transcriptionally inactive structures.

Base Sequence↗
PubMed1991

Aminopeptidase A in human placenta and pregnant serum.

The activities of serum aminopeptidases (APs) derived from the placenta rise during pregnancy. To confirm the localization of AP-A, which liberates N-terminal acidic amino acids, in placental tissue and to identify it in the serum of pregnant women, chromatographic separation was performed. When a placental extract was subjected to DEAE cellulose column chromatography, the Glu-MCA degradative activity, or AP-A, was separated into two entities. Inhibition testing revealed that the enzyme eluted in Fraction No. 20 from the DEAE-cellulose column consisted of typical AP-A with a molecular weight of approximately 500,000. When the Glu-MCA degradative activity was observed by filtering pregnant serum through HPLC TSK G3000 gel, the activity was found to be due to serum CAP rather than AP-A. What had been thought to be a biochemicophysiological action inherent in blood AP-A raised additional questions.

Aminopeptidases↗
PubMed1991

S. pombe gene sds22+ essential for a midmitotic transition encodes a leucine-rich repeat protein that positively modulates protein phosphatase-1.

The fission yeast dis2+ gene encodes one of the two type 1 protein phosphatases (PP1) in this organism. Its semidominant mutant dis2-11 is defective in mitosis. Here we report the characterization of a high dosage suppressor, sds22+, that complements dis2-11. Sequencing of the cloned sds22+ gene predicts a novel 30 kd protein, which consists almost entirely of leucine-rich 22 amino acid repeats and is enriched in the insoluble nuclear fraction. sds22+ is an essential gene required for the mitotic metaphase/anaphase transition; gene disruption causes cell cycle arrest at midmitosis. Unexpectedly, the sds22+ gene becomes dispensable upon high dosage of the PP1 genes. The sds22+ product appears to facilitate PP1-dependent dephosphorylation, but does not substitute PP1. We propose that the sds22+ protein forms a repeating helical rod that is capable of enhancing a PP1-dependent dephosphorylation activity that is essential in midmitosis.

Amino Acid Sequence↗
PubMed1991

Structure of the fission yeast centromere cen3: direct analysis of the reiterated inverted region.

We determined the structure of the Schizosaccharomyces pombe centromere cen3 using direct genomic mapping and cosmid walking. The repetitive region of cen3 is approximately 110 kb, much longer than that of the previously determined cen1 and cen2 regions. The approximately 30 kb long left and approximately 60 kb right repetitive sequences are arranged with an inverted symmetry and flank the 15 approximately 20 kb central domain. The repeat motifs in cen3, although they consist of the common centromeric repeat elements, are slightly different from those in cen1 and cen2. The cen3 repeat motifs appear to be reiterated four times in the left and nine times in the right side repetitive regions. We found that the central domain consists of the common approximately 5 kb core sequence associated with the pair of innermost inverted sequences, most of which are reiterated only twice in the genome. Although their sizes differ significantly, the general features of cen1, cen2 and cen3 are similar, and a prototype, consensus structure for the fission yeast centromere may be deduced.

Base Sequence↗
PubMed1991

Fetal death associated with measles virus infection of the placenta.

A case of fetal death at 25 weeks' gestation, which was associated with maternal measles infection, is described. Immunohistochemical study revealed measles virus antigen in the syncytial trophoblastic cells and decidua but not in the fetus. Results suggest that fetal death was caused by measles virus infection in the placenta. As far as we know, this is the first report in which a measles virus antigen was detected in the placenta by the immunohistochemical method.

Adult↗
PubMed1991

The TPR snap helix: a novel protein repeat motif from mitosis to transcription.

The recently discovered TPR gene family encodes a diverse group of proteins that function in mitosis, transcription, splicing, protein import and neurogenesis. These multi-domain proteins all contain tandemly arranged repeats of a 34-amino acid motif that are presumed to form helix-turn structures, each with a 'knob' and 'hole', acting as helix-associating domains.

Amino Acid Sequence↗
PubMed1991

Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases.

Staurosporine, a potent inhibitor of protein kinase C, arrests fission yeast cell elongation specifically at a stage immediately after cell division. We isolated two genes, which, when carried on multicopy plasmids, confer drug resistance in fission yeast. One, spk1+, encodes a protein kinase highly similar (54% identity) to those encoded by the mammalian ERK1/MAP2 kinase and the budding yeast KSS1 and FUS3 genes. It is not essential for vegetative growth of Schizosaccharomyces pombe cells but is required for conjugation. The spk1+ gene product is a 45-kD protein enriched in the nucleus, and its level increases 10-fold after addition of staurosporine. The other gene pap1+ encodes an AP-1-like transcription factor that contains a region rich in basic amino acids followed by a "leucine zipper" motif. The pap1+ gene is required for spk1(+)-conferred staurosporine resistance. These two genes appear to function as a part of the fission yeast growth control pathway.

Alkaloids↗
PubMed1991

The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control.

The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis.

Amino Acid Sequence↗
PubMed1991

A functional 125-kDa core polypeptide of fission yeast DNA topoisomerase II.

We purified fission yeast DNA topoisomerase II (topo II) to apparent homogeneity. It consists of a single 165-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, upon treatment with a bifunctional reagent, doubles its molecular weight. Limited proteolysis of intact topo II by papain produces a 125-kDa core, which lacks the N-terminal 75 and the C-terminal approximately 260 amino acids but still contains regions similar to those of bacterial or phage T4 topo II subunits. The core retains relaxing and unknotting activities. Further digestion inactivates the core, cleaving it at the middle of the GyrB-like domain and at the beginning of the GyrA-like domain. Therefore, papain appears to cleave spatially distinct subdomains of topo II. We made top2 mutant genes deleted of the C-terminal 286 or N-terminal 74 amino acids, which can substitute for the wild-type top2+ gene in mitosis and meiosis. However, a mutant containing deletions of both termini cannot rescue the top2 null mutant, despite the fact that the product is enzymatically active. Therefore, the top2 product of the doubly truncated gene may not fulfill all of the in vivo requirements for top2+ function.

Amino Acid Sequence↗
PubMed1991

The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers.

gamma-Tubulin exists in fission yeast as the product of an essential gene, encoding a 446 amino acid protein that is 77.3% identical to Aspergillus nidulans gamma-tubulin. The gene disruption caused cell lethality, displaying condensed, undivided chromosomes with aberrant spindle structures. Anti-gamma-tubulin staining showed that gamma-tubulin is located, throughout the wild-type cell cycle, at the spindle pole bodies (SPBs), indicating that gamma-tubulin associates with interphase SPB in the absence of microtubules. In addition, anti-gamma-tubulin immunofluorescence staining revealed cytoplasmic, cell-equatorial putative MTOCs (microtubule organizing centers), which appear only during mitotic telophase and cytokinesis, and are located at the centers for the new cytoplasmic microtubule arrays of the two daughter cells. In the multiple-SPB mutant cut1-cdc11, anti-gamma-tubulin antibodies revealed many dots on the periphery of the nucleus. These results confirm that gamma-tubulin is an important member of the tubulin superfamily, suggest that it may be a universal component of MTOCs, and are consistent with a role for gamma-tubulin in controlling microtubule formation in vivo.

Amino Acid Sequence↗
PubMed1991

Protein phosphatases in cell division: how vital are they?

In contrast to the wealth of information on cellular function of protein kinases, many of which are known to be the products of proto-oncogenes, little is known about how protein dephosphorylation is involved in growth control of normal and malignant cells. In the present study, roles of protein phosphatases in cell division cycle control were examined by molecular genetic approaches using a lower eukaryote, the fission yeast Schizosaccharomyces pombe. Nine protein phosphatase genes have been so far identified and characterized in this organism. Each of two (dis2+, sds21+, and ppa1+, ppa2+) gene products is highly similar to mammalian type 1 and 2A ser/thr phosphatases, respectively. The ppx1+ product is an intermediate of type 1 and 2A, while the ppb1+ product is similar to Ca(2+)-dependent type 2B. At least two protein tyrosine phosphatase genes (pyp1+ and pyp2+) exist. The cdc25 protein is now established to be a tyrosine phosphatase that activates cdc2 kinase. Some of these phosphatase genes are interrelated but have distinct, essential functions in cell cycle control. Missense mutations, deletions or high dosage expression of these phosphatase genes affect entry into and exit from mitosis, mitotic chromosome disjunction, cell size and cell shape. They seem to interact with the main regulators of mitosis, cdc2, cdc13/cyclin, cdc25 and weel, or with mitotic structural components, such as condensed chromosomes or the spindle apparatus. We show that the product of an essential gene, sds22+, is an important, positive factor in controlling the expression and modulating the activity of dis2 phosphatase.

Amino Acid Sequence↗