Yeast cyclophilin-related gene encodes a nonessential second peptidyl-prolyl cis-trans isomerase associated with the secretory pathway.
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Amino Acid Isomerases↗
Biomedical subjects
M Yanagida
Publications and source records attributed to M Yanagida.
[Control and nutritional treatment of diabetics with renal failure].
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Aged↗
Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle.
The activities of type 1 protein phosphatase (PP1) and 2A (PP2A) have distinct, essential roles in cell cycle control. Two previously identified PP1 genes (dis2+ and sds21+) and two PP2A genes (ppa1+ and ppa2+), highly homologous to mammalian PP2A, have been isolated from fission yeast. Only double gene disruption of both PP2A genes results in lethality, as is the case for PP1 genes. By fractionating and assaying PPases in wild-type, various deletion, and point mutant strains, the decrease of PP1 or PP2A activity is shown to cause mitotic defects, exhibiting strikingly different cell cycle phenotypes: cold-sensitive mutations in the same amino acid lesion of PP1 and PP2A produce chromosome nondisjunction and premature mitosis, respectively. Consistently, PP1 and PP2A genes cannot be functionally substituted. Although the overall levels of PP1 and PP2A activities do not fluctuate during the cell cycle, subpopulations might be regulated.
Amino Acid Sequence↗
Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene.
The structure equivalent to higher eukaryotic centrosomes in fission yeast, the nuclear membrane-bound spindle pole body, is inactive during interphase. On transition from G2 to M phase of the cell cycle, the spindle pole body duplicates; the daughter pole bodies seed microtubules which interdigitate to form a short spindle that elongates to span the nucleus at metaphase. We have identified two loci which, when mutated, block spindle formation. The predicted product of one of these genes, cut7+, contains an amino-terminal domain similar to the kinesin heavy chain head domain, indicating that the cut7+ product could be a spindle motor. The cut7+ gene resembles the Aspergillus nidulans putative spindle motor gene bimC, both in terms of its organization with a homologous amino-terminal head and no obvious heptad repeats and in the morphology of the mutant phenotype. But we find no similarity between the carboxy termini of these genes, suggested that either the cut7+ gene represents a new class of kinesin genes and that fission yeast may in addition contain a bimC homologue, or that the carboxy termini of these mitotic kinesins are not evolutionarily conserved and that the cut7+ gene belongs to a subgroup of bimC-related kinesins.
Adenosine Triphosphatases↗
The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene.
Mutations in the fission yeast cut1+, cut2+, and cut10+ genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis. DNA synthesis continues, making polyploid nuclei with several spindles. Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells. The cut1+ and cut2+ genes are cloned by transformation. High gene dosage of cut1+ also complements cut2 and cut10 mutants. The cut2+ gene, however, complements only cut2. The 210 kd cut1+ gene product contains putative ATP binding and helical coil regions followed by a COOH-terminal domain homologous to the S. cerevisiae gene ESP1. Mutations in the ESP1 gene also result in many SPBs. The cut1+ product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction. It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.
Amino Acid Sequence↗
Dibutyrylcytidine 3',5'-cyclic monophosphate stimulates neurite outgrowth in rat pheochromocytoma PC12.
Dibutyrylcytidine 3',5'-cyclic monophosphate (Bt2cCMP) stimulated neurite outgrowth in rat pheochromocytoma PC12 cells in a dose-dependent manner at the dose range from 0.5 mM to 6 mM. About 25% of the cells had neurites in response to 6 mM Bt2cCMP. In contrast with the effect of nerve growth factor (NGF) which increased the percentage of cells with neurites gradually during a 6-day culture period, the stimulating effect of Bt2cCMP reached a plateau 2 days after plating. Staurosporine inhibited the neurite outgrowth induced by NGF, but not that by Bt2cCMP. These results suggest that Bt2cCMP stimulates neuronal differentiation by a mechanism different from that by NGF.
Adrenal Gland Neoplasms↗
Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+.
The S. pombe nuc2+ gene is required for mitotic chromosome disjunction. Its mutation arrests mitosis at the metaphase. The gene product is present in the nuclear scaffold-like fraction. The nuc2+ protein contains a domain, separated from ten 34 amino acid repeat segments, that is capable of binding AT-rich DNA in vitro. The ts mutation resides in one of the 34 amino acid repeats. Circular dichroism, limited proteolysis of the repeats, and model fitting indicate the presence of helical segments connected by protease-sensitive hinges. We propose that these repeats form a novel secondary structure (snap helix) having "knob and hole" helix-associating motifs. The packing of the snap helices would be stabilized by the bonding between the hydrophobic amino acids surrounding the knobs and holes. The nuc2+ protein may in one way bind to DNA and in another way mutually associate to form a part of the chromosome scaffold.
Amino Acid Sequence↗
In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells.
To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.
Adenylyl Imidodiphosphate↗
Higher-order chromosome structure in yeast.
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Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
The transition of cells of the fission yeast beta-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy. Requirement of functional tubulin for spindle pole body duplication.
A previous fluorescence light-microscopic study showed that the fission yeast cold-sensitive beta-tubulin mutant nda3-311 was arrested with rod-like condensed chromosomes in a mitotic state at the restrictive temperature. Upon transfer to the permissive temperature, a spindle was formed and the nucleus was divided. In the present study, we employed freeze-substitution electron microscopy to examine the ultrastructure of arrested and released nda3-311 cells. In arrested cells, a single, displaced nucleus was seen with a single spindle pole body. Therefore, spindle pole body duplication seemed to require functional beta-tubulin. The nuclear membrane was highly deformed with a leaf-like profile in cross-section, possibly due to an interaction with the rod-like, condensed chromosomes. Upon transfer to the permissive temperature, the spindle pole duplicated and the daughter spindle pole bodies rapidly migrated to the opposite ends of the nucleus, accompanied by the formation of the mitotic spindle. Elongation of the nuclear envelope occurred with concomitant spindle extension, as in a wild-type mitosis. The deformed nuclear membrane became smooth and described a convex curve. The numerous vacuoles that are seen in the arrested cells decreased in number and increased in size. Septation was completed, leaving the two divided nuclei in one half of the cell. Hexagonally arranged microtubules, apparently forming the mitotic spindle, were observed in a cross-section of a cell after return to the permissive conditions.
Freezing↗
[The balance of occlusal contacts during intercuspation using T-scan system].
Many methods have been clinically applied for the occlusal analysis in the intercuspal position. However, none of these methods are quantitative. This study described the new software version of the T-Scan system to record and analyze occlusal contact balance in the anterior-posterior and right-left directions. Six time moment statistics and five force moment statistics were calculated in the midsagittal and the incisal axes of the occlusal plane. In the present study, informed consent was obtained from 60 subjects with natural dentitions and from two patients with craniomandibular disorders. In normal subjects, the time moments and the force moments of occlusal contacts were symmetrical about the midsagittal axis of occlusal plane. The location of the center of effort for the antero-posterior occlusal contacts, which was measured from the incisal axis of occlusal plane, was also in the first molar region. However, any of the above mentioned statistics was abnormal in the patients with craniomandibular disorders.
Dental Occlusion↗
Characterization of detergent dispersed cytidylate cyclase of rat brain.
Cytidylate cyclase was demonstrated to be distributed in various tissues of rat, with the highest activity in brain, and it was shown to be a membrane-bound type enzyme. The enzyme was effectively dispersed from the membrane fraction of rat brain with 0.3% (w/v) Triton X-100. The dispersed cytidylate cyclase had an optimal pH of 9.4 and the activity at a physiological pH of 7.5 was less than 20% of the maximum value. This enzyme completely lost its activity in the absence of divalent cation such as Mn2+ and Mg2+. The Km value for CTP was calculated as 0.0156 mM, by Lineweaver-Burk analysis. It was also found that activity of dispersed enzyme was inhibited by ATP, but not GTP. Both forskolin and lanthanum chloride, which affect adenylate cyclase, showed no effect on cytidylate cyclase. These results indicate that cytidylate cyclase is a unique membrane-bound enzyme distinct from purine nucleotide cyclases, adenylate cyclase and guanylate cyclase.
Adenosine Triphosphate↗
The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases.
S. pombe dis mutants block mitotic chromosome disjunction in a manner reminiscent of aneuploidy formation, and belong to three distinct genes, dis1-dis3. We cloned two independent genomic DNAs that complemented both the cold-sensitive and caffeine-hypersensitive phenotype of dis2-11. These genes, dis2+ and a suppressor sds21+, encode proteins (calculated MW 37,000) with similar predicted amino acid sequences. dis2+ and sds21+ have overlapping functions, and disruptants are lethal only when both genes are disrupted. The gene products identified by anti-dis2 serum are enriched in nuclei. By hybridization, we obtained two cDNA clones from mouse and one genomic clone from S. cerevisiae; the latter complements S. pombe dis2-11. These dis2+ and similar polypeptides of yeasts and mouse are found to be highly homologous (75%-90% identical) to rabbit protein phosphatase 1. The implications of these findings are discussed with regard to mitotic control.
Alleles↗
Composite motifs and repeat symmetry in S. pombe centromeres: direct analysis by integration of NotI restriction sites.
S. pombe centromeres are large and complex. We introduced a method that enables us to characterize directly centromere DNAs. Genomic DNA fragments containing cen1, cen2, or cen3, respectively, are made by cleaving NotI sites integrated on target sites and are partially restricted for long-range mapping in PFG electrophoresis. The 40 kb long cen1 consists of two inverted approximately 10 kb motifs, each containing centromeric elements dg and dh, flanked by a central region. In cen2, three motifs are arranged in inverted and direct orientations with flanking domains, making up the approximately 70 kb long repetitious region. In cen3, approximately 15 copies of dg-dh constitute a region longer than 100 kb. A set of inverted motifs with an approximately 15 kb central region might be a prototype for the S. pombe centromeres. The motifs appear to play a role in chromosome stability and segregation. Their action may be additive, and the mutual directions of dg and dh inside a motif may not be essential for function.
Blotting, Southern↗
Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome.
Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe. There are 14 detectable Not I sites in S. pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III. The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis. These fragments ranged in size from 4.5 kb to approximately 3.5 Mb. Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes. The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb. These are generally somewhat smaller than estimated previously.
Blotting, Southern↗
Characterization of Schizosaccharomyces pombe minichromosome deletion derivatives and a functional allocation of their centromere.
A 530 kb long Schizosaccharomyces pombe linear minichromosome, Ch16, containing a centric region of chromosome III, has previously been made. In the present study, we constructed a number of deletions in the right and/or left arms of Ch16, and compared their structure and behaviour with Ch16. The functional centromere, cen3, is allocated within a 120 kb long region which is covered by the shortest derivative, Ch10, and is comprised mostly of centromeric repeating sequences. The shortest minichromosome is stable in mitosis and the copy number control is apparently precise. In monosomic meiosis it segregates normally. In disomic meioses, however, the frequency of non-disjunction is very high, suggesting that it may not form a pair. The mitotic loss rate of one of the left-arm deletions, ChR32, which lacks a part of the centromeric repeating sequence, is the highest of all the deletions. This deletion also exhibits the highest precocious sister chromatid separation in meiosis I, suggesting that sister chromatid association might become weakened in ChR32. Our results indicate that the proper meiotic segregation of S.pombe minichromosomes is dependent upon the formation of a bivalent. S.pombe may not have the 'distributive segregation' found with Saccharomyces cerevisiae minichromosomes.
Blotting, Southern↗
Inhibition of DNA synthesis of adult rat hepatocytes in primary culture by dibutyrylcytidine 3', 5'-cyclic monophosphate.
The effect of dibutyrylcytidine 3',5'-cyclic monophosphate (Bt2cCMP) on DNA synthesis of adult rat hepatocytes in primary culture was examined. Bt2cCMP caused dose-dependent inhibition of the DNA syntheses stimulated by various growth factors including human hepatocyte growth factor (hHGF). Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) inhibited the DNA synthesis more effectively than Bt2cCMP, but dibutyrylguanosine 3',5'-cyclic monophosphate (Bt2cGMP) and n-butyrate had a slight or null inhibitory effect. When added at the onset of DNA synthesis, Bt2cAMP was much less effective, but Bt2cCMP was still effective. Thus Bt2cCMP is able to inhibit growth factor-stimulated hepatocyte proliferation.
1-Methyl-3-isobutylxanthine↗
Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the formation of fission yeast nucleolus.
A temperature-sensitive lethal mutant nuc1-632 of Schizosaccharomyces pombe shows marked reduction in macromolecular synthesis and a defective nuclear phenotype with an aberrant nucleolus, indicating a structural role of the nuc1+ gene product in nucleolar organization. We cloned the nuc1+ gene by transformation and found that it appears to encode the largest subunit of RNA polymerase I. We raised antisera against nuc1+ fusion polypeptides and detected a polypeptide (approximately 190 kD and 2 x 10(4) copies/cell) in the S. pombe nuclear fraction. By immunofluorescence microscopy, anti-nuc1+ antibody revealed intense staining at a particular nuclear domain previously defined as the nucleolus. The nucleolar immunofluorescence by anti-nuc1+ was faded in nuc1-632 at restrictive temperature and dramatically diminished in the absence of DNA topoisomerases I and II. Thus active RNA polymerase I appears to be required for the formation of the nucleolus as its major component, and DNA topoisomerases appear to be required for the folding of rDNA and RNA polymerase I molecules into the functional organization of nucleolar genes.
Amino Acid Sequence↗