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1.
Construction of a Schizosaccharomyces pombe gene bank in a yeast bacterial shuttle vector and its use to isolate genes by complementation 总被引:57,自引:0,他引:57
Summary A gene bank of partial Sau3A restriction fragments of S. pombe DNA has been constructed in the plasmid vector, pDB248, which is capable of high frequency transformation of S. pombe. Procedures are described which enable plasmids to be recovered from S. pombe by their reintroduction into E. coli. These methods have been used to detect the S. pombe genes lys 1+, ade 6+ and his 2+ in the gene bank by complementation of mutant gene functions, and to physically isolate the lys 1+ gene. 相似文献
2.
Identification of thermostable glyoxalase I in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> 总被引:1,自引:0,他引:1
Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I (glo1+) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1+ enhanced susceptibility to methylglyoxal, while expression of glo1+ in a glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1+ gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34,000 and the kcat/Km value for methylglyoxal was 4.3×107 M–1 min–1. Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn2+, Co2+, Ni2+ and Mn2+. The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60°C for 4 h. 相似文献
3.
Tomohiko Matsuzawa Takao Ohashi Akira Hosomi Naotaka Tanaka Hideki Tohda Kaoru Takegawa 《Applied microbiology and biotechnology》2010,87(2):715-727
The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone
[DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such
as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1
+), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1Δ cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1
+ is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1
+ and dak2
+ encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1Δ strain showed a more severe reduction of growth on glycerol and DHA than the dak2Δ strain because the expression of dak1
+ mRNA was higher than that of dak2
+. In wild-type S. pombe, expression of the gld1
+, dak1
+, and dak2
+ genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1
+ was regulated by glucose repression and that it was derepressed in scr1Δ and tup12Δ strains. 相似文献
4.
J. T. Kemp M. K. Balasubramanian K. L. Gould 《Molecular & general genetics : MGG》1997,254(2):127-138
The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated
a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing
of wat1
+ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1
+ are slow growing but viable at 25° C and temperature-sensitive for growth above 33° C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1
+ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This
analysis suggests that wat1
+ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology.
Received: 18 September 1996 / Accepted: 22 October 1996 相似文献
5.
In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle). The Ura+ Mal+ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura+ Mal +, others Ura+ Mal- and others Ura - Mal+. The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.—Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic break-age of the dicentric chromosome, followed by one of several different healing events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome III partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres. 相似文献
6.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1454-1463
Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four α-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3 + resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3 + deletion, but the ascal wall of aah3Δ cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Δ cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe. 相似文献
7.
Benjamin C KirkupJr LeeAnn Chang Sarah Chang Dirk Gevers Martin F Polz 《BMC microbiology》2010,10(1):137
Background
While most gamma proteobacteria have a single circular chromosome, Vibrionales have two circular chromosomes. Horizontal gene transfer is common among Vibrios, and in light of this genetic mobility, it is an open question to what extent the two chromosomes themselves share a common history since their formation. 相似文献8.
M. Shimanuki F. Miki D.-Q. Ding Y. Chikashige Y. Hiraoka T. Horio O. Niwa 《Molecular & general genetics : MGG》1997,254(3):238-249
In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while
centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB
and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis
showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic
segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution
that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous
chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1
+ gene is involved in SPB function. However, the kms1
+ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity
to known proteins.
Received: 5 September 1996 / Accepted: 21 November 1996 相似文献
9.
10.
Sobel ES Brusko TM Butfiloski EJ Hou W Li S Cuda CM Abid AN Reeves WH Morel L 《Arthritis research & therapy》2011,13(3):R106
Introduction
CD25+ FOXP3+ CD4+ regulatory T cells (Tregs) are induced by transforming growth factor β (TGFβ) and further expanded by retinoic acid (RA). We have previously shown that this process was defective in T cells from lupus-prone mice expressing the novel isoform of the Pbx1 gene, Pbx1-d. This study tested the hypothesis that CD4+ T cells from systemic lupus erythematosus (SLE) patients exhibited similar defects in Treg induction in response to TGFβ and RA, and that PBX1-d expression is associated with this defect. 相似文献11.
The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the
consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop
of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles
and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However,
T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167
E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors
of the dominant negative phenotype were isolated, and identified as cdc13
+
and suc1
+
. Overexpression of suc1
+
suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through
mitosis.
Received: 3 April 1998 / Accepted: 23 May 1998 相似文献
12.
We have constructed circular minichromosomes, ranging in size from 36 to 110 kb, containing the centromeric repeats of Schizosaccharomyces pombe cen3. Comparison of their mitotic stability showed that the circular minichromosomes became more unstable with increasing in size, however, a linear cen3 minichromosome, which is almost the same size as the largest circular one tested, does not show such instability. High levels of expression of the top2
+ (type II DNA topoisomerase; topo II) but not top1
+ gene (type I DNA topoisomerase) suppressed the instability of the largest circular minichromosome, whereas partial inactivation of topo II dramatically destabilized the minichromosome. A mutant topo II, defective in nuclear localization but still retaining its in vitro relaxation activity, did not stabilize the circular minichromosome. These results indicate that endogenous type II DNA topoisomerase is insufficient for accurate segregation of the circular minichromosome. In addition, the replication of the minichromosomal DNA appears to proceed normally, because the presence of the unstable minichromosome did not cause G2 delay. A likely cause of the instability is intertwining of the minichromosome DNA possibly occuring after DNA replication. An interaction between topo II and the centromeric repeats is implied by the finding that multiple copies of the centromeric repeat, dg-dh, affect stability of the minichromosome similarly to top2
+ gene dosage. 相似文献
13.
We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms.
This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a
coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the
medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4
+
disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4
+
diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous
alm1
+
/ alm1::ura4
+
diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the
medial region in S. pombe.
Received: 9 April 1999 / Accepted: 22 October 1999 相似文献
14.
Manuel Arellano Pedro M. Coll Wenli Yang Angel Duran Fuyuhiko Tamanoi & Pilar Perez 《Molecular microbiology》1998,29(6):1357-1367
The Schizosaccharomyces pombe cwg2+ gene encodes the β-subunit of geranylgeranyl transferase I (GGTase I), which participates in the post-translational C-terminal modification of several small GTPases, allowing their targeting to the membrane. Using the two-hybrid system, we have identified the cwp1+ gene that encodes the α-subunit of the GGTase I. cwp1p interaction with cwg2p was mapped to amino acids 1–244 or 137–294 but was not restricted to amino acids 137–244. The genomic cwp1+ was isolated and sequenced. It has two putative open reading frames of 677 and 218 bp, separated by a 51 bp intron. The predicted amino acid sequence shows significant similarity to GGTase I α-subunits from different species. However, complementation of Saccharomyces cerevisiae ram2-1 mutant by overexpressing the cwp1+ gene was not possible. Expression of both cwg2+ and cwp1+ in Escherichia coli allowed ‘in vitro’ reconstitution of the GGTase I activity. S. pombe cells expressing the mutant enzyme containing the cwg2-1 mutation do not grow at 37°C, but the growth defect can be suppressed by the addition of sorbitol. Actin immunostaining of the cwg2-1 mutant strain grown at 37°C showed an abnormal distribution of actin patches. The cwg2-1 mutation was identified as a guanine to adenine substitution at nucleotide 604 of the coding region, originating the change A202T in the cwg2p. Deletion of the cwg2 gene is lethal; Δcwg2 spores can divide two or three times before losing viability. Most cells have aberrant morphology and septation defects. Overexpression of the rho1G15VC199R double-mutant allele in S. pombe caused loss of polarity but was not lethal and did not render the (1–3)β-D -glucan synthase activity independent of GTP. Therefore, geranylgeranylation of rho1p is required for the appropriate function of this GTPase. 相似文献
15.
16.
de Medina-Redondo M Arnáiz-Pita Y Clavaud C Fontaine T del Rey F Latgé JP Vázquez de Aldana CR 《PloS one》2010,5(11):e14046
Background
The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed.Methodology/Principal Findings
Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast.Conclusions/Significance
We conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth. 相似文献17.
18.
The Schizosaccharomyces pombe maltase structural gene (SPMAL1+) was amplified from genomic DNA of S. pombe by PCR. An open reading frame of 1740 bp, encoding a putative 579 amino-acid protein with a calculated molecular mass of 67.7 kDa was characterized in the genomic DNA insert of plasmid pQE30. The specific maltase activity in the induced transformants was 21 times higher than that in wild-type. However, the estimated molecular mass of the purified recombinant maltase was 44.3 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified recombinant maltase were 40 °C and 6, respectively. The recombinant maltase was weakly activated by Mg2+, Ca2+, Na+, and Ba2+, but was strongly inhibited by Hg2+, Ag+ and Cu2+, EDTA, and PMSF. The purified maltase could actively hydrolyse ρ-nitrophenyl glucoside (PNPG), maltose, dextrin, and soluble starch. The results demonstrate that maltase from S. pombe was different from that from other yeasts, and might be usefully exploited in the future by the biotechnology industry or lead to the development of new molecular genetic tools. 相似文献
19.
The methionine salvage pathway (MSP) regenerates methionine from 5′-methylthioadenosine (MTA). Aerobic MSP consists of six enzymatic steps. The mug14+ and adi1+ genes that are involved in the third and fifth steps of the pathway are repressed when Schizosaccharomyces pombe undergoes a transition from high- to low-iron conditions. Results consistently show that methionine auxotrophic cells (met6Δ) require iron for growth in the presence of MTA as the sole source of methionine. Inactivation of the iron-using protein Adi1 leads to defects in the utilization of MTA. In the case of the third step of the pathway, co-expression of two distinct proteins, Mta3 and Mde1, is required. These proteins are interdependent to rescue MTA-dependent growth deficit of met6Δ cells. Coimmunoprecipitation experiments showed that Mta3 is a binding partner of Mde1. Meiotic met6Δ cells co-expressing mta3+ and mde1+ or mta3+ and mug14+ produce comparable levels of spores in the presence of MTA, revealing that Mde1 and Mug14 share a common function when co-expressed with Mta3 in sporulating cells. In sum, our findings unveil several novel features of MSP, especially with respect to its regulation by iron and the discovery of a non-canonical third enzymatic step in the fission yeast. 相似文献
20.
Hiromi Maekawa Tomoko Nakagawa Yoko Uno Kenji Kitamura Chikashi Shimoda 《Molecular & general genetics : MGG》1994,244(5):456-464
When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13
+ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13
+ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13
+ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13
+ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development. 相似文献