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ABSTRACT: Two versatile shuttle vectors for Thermus thermophilus and Escherichia coli were developed on the basis of the T. thermophilus cryptic plasmid pTT8 and E. coli vector pUC13. These shuttle vectors, pTRK1T and pTRH1T, carry a gene encoding a protein homologous to replication protein derived from pTT8, a replicon for E. coli, new multiple cloning sites and a lacZα gene from E. coli vector pUC13, and also have a gene encoding a thermostable protein that confers resistance to kanamycin or hygromycin, which can be used as a selection marker in T. thermophilus. These shuttle vectors are useful to develop enzymes and proteins of biotechnological interest. We also constructed a plasmid, pUC13T, which carries the same multiple cloning sites of pTRK1T and pTRH1T. These vectors should facilitate cloning procedures both in E. coli and T. thermophilus.
Plasmid 01/2012; 67(3):272-5. · 1.52 Impact Factor
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ABSTRACT: In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). We previously found that Arf6, a member (class III) of the ADP-ribosylation factor GTPase (Arf) family, is necessary for NETO in fission yeast. Here we report the characterization of a S. pombe gene, ucp3, encoding a putative Arf GTPase-activating protein (GAP) for Arf6. The Ucp3 contains Arf GAP domain, and has a high similarity to Gts1, which was identified as a GAP for Arf3 (class III Arf) in Saccharomyces cerevisiae. Overexpression of ucp3 inhibited growth from new end possibly by disturbing the GDP/GTP-cycling of Arf6. Gene disruption of ucp3 revealed that Ucp3 is essential for cell viability. Ucp3 uniformly localizes to the cell periphery. And its localization is not dependent on microtubules, actin cytoskeletons, Arf6 and Syt22 (guanine nucleotide exchange factor for Arf6). We hypothesize that Ucp3 functions as a GAP for Arf6. Moreover, Ucp3 might have another function important for cell viability.
Molecular Biology Reports 11/2010; 38(6):3875-82. · 2.93 Impact Factor
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ABSTRACT: In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from monopolar to bipolar in character, which is known as 'new end take off ' (NETO). We previously found that arf6p, a member (class III) of the ADP-ribosylation factor (Arf) family, is necessary for NETO in fission yeast. Here we report the characterization of an S. pombe gene, syt22(+), encoding a putative Arf guanine nucleotide exchange factor (GEF). The syt22 protein contains a Sec7 domain and a PH domain conserved in the mammalian EFA6 GEF family, and has high similarity to Yel1p, which was identified as a GEF for Arf3p (class III Arf) in Saccharomyces cerevisiae. syt22Delta cells, like arf6Delta cells, completely failed to undergo NETO. Syt22p uniformly localizes to the cell periphery. Its localization is not dependent on microtubules, actin cytoskeletons or arf6p. We hypothesize that syt22p functions as a GEF for arf6p.
FEMS Microbiology Letters 06/2009; 294(2):191-7. · 2.04 Impact Factor
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Atsushi Fujita
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ABSTRACT: Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6(+), encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6Delta cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.
Biochemical and Biophysical Research Communications 03/2008; 366(1):193-8. · 2.48 Impact Factor
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ABSTRACT: In response to nitrogen limitation, diploid yeast strains of Saccharomyces cerevisiae undergo a dimorphic transition to a filamentous growth form known as pseudohyphal growth. This developmental change can be classified into two distinct growing forms: invasive pseudohyphal growth and superficial pseudohyphal growth. We identified a yeast gene, SFG1, whose overexpression predominantly enhances superficial pseudohyphal growth when starved for nitrogen. Sfg1 has a sequence similarity to members of a family of transcriptional regulators of fungal development. Cells of a homozygous sfg1/sfg1 diploid strain have a serious defect in pseudohyphal growth, indicating that Sfg1 has an essential function for pseudohyphal development. Our analyses show that Sfg1 may act separately from mitogen-activated protein kinase (MAPK) pathway and cAMP-dependent protein kinase A (PKA) pathway.
Gene 01/2006; 363:97-104. · 2.34 Impact Factor
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ABSTRACT: In Saccharomyces cerevisiae, cell type determines two distinct spatial budding patterns. Haploid cells exhibit an axial pattern, whereas diploid cells exhibit a bipolar pattern. Axl1, a member of the insulin-degrading enzyme (IDE) family, is the key morphological determinant for the haploid axial pattern. Here we identified a novel gene, RAX1, specifically required for the bipolar budding pattern. Loss of RAX1 alters the bipolar pattern of axl1 haploids resulting in reversion to the axial pattern, and also alters the bipolar patterns of bud3 and bud4 haploids. However, bud10 rax1 haploids exhibit a random budding pattern, suggesting Bud10 acts as the key proximal landmark in axial budding. Rax1 is required for the localization of Bud8, the distal bipolar budding landmark. Interestingly, Rax1 contains a C-terminal domain possessing some similarity to insulin-related peptides. Our results suggest that Rax1 is necessary for the establishment of the bipolar budding landmark.
Gene 04/2004; 327(2):161-9. · 2.34 Impact Factor
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ABSTRACT: Bud-site selection in yeast offers an attractive system for studying cell polarity and asymmetric division. Haploids divide in an axial pattern, whereas diploids divide in a bipolar pattern. AXL1 is expressed in haploids but not diploids, and ectopic expression of AXL1 in diploids converts their bipolar budding pattern to an axial pattern. How Axl1 acts as a switch between the bipolar and axial patterns is not understood. Here we report that Axl1 localizes to the mother-bud neck and division site remnants of haploids. Axl1 is absent from diploids. Axl1 colocalizes with Bud3, Bud4, and Bud10, components of the axial landmark structure. This localization suggests that Axl1 couples the axial landmark with downstream polarity establishment factors. Consistent with such a role, Axl1 associated biochemically with Bud4 and Bud5. Genetic evidence suggests that Axl1 works with Bud3 and Bud4 to promote the activity of the Bud10 membrane protein. Given Axl1's suggested role in morphogenesis and cell fusion during mating, we also examined its localization during this process. Axl1 redistributes independently of the axial landmark to a tight cell surface dot at the tip of each mating projection. These dots are rapidly lost as prezygotes form.
Current Biology 09/2002; 12(15):1347-52. · 9.65 Impact Factor
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Methods in Enzymology 02/2002; 350:131-41. · 2.04 Impact Factor
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ABSTRACT: Diploid yeast cells repeatedly polarize and bud from their poles, probably because of highly stable marks of unknown composition.
Here, Rax2, a membrane protein, was shown to behave as such a mark. The Rax2 protein itself was inherited immutably at the
cell cortex for multiple generations, and Rax2 was shown to have a half-life exceeding several generations. The persistent
inheritance of cortical protein markers would provide a means to couple a cell's history to the future development of a precise
morphogenetic form.
Science 12/2000; 290(5498):1975-1978. · 31.20 Impact Factor
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ABSTRACT: We identified a yeast gene encoding Flocculent Specific Protein (FSP) produced excessively in the SFL1 gene-disrupted flocculent strain. The sequenced gene encodes a 430 amino acid protein and is mainly composed of multiple repeats of Ser-Asn-Asn-X-Asp-Ser-Tyr-Gly. The FSP gene disruption of the flocculent strain decreased the degree of flocculation, so FSP may be one factor concerned with yeast flocculation. A gene database search indicated that the FSP gene is identical with the DDR48 gene.
