High expression vectors for a Zygosaccharomyces rouxii host.

Imura, Toshiaki; Utatsu, Ikuyo; Toh‐e, Akio

doi:10.1271/bbb1961.53.813

Cited by 2 publications

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“…The two DNA fragments thus amplified were annealed and used as template for the second PCR, in which the forward primer and the reverse primer described above were used. The amplified DNA segments were digested with XbaI and SalI, and then ligated with similarly digested pRS306 (Sikorski and Hieter, 1989) (Casadaban et al, 1980) excised from pAT196 (Imura et al, 1989) by digesting with BamHI and NruI, to generate TOp812. TOp812 was linearized at the unique XbaI site in the promoter region of the CUP1 gene and targeted to the CUP1 locus of W303-1B.…”

Section: Methodsmentioning

confidence: 99%

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae.

Toh‐e

Oguchi

2001

Genes Genet. Syst.

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Las21/Gpi7 contains a heavy-metal-associated motif at its N-terminus. When this motif was disrupted by amino acid substitution, the cells acquired weak copperresistance. We found that the previously isolated las21 mutants were strongly resistant to copper. Metallothionein is necessary for the expression of the copperresistance of the las21 mutants. However, hyper-production of metallothionein is unlikely to be the cause of copper-resistance of the las21 mutants. Copper-sensitive mutants (collectively called Cus mutants) were isolated from the las21∆ and characterized. One of the Cus genes was found to be PBS2, which encodes Hog1 MAP kinase kinase, indicating that the Hog1 MAP kinase pathway is needed for the expression of copper-resistance of the las21 mutants. As expected, the las21∆ hog1∆ strain was no longer copper-resistant. We found that Hog1 was constitutively activated in las21∆ cells and in ssk1∆ las21∆ cells but not in sho1∆ las21∆ cells. Inactivation of either FSR2/MCD4 or MPC1/GPI13, both of which are involved in GPI anchor synthesis, like LAS21, caused a similar level of constitutive activation of Hog1 kinase and copper-resistance as found in the las21∆ strain. The constitutive activation was canceled by introducing the ssk1 mutation, but not the sho1 mutation, in each GPI anchor mutant tested, suggesting that the defect in GPI anchor synthesis specifically affects the Sln1 branch of the MAP kinase pathway. Since the wild-type cells grown in YPD containing 0.5 M NaCl do not show copper-resistance, mere activation of Hog1 is not sufficient for expression of copper-resistance. We propose that a defect in GPI anchor synthesis has multiple consequences, including activation of the Hog1 MAP kinase cascade and conferring copper-resistance.

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Section: Methodsmentioning

confidence: 99%

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae.

Toh‐e

Oguchi

2001

Genes Genet. Syst.

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“…The resulting fragment was inserted into the NotI-BamHI gap of pRS306 to generate TOp670. The lacZ fragment without the nucleotide sequence corresponding to the the Nterminal eight codons (Casadaban, 1983) was excised from pAT196 (Imura et al, 1989) Sensitivity of spores to Zymolyase treatment. W303D (+/+) and YAT2290/YAT2498 (las21∆/las21∆) diploids were smeared on sporulation medium and incubated at 25°C for 3 days.…”

Section: Methodsmentioning

confidence: 99%

Las21 participates in extracellular/cell surface phenomena in Saccharomyces cerevisiae.

Toh‐e

Oguchi

1999

Genes Genet. Syst.

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Las21 (Yjl062W) is a member of the major facilitator super family, possessing multimembrane spanning domains. The LAS21 gene was identified as a responsible gene for a Saccharomyces cerevisiae mutan which shows sensitivity to a local anestheticum, tetracaine. The null las21 mutant (las21∆) is viable but shows temperature sensitive growth. We found, in addition to this phenotype, that the las21∆ strain shows a number of defects; mating deficiency, calcofluor resistance, and formation of Zymolyase sensitive spores. Temperature sensitive growth of the las21∆ mutant was found to be suppressed by 0.1 M MgSO 4 . Two multicopy suppressors were obtained. They are ECM33 (YBR078W) and PIR2/HSP150 (YJR159W) both have some roles in an extracellular function. The common features of the suppressors, genetic and physiological, of the las21∆ mutation suggest that Las21 participates in a global activity of extracellular phenomena. The las 21 phenotypes are consistent with the idea that Las21/Gpi7 acts in metabolism of glycosylphosphatidylinositol.

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High expression vectors for a Zygosaccharomyces rouxii host.

Cited by 2 publications

References 0 publications

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae.

Defects in glycosylphosphatidylinositol (GPI) anchor synthesis activate Hog1 kinase and confer copper-resistance in Saccharomyces cerevisisae.

Las21 participates in extracellular/cell surface phenomena in Saccharomyces cerevisiae.

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