Cloning of the SAT1 gene concerned with salt tolerance of the yeast Zygosaccharomyces rouxii

Ushio, Kohei; Otsuka, Harumi; Yoshikawa, Shigetoshi; Taguchi, Goro; Shimosaka, Makoto; Mitsui, Nobuo; Okazaki, Mitsuo

doi:10.1016/0922-338x(96)89448-7

Cited by 4 publications

(1 citation statement)

References 13 publications

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“…PCR amplification was carried out in a 50 µl reaction mixture containing 0.5 µmol Tris-HCl buffer (pH 8.3), 2.5 µmol KCl, 125 nmol MgCl 2 , 5 nmol each dNTP, 100 pmol each primer, 1 µg chromosomal DNA of Z. rouxii NRRL2547 and 1.25 U Taq DNA polymerase (Promega, USA) at 95 • C for 1 min, 42 • C for 2 min and 65 • C for 3 min for a total of 35 cycles, with chromosomal DNA of Z. rouxii as template. The 0.54 kb PCR product (DAK 540 ) was purified (Qiagen kit) and cloned into pGEM-T-Easy vector for sequencing or labelled with dioxigenin (Dig Labeling Kit, Boehringer-Mannheim) as a DNA probe for screening the genomic library of Z. rouxii NRRL 2547 (Ushio et al, 1996). Both strands of the subclone were sequenced with an automated sequencer (ABI Prism 377, PerkinElmer) using the thermosequenase dye terminator cycle sequencing kit (Amersham).…”

Section: Gene Cloning From Z Rouxiimentioning

confidence: 99%

Cloning, sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547

Wang

Kayingo

Blomberg

et al. 2002

Yeast

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The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1 dak2 strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1 dak2 strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1 are deposited as Accession Nos AJ294739 and AJ294720, respectively.

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Section: Gene Cloning From Z Rouxiimentioning

confidence: 99%