Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure

Gietz, R. Daniel; Schiestl, Robert H.; Willems, Andreas; Woods, Robin A.

doi:10.1002/yea.320110408

Cited by 1,947 publications

(1,427 citation statements)

References 32 publications

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“…Standard methods were used for genetic analysis (Guthrie and Fink, 1991), cloning (Sambrook et al, 1989;Ausubel et al, 1995) and transformation (Gietz et al, 1995).…”

Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

YAP4 gene expression is induced in response to several forms of stress in Saccharomyces cerevisiae

Nevitt

Rodrigues-Pousada

2004

Yeast

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Exposure of Saccharomyces cerevisiae to several environmental insults, including conditions of oxidative, heavy metal, metalloid and heat stress, induces the expression of the YAP4 gene, previously shown to play a role in the response to hyperosmotic stress. Expression analyses in several mutant strains under pro-oxidant conditions have determined that YAP4 is regulated by the transactivators Yap1p and Msn2p. Mutation of either the Yap1p-response element (YRE), located at −517 bp from the ATG, or the most proximal stress response element (STRE) at −430 bp, is shown to strongly compromise YAP4 gene expression under these conditions. Furthermore, these two mutations in combination lead to a severe depletion of detectable mRNA levels, indicating interplay between the transcription factors Yap1p and Msn2p in the regulation of YAP4 transcription. Transcriptional activation of this gene reflects a concomitant increase in Yap4p protein levels that appear phosphorylated upon stress and negatively regulated by protein kinase A. Yap4p amino acid residues Ser89, Ser196 and Thr241 are shown to be required for protein phosphorylation and/or protein stability.

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“…Standard methods were used for genetic analysis (Guthrie and Fink, 1991), cloning (Sambrook et al, 1989;Ausubel et al, 1995) and transformation (Gietz et al, 1995).…”

Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

YAP4 gene expression is induced in response to several forms of stress in Saccharomyces cerevisiae

Nevitt

Rodrigues-Pousada

2004

Yeast

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“…Yeast strains used in this study are listed in Table 1 and are all derived from the W303-1A or W303-1B background (Wallis et al, 1989) by transformation with the lithium acetate protocol according to Gietz et al (1995). Strains SKY697 and AG86 have been obtained by disrupting the ENA1,2,3,4 open reading frames (ORFs) and have been described elsewhere (Goossens et al, 2000).…”

Section: Plasmids and Strainsmentioning

confidence: 99%

Involvement of Nst1p/YNL091w and Msl1p, a U2B″ splicing factor, in Saccharomyces cerevisiae salt tolerance

Goossens¹,

Forment²,

Serrano³

2002

Yeast

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Cell tolerance to salt stress depends on many physiological functions, including the best characterized of osmotic adjustment, ion transport and sodium-sensitive sulphate metabolism. From a screening designed to identify novel determinants of salt tolerance we have isolated the YNL091w gene, probably an Ascomycete-specific gene encoding a protein of unknown function. This gene negatively affects salt tolerance and therefore has been designated NST1. The salt tolerance mechanism of nst1 mutants is novel because it is not related to osmoregulation, altered cation accumulation or sulphate metabolism. Genome-wide two-hybrid analysis has suggested that Nst1p interacts with the splicing factor Msl1p and, accordingly, the impact of NST1 on salt tolerance is dependent on a functional MSL1 gene. Loss of MSL1 and NST1 function has pleiotropic phenotypes including increased sensitivity to divalent cations (manganese and zinc) and to caffeine (a cell wall-weakening agent). On the other hand, msl1 mutants but not nst1 mutants are sensitive to thiabendazole (a microtubule-destabilizing agent) and to osmotic stress.

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“…All yeast cell transformations were carried out according to the lithium acetate method [17]. Briefly, an overnight yeast SFY526 cell culture (20 ml) in YPD medium (1% w/v yeast extract, 2% w/v peptone, and 2% w/v dextrose, pH 5.8) was introduced into 300 ml of YPD medium.…”

Section: Transformation Of Yeastmentioning

confidence: 99%

Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization

et al. 2001

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We have developed a laboratory method combining a random mutagenesis method and a yeast two-hybrid system to study effects of mutation on human erythrocyte spectrin tetramerization. A PCR-based procedure was used to generate random mutations in DNA fragments of the first 55 residues of ␣-spectrin. Each of the DNA fragments from random mutagenesis was fused with a DNA fragment of native spectrin consisting of residues 56 to 368 to give a DNA fragment of the first 368 residues in ␣-spectrin. The ␣-spectrin DNA fragment and a DNA fragment containing the last 449 residues in ␤-spectrin were introduced into the yeast two-hybrid system for rapid screening of ␣-and ␤-spectrin interaction. Yeast colonies with interacting ␣-and ␤-peptides were blue, and those with noninteracting ␣-and ␤-peptides were white. Six single amino acid mutations (R27G, Y35N, F38S, L49H, Y53N, and Y53C) and a double amino acid mutation (K16M, I24N) were identified from 8 white colonies, but no mutations were found in the DNA fragments of 14 blue colonies. Thus this simple laboratory method allows us to study effects of mutation on interactions of ␣-and ␤-spectrin at the tetramerization site. Am. J. Hematol. 67:247-251, 2001.

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Studies on the transformation of intact yeast cells by the LiAc/SS‐DNA/PEG procedure

Cited by 1,947 publications

References 32 publications

YAP4 gene expression is induced in response to several forms of stress in Saccharomyces cerevisiae

YAP4 gene expression is induced in response to several forms of stress in Saccharomyces cerevisiae

Involvement of Nst1p/YNL091w and Msl1p, a U2B″ splicing factor, in Saccharomyces cerevisiae salt tolerance

Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization

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