Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions

Myers, Alan M.; Tzagoloff, Alexander; Kinney, D M; Lusty, Carol J.

doi:10.1016/0378-1119(86)90028-4

Cited by 625 publications

(422 citation statements)

References 15 publications

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“…Copy number and orientation of inserts were determined by DNA sequencing. The complete ADH2 control region (k926\j3) was amplified as a HindIII\ BamHI fragment by PCR and subsequently inserted into lacZ fusion vector YEp356R (Myers et al, 1986) to give pKW13 (ADH2-lacZ URA3 2µ). Point mutations within UAS2 (alteration of 4 nt essential for a functional CSRE) were generated with the QuikChange site-directed mutagenesis kit (Stratagene), using mutagenic primers ADH2-Mut1 (5h-CTCCTCTGCGTCGACACCGGGCATCT-3h ; artificial SalI site underlined) and ADH2-Mut2 (reverse complement).…”

Section: Methodsmentioning

confidence: 99%

Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae

Walther¹,

Schüller²

2001

Microbiology

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Glucose-repressible alcohol dehydrogenase II, encoded by the ADH2 gene of the yeast Saccharomyces cerevisiae, is transcriptionally controlled by the activator Adr1, binding UAS1 of the control region. However, even in an adr1 null mutant, a substantial level of gene derepression can be detected, arguing for the existence of a further mechanism of activation. Here it is shown that the previously identified UAS2 contains a distantly related variant of the carbon source-responsive element (CSRE) initially found upstream of gluconeogenic genes. In a mutant defective for the CSRE-binding factor Cat8, derepression of an ADH2-lacZ fusion was reduced to about 12 % of the wildtype level. Gene expression in a cat8 adr1 double mutant decreased almost to the basal level of the glucose-repressed promoter. CSRE ADH2 present in a single copy turned out to be a weak UAS element, while a significant synergism of gene activation was found in the presence of at least two copies. Its importance for regulated gene activation was confirmed by site-directed mutagenesis of the CSRE in the natural ADH2 control region. Direct binding of Cat8 to CSRE ADH2 could be shown by electrophoretic retardation of the corresponding protein/DNA complex in the presence of a specific antibody. In contrast to what was shown previously for CSRE sequence variants, no significant influence of the isofunctional activator Sip4 on CSRE ADH2 was detected. In conclusion, these results show a derepression of ADH2 by synergistically acting regulators Adr1 (interacting with UAS1) and Cat8, binding to UAS2 (l CSRE ADH2 ).

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Section: Methodsmentioning

confidence: 99%

Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae

Walther¹,

Schüller²

2001

Microbiology

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“…The BamHI/EcoRI promoter fragments were subcloned into YIp357R upstream of the LacZ gene (Myers et al, 1986) and introduction of the desired mutation was confirmed by sequencing (BaseClear, Leiden, The Netherlands). Using the procedure described above, mutations of both STRE and both HAP elements were obtained.…”

Section: Plasmids and Site-directed Mutagenesismentioning

confidence: 99%

HXT5 expression is under control of STRE and HAP elements in the HXT5 promoter

Verwaal

Arako

Kapur

et al. 2004

Yeast

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Hexose transporter (Hxt) proteins transport glucose across the plasma membrane in the yeast Saccharomyces cerevisiae. Recently, we have shown that expression of HXT5 is regulated by the growth rate of the cells. Because gene expression is regulated by binding of specific transcription factors to regulatory elements in the promoters of genes, the presence of putative regulatory elements in the promoter of HXT5 was determined by computer-assisted analysis. This revealed the presence of two putative stress-responsive elements (STREs), one putative post-diauxic shift (PDS) element and two putative Hap2/3/4/5p (HAP) complex binding elements. The involvement of these elements was studied by using mutations in a HXT5 promoter-LacZ fusion construct. Growth during various conditions that result in low growth rates of yeast cells revealed that the STRE most proximal to the translation initiation site seemed to be involved in particular in regulation of HXT5 expression during growth at decreased growth rates. In addition, the HAP elements seemed to be required during growth on non-fermentable carbon sources. The PDS element and, to a lesser extent, the other STRE showed particular involvement in regulation of HXT5 expression during growth on ethanol. Finally, it was shown that the PKA pathway, which is known to be involved in expression of STRE-regulated genes, was also involved in regulation of HXT5 expression. A possible mechanism by which expression of HXT5 could be regulated by the transcriptional regulatory elements in the promoter is discussed.

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“…A fragment containing the SRP1 promoter and initiator methionine was amplified by PCR and cloned into YIp368R (41) to produce an in-frame fusion with LacZ. Next, the region of SRP1 encoding residues 463-542 was amplified and inserted into this plasmid.…”

Section: Yeast Expression Plasmidsmentioning

confidence: 99%

The Yeast Nucleoporin Nup2p Is Involved in Nuclear Export of Importin α/Srp1p

Booth

Belanger

Sannella

et al. 1999

Journal of Biological Chemistry

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The importin ␣⅐␤ heterodimer mediates nuclear import of proteins containing classical nuclear localization signals. After carrying its cargo into the nucleus, the importin dimer dissociates, and Srp1p (the yeast importin ␣ subunit) is recycled to the cytoplasm in a complex with Cse1p and RanGTP. Nup2p is a yeast FXFG nucleoporin that contains a Ran-binding domain. We find that export of Srp1p from the nucleus is impaired in ⌬nup2 mutants. Also, Srp1p fusion proteins accumulate at the nuclear rim in wild-type cells but accumulate in the nuclear interior in ⌬nup2 cells. A deletion of NUP2 shows genetic interactions with mutants in SRP1 and PRP20, which encodes the Ran nucleotide exchange factor. Srp1p binds directly to an Nterminal domain of Nup2p. This region of Nup2p is sufficient to allow accumulation of an Srp1p fusion protein at the nuclear rim, but the C-terminal Ran-binding domain of Nup2p is required for efficient Srp1p export.

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Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions

Cited by 625 publications

References 15 publications

Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae

Adr1 and Cat8 synergistically activate the glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast Saccharomyces cerevisiae

HXT5 expression is under control of STRE and HAP elements in the HXT5 promoter

The Yeast Nucleoporin Nup2p Is Involved in Nuclear Export of Importin α/Srp1p

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