Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.

Thill, G P; Kramer, Richard A.; Turner, Katie J.; Bostian, Keith A.

doi:10.1128/mcb.3.4.570

Cited by 48 publications

(26 citation statements)

References 12 publications

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“…A pyrimidine-rich sequence, CTTTATTCTTC, was found between -162 and -152, implicated to be the mRNA capping site (55). The sequence CAAG, located at position -71, is consistent with the sequence reported to be involved in transcription initiation (10).…”

Section: Resultssupporting

confidence: 66%

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

et al. 1991

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Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S.cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thius, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.Saccharomyces cerevisiae, unlike higher eukaryotes, can metabolize as well as produce ethanol. During fermentation, the consumption of sugars results in the accumulation of ethanol. When glucose or any other fermentable sugar is absent from the culture medium, S. cerevisiae can utilize ethanol as the carbon source aerobically. The metabolism of ethanol is well understood in mammals. In the liver, cytosolic alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde and then mitochondrial aldehyde dehydrogenase (ALDH) oxidizes the intermediate to acetate (14). In S. cerevisiae, it appears that a mitochondrial alcohol dehydrogenase (ADH3) is responsible for the initial oxidation (65,67). Little is known about the role of ALDHs in the next step.Various groups have purified and characterized yeast ALDH (12,49,54). It was reported to be a tetrameric enzyme with a subunit molecular mass of 60 kDa and a low Km for acetaldehyde. Unlike the liver enzymes, the yeast enzyme is activated by K+ ions (4, 32). Whereas the mammalian enzymes have been sequenced at the cDNA (16,19,29,34) as well as the protein (24, 33, 60) level, no sequence work has been reported for the yeast enzyme.One of our interests was to study the subcellular localization of acetaldehyde metabolism in S. cerevisiae. In the mammalian liver tissue, this was studied by selectively inhibiting the cytosolic or the mitochondrial isozyme of ALDH (11,52). An alternative approach would be to introduce the enzyme into a cell deficient in ALDH activity. S. cerevisiae would be a suitable model system, as it can metabolize ethanol and could serve as a host for the expression of foreign g...

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Section: Resultssupporting

confidence: 66%

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

et al. 1991

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“…Finally, it is interesting to speculate whether a relationship exists between the topological structure (and the sequences which mediate this effect) and the precise positioning of the nucleosomes along the PH05 sequence. Futhermore, these results suggest that the binding site for the PH05 activator complex (19,21) may be inaccessible under repressed conditions due to the increased chromatin condensation and becomes available for binding on transfer to conditions of derepression only after a relaxation of the chromatin structure within this region.…”

Section: Discussionmentioning

confidence: 92%

“…Finally, to further confirm that this process which causes a change in plasmid linking number is associated with the mechanism of regulation of PHO5 and not transcription itself, we examined the topological state of a plasmid under high and low Pi-containing growth conditions containing a 353-bp Sau3A fragment which spans a region from -167 to -520 bp upstream from the 5' end of the mRNA (19) (Fig. 1).…”

Section: Oed1mentioning

confidence: 99%

Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

Bergman¹,

Stranathan²,

Preis³

1986

Mol. Cell. Biol.

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We developed a high-copy-number plasmid system containing the entire structural and regulatory sequences of the phosphate-repressible acid phosphatase (PHO5) gene and the TRP1/ARS1 replicator sequences of the yeast Saccharomyces cerevisiae to investigate the mechanism of repression-derepression of transcription. The resulting plasmid was used to transform either wild-type cells or a number of strains which contain mutations in various trans-acting regulatory loci for the production of acid phosphatase. Results of analysis of mRNA levels isolated from the transformed strains grown under repressed or derepressed conditions suggested that normal transcriptional regulation of the gene persisted, although gene copy number was significantly increased. Analysis of changes in linking number (i.e., the number of negative supercoils) of the plasmid isolated under repressed and derepressed growth conditions revealed that the transcriptionally inactive plasmid contained approximately three more negative supercoils than the transcriptionally active plasmid. This difference in topological state was similarly seen in a plasmid containing a sequence-related acid phosphatase gene (PHO11) under the same regulatory control system, but it was not seen in plasmids isolated from some strains containing mutations which caused either fully constitutive or nonderepressible production of acid phosphatase. Finally, analysis of the nucleosome positioning along the inactive gene sequence revealed that an abnormally broad internucleosomal spacer is present in a region presumed to function in the regulation of transcription by the level of Pi in the growth media.

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“…Oligonucleotides were designed as shown in Figure 1 based on the known coding regions for the yeast invertase signal peptide (5) and the yeast acid phosphatase signal peptide (33). The two sets of complementary oligonucleotides in TE buffer (10 mM Tris-C1 pH 7.8, 1 mM EDTA) were heated to 95 ~ and slowly cooled down to room temperature and ligated (using T4 DNA ligase) onto the ApaI site of the gel-purified large fragment of BamHI and ApaI cut vector pAG ( Fig.…”

Section: Oligonucleotidesmentioning

confidence: 99%

Processing and secretion of barley (1–3, 1–4)-β-glucanase in yeast

Olsen

Thomsen

1989

Carlsberg Res. Commun.

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Comparative analysis of the 5'-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.

Cited by 48 publications

References 12 publications

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation

Structure of the transcriptionally repressed phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae.

Processing and secretion of barley (1–3, 1–4)-β-glucanase in yeast

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