TDH2 is linked to MET3 on chromosome X of Saccharomyces cerevisiaeMountain, Harry A.; Korch, Christopher
doi: 10.1002/yea.320070814pmid: 1789010
The MET3 gene of Saccharomyces cerevisiae was cloned and its restriction map was found to differ in the upstream region from an earlier published map (Cherest et al. Gene 34, 269–281, 1985) and nucleotide sequence (Cherest et al. Mol. Gen. Genet. 210, 307–313, 1987). Southern blot analysis of genomic DNA from strains S288C and FL100 (the genetic backgrounds from which these different copies of the gene had been cloned) showed that our clone from a S288C‐based library had the same restriction map as the chromosomal DNA from both of the strains. Comarison of the nucleotide sequence of the two clones indicated that the earlier published clone probably represented a cloning artifact. In our clone, we found upstream of MET3, the nucleotide sequence of the TDH2 gene (Holland and Holland, J. Biol. Chem. 255, 2596–2605, 1980). The chromosomal orientation of the two genes was determined to be MET3–TDH2–CEN10.
Electroporation‐stimulated recombination in yeastHiggins, David R.; Strathern, Jeffrey N.
doi: 10.1002/yea.320070807pmid: 1789003
Saccharomyces cerevisiae cells treated by high voltage and made transformation‐competent (electroporation) are also made hyper‐recombinational as determined by an assay that measures interchromosomal mitotic recombination between chromosome III homologs, each containing mutant heteroallelic copies of the trp1 and his3 genes. There is a 10‐fold stimulation of Trp+ and 21‐fold stimulation of His+ prototrophs. Although this stimulation coincides with conditions for maximal transformation competence it is independent of the presence of transforming plasmid DNA. Electroporation does not increase the reversion frequency of these mutations, nor is there a stimulation in Ty transposition. Among the electroporation‐stimulated Trp+ and His+ recombinants there is no dramatic difference in the pattern of events: that is to say that, while there is an increase in the number of recombinants, the distribution of gene conversion and cross–over events among the stimulated recombinants is not significantly altered compared to spontaneously arising Trp+ and His+ recombinants. This electroporation‐stimulated recombination is abolished in an isogenic rad52 mutant strain consistent with the increase in Trp+ and His+ prototrophs being the result of a stimulation of a RAD52‐dependent recombination pathway.
Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiaeMountain, Harry A.; Byström, Anders S.; Larsen, Jörgen Tang; Korch, Christopher
doi: 10.1002/yea.320070804pmid: 1789001
Genes encoding enzymes in the threonin/methionine biosynthetic pathwa were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific‐activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.
Transport of lactic acid in Kluyveromyces marxianus: Evidence for a monocarboxylate uniportFonseca, A.; Spencer‐Martins, I.; Van Uden, N.
doi: 10.1002/yea.320070803pmid: 1789000
Lactic acid‐grown cells of a strain of Kluyveromyces marxianus transported D‐and L‐lactic acid by a saturable mechanism that was partially inducible and subject to glucose repression, with the following kinetic parameters at pH 5·4: Vmax = 1·00 (±0·13) mmol h−1 per g dry weight and Ks = 0·42 (±0·08) mM. Lactic acid transport was competitively inhibited by pyruvic, glycolic, acetic and bromoacetic acids. The latter, a non‐metabolizable analogue, was transiently accumulated, the extent depending on the extracellular pH. The pH dependence of the Ks values for undissociated lactic acid and for the lactate anion indicated that the latter was the transported species. Lactate uptake was not accompanied by the simulatate uptake of protons, potassium ions or sodium ions excluding symport mechanisms. Initial lactic acid uptake led to transient membrane hyperpolarization as measured with a fluorescent dye excluding also an electroneutral anion antiport mechanism. It was concluded that lactate anions use a monocarboxylate uniport and that the counter anion, possibly bicarbonate, uses a separate channel, the coupling being electrical and loose.
Selective inactivation of alcohol oxidase in two peroxisome‐deficient mutants of the yeast Hansenula polymorphaVan Der Klei, I. J.; Harder, W.; Veenhuis, M.
doi: 10.1002/yea.320070806pmid: 1789002
We have studied selective inactivation of alcohol oxidase (AO) in two peroxisome‐deficient (PER) mutants of the yeast Hansenula polymorpha. In these mutants high activities of cytosolic AO are induced by different growth conditions. At enhanced expression rates AO is arranged in large crystalloids in the cytosol, whereas smaller crystalloids are often observed inside the nucleus. Transfer of cells of the PER mutant 125‐2E, which completely lacks peroxisomes, to glucose‐excess conditions did not lead to degradative inactivation of AO and catalase as observed in wild‐type (WT) cells used as a control. The gradual decrease in enzyme activities in the PER mutant could be accounted for by dilution of existing enzyme into newly formed cells as a result of growth. Morphologically, degradation of the cytosolic crystalloids was also not observed. Similar results were obtained with a second PER mutant (strain 124‐2D), impaired in the import of peroxisomal matrix proteins. This mutant is characterized by the presence of small peroxisomes and large cytosolic AO crystalloids. Upon a shift of cells to glucose‐excess conditions only part of the small peroxisomes present in these cells were degraded by mechanisms similar to those observed in WT cells placed under identical conditions. These results indicate that degradative inactivation of AO in H. polymorpha is strictly dependent on the localization of the enzyme inside peroxisomes and furthermore suggests that the mechanisms triggering this process are not directed against AO protein, but instead, to the membrane surrounding the organelle. Transfer of cells to methanolor ethanol‐containing media both resulted in modification inactivation of AO. Under these conditions also the AO crystalloids remained unaffected by incubation in the new environment.