The Spoilage Yeast Zygosaccharomyces bailii Forms Mitotic Spores: a Screening Method for Haploidization

Zygosaccharomyces bailii ISA 1307 and the type strain of this spoilage yeast show a diploid DNA content. Together with a rather peculiar life cycle in which mitotic but no meiotic spores appear to be formed, the diploid DNA content explains the observed difficulties in obtaining auxotrophic mutants. Mitotic chromosome loss induced by benomyl and selection on canavanine media resulted in three haploid strains of Z. bailii. This new set of Z. bailii strains allows the easy isolation of recessive mutants and is suitable for further molecular genetic studies.     

A spectrum of mutations in the second gene for autosomal dominant polycystic kidney disease (PKD2).

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.     

Isolation and characterization of mutants of the facultative methylotroph Arthrobacter P1 blocked in one-carbon metabolism

Among methylamine and/or ethylamine minus mutants of Arthrobacter P1 four different classes were identified, which were blocked either in the methylamine transport system, amine oxidase, hexulose phosphate synthase or acetaldehyde dehydrogenase. The results indicated that a common primary amine oxidase is involved in the metabolism of methylamine and ethylamine. Growth on ethylamine, however, was not dependent on the presence of the methylamine transport system. In mutants lacking amine oxidase, methylamine was unable to induce the synthesis of hexulose phosphate synthase, thus confirming the view that the actual inducer for the latter enzyme is not methylamine, but its oxidation product formaldehyde. Contrary to expectation, when the formaldehyde fixing enzyme hexulose phosphate synthase was deleted (mutant Art 11), accumulation of formaldehyde during growth on choline or on glucose plus methylamine as a nitrogen source did not occur. Evidence was obtained to indicate that under these conditions formaldehyde may be oxidized to carbon dioxide via formate, a sequence in which peroxidative reactions mediated by catalase are involved. In addition, a specific NAD-dependent formaldehyde dehydrogenase was detected in choline-grown cells of wild type Arthrobacter P1 and strain Art 11. This enzyme, however, does not play a role in methylamine or formaldehyde metabolism, apparently because these compounds do not induce its synthesis.Abbreviations RuMP ribulose monophosphate - HPS hexulose phosphate synthase - HPI hexulose phosphate isomerase     

The consensus sequence of Kluyveromyces lactis centromeres shows homology to functional centromeric DNA from Saccharomyces cerevisiae

Summary The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5-ATCACGTGA-3) flanked by an AT-rich (±90%) stretch of ± 160 by followed by another conserved box (5-TNNTTTATGTTTCCGAAAATTAATAT-3).These three elements were named K1CDEI, K1CDEII, and K1CDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 by AT-rich (±90%) element, named K1CDE0, was found ± 150 by upstream of K1CDEI. The sequences of both K1CDEI and K1CDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161–164 by in K. lactis versus 78–86 by in S. cerevisiae and the presence in K. lactis of K1CDEO, which is not found in S. cerevisiae.     

Molecular cloning of the gene for the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae

The E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector lambda gt11. The gene encoding the E1 alpha subunit was unique and localized on a 1.7-kb HindIII fragment from chromosome V. The identify of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti-E1 alpha serum. (b) Gene replacement at the 1.7-kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti-E1 alpha serum in haploid cells. In addition, the 1.7-kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N-terminal and central regions of the human E1 alpha peptide. We propose to call the gene encoding the E1 alpha subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the lambda gt11 library using the anti-E1 beta serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.     

Mutational analysis of centromeric DNA elements of Klayveromyces lactis and their role in determining the species specificity of the highly homologous centromeres from K. lactis and Saccharomyces cerevisiae

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KICDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KICDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20–80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of K1CDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level then a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.     

Cell stress induced HSP are targets of regulatory T cells: a role for HSP inducing compounds as anti-inflammatory immuno-modulators?

T cell responses to heat shock proteins (HSP) have disease suppressive activities through production of anti-inflammatory cytokines in patients and in models of inflammatory diseases. There is evidence that the anti-inflammatory activity of HSP-specific T cells depends on their recognition of endogenous HSP epitopes as expressed by stressed cells at sites of inflammation. Previously, we have demonstrated that such T cells can be induced by conserved sequences of microbial HSP. Now we propose that drug induced up-regulation of endogenous HSP can contribute to anti-inflammatory T cell regulation.