Anand Kumar Bachhawat

HGT1 encodes a high-affinity glutathione transporter in the yeast Saccharomyces cerevisiae that is induced under sulphur limitation. The present work demonstrates that repression by organic sulphur sources is under the control of the classic sulphur regulatory network, as seen by the absence of expression in a met4Δ background. Cysteine appeared to be the principal regulatory molecule, since elevated levels were seen in str4Δ strains (deficient in cysteine biosynthesis) that could be repressed by elevated levels of cysteine, but not by methionine or glutathione. Investigations into cis-regulatory elements revealed that the previously described motif, a 9-bp cis element, CCGCCACAC, located at the −356 to −364 region of the promoter could in fact be refined to a 7-bp CGCCACA motif that is also repeated at −333 to −340. The second copy of this motif was essential for activity, since mutations in the core region of the second copy completely abolished activity and regulation by sulphur sources. Activity, but not regulation, could be restored by reintroducing an additional copy upstream of the first copy. A third region, GCCGTCTGCAAGGCA, conserved in the HGT1 promoters of the different Saccharomyces spp, was observed at −300 to −285 but, while mutations in this region did not lead to any loss in repression, the basal and induced levels were significantly increased. In contrast to a previous report, no evidence was found for regulation by the VDE endonuclease. The strong repression at the transport level by glutathione seen in strains overexpressing HGT1 was due to a glutathione-dependent toxicity in these cells.

Pichia anomalais an emerging nosocomial pathogen and there is a need for methods that distinguish between differentP. anomalastrains. In the typing of several clinical as well as non-clinicalP. anomalastrains, the sequence variation of the internal transcribed spacer (ITS) was found to be inadequate for typing purposes. The intergenic spacer 1 (IGS1) region of the rDNA of severalP. anomalastrains was therefore investigated in detail. The IGS1 region (which varied from 1213 to 1231 bp in length) was interspersed with repeats and had more variation than the ITS regions. Comparative analysis in cases where analysis by the ITS was ambiguous clearly revealed the IGS1 region to be a more discriminatory tool in the typing ofP. anomalastrains.

The glutathione-mediated pathway for the detoxification of endogenously and exogenously derived toxic compounds was investigated using a pigment that accumulates in certain adenine biosynthetic mutants of yeasts. The ade1 / ade2 mutants of Saccharomyces cerevisiae, when grown on adenine-limiting medium, accumulate a characteristic red pigment (ade pigment) in their vacuoles. The precursors of the ade pigments are toxic intermediates that form conjugates with glutathione, followed by their transport inside the vacuole. In this study, this red pigment was used as a phenotypic screen to obtain insight regarding new genes involved in the three phases of this detoxification pathway: the activation phase (phase I), the conjugation phase (phase II), and the efflux phase (phase III). Components of the phase III (efflux) pathway which includes, in addition to the previously characterized Ycf1p and Bpt1p, another member of the 'Ycf1p family', Bat1p, as well as a vacuolar H⁺-ATPase-dependent transport were identified. In the investigation of phase II (conjugation), it was found that glutathione S-transferases, encoded by GTT1 and GTT2,do not appear to play a role in this process. By contrast, two other previously characterized genes, the oxidative stress transcription factor gene, SKN7 , and the yeast caesin protein kinase gene, YCK1, of S. cerevisiae do participate in this pathway.

We describe a program (and a website) to reformat the ClustalX/ClustalW outputs to a format that is widely used in the presentation of sequence alignment data in SNP analysis and molecular systematic studies. This program, CLOURE, CLustal OUtput REformatter, takes the multiple sequence alignment file (nucleic acid or protein) generated from Clustal as input files. The CLOURE-D format presents the Clustal alignment in a format that highlights only the different nucleotides/residues relative to the first query sequence. The program has been written in Visual Basic and will run on a Windows platform. The downloadable program, as well as a web-based server which has also been developed, can be accessed at http://imtech.res.in/~anand/cloure.html.

The ECM38 gene encodes the γ‐glutamyl transpeptidase enzyme, an enzyme involved in glutathione turnover. The enzyme was found to be present in the S288C strain, BY4742, but absent in another widely used strain congenic to S288C, YPH499. Cloning and sequencing the genes from these yeasts indicated the presence of 11 single nucleotide polymorphisms in the coding region and eight single nucleotide polymorphisms in the promoter region of the ECM38 gene of YPH499 (but none in that of BY4742). One of the SNPs in the ECM38 ORF led to a G → D conversion in a region conserved in all γ‐GT enzymes and was found to be responsible for the loss of activity in this strain. The presence of γ‐GT activity in other YPH strains led us to trace the origins of the polymorphisms in YPH499. Our results indicated that among the progenitor strains, YPH1 and YPH2, YPH1 carried the polymorphisms seen in YPH499 and also lacked the γ‐GT activity. The implications of these results for the use of these widely used S288C strains and the origin of these single nucleotide polymorphisms are presented. Copyright © 2003 John Wiley & Sons, Ltd.

γ-Glutamyl transpeptidase (γ-GT) is the only enzyme known to be responsible for glutathione degradation in living cells. In the present study we provide evidence that the utilization of glutathione can occur in the absence of γ-GT. When disruptions in the CIS2 gene encoding γ-GT were created in met15Δ strains, which require organic sulfur sources for growth, the cells were able to grow well with glutathione as the sole sulfur source suggesting that a γ-GT-independent pathway for glutathione degradation exists in yeast cells. The CIS2 gene was strongly repressed by ammonium and derepressed in glutamate medium, and was found to be regulated by the nitrogen regulatory circuit. The utilization of glutathione as a sulfur source was, however, independent of the nitrogen source in the medium, further underlining that the two degradatory pathways were distinct.