Bun-ichiro Ono

SACCHAROMYCES CEREVISIAE CULTURED UNDER AEROBIC AND ANAEROBIC CONDITIONS :AIR-LEVEL OXYGEN STRESS AND PROTECTION AGAINST STRESS

Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells. Aerobiosis increased both the compounds and enzyme activities. The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine beta-synthase.

Involvement of the VDE Homing Endonuclease and Rapamycin in Regulation of the Saccharomyces cerevisiae GSH11 Gene Encoding the High Affinity Glutathione Transporter

The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5′-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004–1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

Transcriptional regulation of Saccharomyces cerevisiaeCYS3 encoding cystathionine γ-lyase

In studying the regulation of GSH11, the structural gene of the high-affinity glutathione transporter (GSH-P1) in Saccharomyces cerevisiae, a cis-acting cysteine responsive element, CCGCCACAC (CCG motif), was detected. Like GSH-P1, the cystathionine γ-lyase encoded by CYS3 is induced by sulfur starvation and repressed by addition of cysteine to the growth medium. We detected a CCG motif (−311 to −303) and a CGC motif (CGCCACAC; −193 to −186), which is one base shorter than the CCG motif, in the 5′-upstream region of CYS3. One copy of the centromere determining element 1, CDE1 (TCACGTGA; −217 to −210), being responsible for regulation of the sulfate assimilation pathway genes, was also detected. We tested the roles of these three elements in the regulation of CYS3. Using a lacZ-reporter assay system, we found that the CCG/CGC motif is required for activation of CYS3, as well as for its repression by cysteine. In contrast, the CDE1 motif was responsible for only activation of CYS3. We also found that two transcription factors, Met4 and VDE, are responsible for activation of CYS3 through the CCG/CGC and CDE1 motifs. These observations suggest a dual regulation of CYS3 by factors that interact with the CDE1 motif and the CCG/CGC motifs.

Role of the ompT Mutation in Stimulated Decrease in Colony-Forming Ability Due to Intracellular Protein Aggregate Formation in Escherichia coli Strain BL21

Recently we found that the cells of Escherichia coli strain BL21 producing a fusion protein, GST-Sup35NM, show a much more rapid decrease in colony-forming ability in the stationary phase than control cells. In this study, it was found that an extract of the cells producing GST-Sup35NM forms fibrous protein polymers containing GST-Sup35NM. In the course of the study, we realized that strain BL21 carried the ompT mutation. We suspected that the deficiency in OmpT protease was responsible for the observed phenotype. To test this, we introduced the wild-type ompT gene into strain BL21, and found that the transformed cells recovered the wild-type phenotype. We concluded that OmpT protease, though known to localize on the cell surface, is involved in protein quality control within the cell.

Studies on the ATP3 gene of Saccharomyces cerevisiae: presence of two closely linked copies, ATP3a and ATP3b, on the right arm of chromosome II.

In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the γ subunit of the F1F0‐ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single‐copy gene on chromosome II. We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F1‐α and F1‐β, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F1F0‐ATPase. By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b. The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303‐1A, W303‐1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a. With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp. The differences between ATP3 with the single base substitution (Ser308 to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions. However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3. It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild‐type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone. Copyright

Isolation and characterization of a virus (CvV-BW1) that infects symbiotic algae of Paramecium bursaria in Lake Biwa, Japan

BackgroundWe performed an environmental study of viruses infecting the symbiotic single-celled algae of Paramecium bursaria (Paramecium bursaria Chlorella virus, PBCV) in Lake Biwa, the largest lake in Japan. The viruses detected were all Chlorella variabilis virus (CvV = NC64A virus). One of them, designated CvV-BW1, was subjected to further characterization.ResultsCvV-BW1 formed small plaques and had a linear DNA genome of 370 kb, as judged by pulsed-field gel electrophoresis. Restriction analysis indicated that CvV-BW1 DNA belongs to group H, one of the most resistant groups among CvV DNAs. Based on a phylogenetic tree constructed using the dnapol gene, CvV was classified into two clades, A and B. CvV-BW1 belonged to clade B, in contrast to all previously identified virus strains of group H that belonged to clade A.ConclusionsWe conclude that CvV-BW1 composes a distinct species within C. variabilis virus.

The Saccharomyces cerevisiae ESU1 gene, which is responsible for enhancement of termination suppression, corresponds to the 3'-terminal half of GAL11.

A DNA fragment enhancing efficiency of [PSI+]‐dependent termination suppressor, sup111, was isolated from a genomic library of Saccharomyces cerevisiae and its function was attributed to an ORF of 1272 bp. This ORF, designated ESU1 (enhancer of termination suppression), corresponded to the 3′‐terminal portion of GAL11. Contrasting to ESU1, GAL11 lowered the suppression efficiency of [PSI+] sup111. ESU1 possesses a TATA‐like sequence of its own and three ATG codons following it within a distance of about 70 bp and all in the same reading frame as GAL11. A 52.7 kDa protein corresponding in size to the predicted Esu1 protein is detected by western blot analysis using anti‐Gal11 antiserum. We therefore conclude that ESU1 is the gene that encodes a polypeptide corresponding to the C‐terminal 424 amino acids of Gal11. It was further found that ESU1 increases the level of GAL11 mRNA and probably also of its own mRNA. Moreover, ESU1 increased the cellular level of mRNA transcribed from the leu2‐1UAA mutant gene, while GAL11 did not. Based on these findings, we propose the following scheme for the events taking place in the [PSI+] sup111 cell that is transformed with an ESU1‐bearing plasmid: (a) ESU1 stimulates transcription of leu2‐1; (b) leu2‐1 mRNA is not effectively degraded because of the possession of sup111, which belongs to the upf group; (c) [PSI+] causes increased mis‐termination due to depletion of eRF3; (d) functional Leu2 product is made using leu2‐1 mRNA; and (d) suppression of leu2‐1 is eventually accomplished. Copyright

Effects of mutations in yeast prion [PSI+] on amyloid toxicity manifested in Escherichia coli strain BL21.

We previously showed that over production of a fusion protein in which the prion domain of Saccharomyces cerevisiae [PSI+] is connected to glutathione S-transferase (GST-Sup35NM) causes a marked decrease in the colony forming ability of Escherichia coli strain BL21 after reaching stationary phase. Evidence indicated that the observed toxicity was attributable to intracellular formation of fibrous aggregates of GST-Sup35NM. In this report, we describe the isolation of plasmids that encode mutant forms of GST-Sup35NM which do not confer the toxicity to E. coli strain BL21. Each of the four spontaneous mutant-forms of GST-Sup35NM obtained revealed amino acid substitutions. One substitution was located in the N domain, and the others in the M domain. Congo red binding assay indicated that none of these mutant protein underwent conformational alteration in vitro. From these results, we conclude that the M domain, in collaboration with the N domain, plays an essential role in aggregation of Sup35NM. In addition, our data demonstrate the usefulness of the E. coli expression system in studying aggregate-forming proteins.

Production of a Polymer-Forming Fusion Protein in Escerichia coli Strain BL21

In the course of studying [PSI +], a yeast prion, we found inadvertently that Escherichia coli strain BL21 overproducing a fusion protein, in which the prion-domain of Sup35 was connected to the C terminus of glutathione S-transferase, grew normally to the stationary phase and rapidly decreased in colony-forming ability thereafter. Evidence indicated that protein polymers consisting mainly of the fusion protein GST-Sup35NM (about 70% of the mass) and its N-terminal fragments were formed in extract prepared from the cells producing GST-Sup35NM. It was further found that cells of strain BL21 accumulated the protein polymers during prolonged cultivation. Based on these results, we contend that the initially observed defect in colony forming ability is the direct or indirect consequence of intracellular formation and accumulation of the protein polymers.

Inverted repeat of a large segment unveiled on the right arm of Saccharomyces cerevisiae chromosome II.

By means of gene disruption analyses, Saccharomyces cerevisiae strain YPH499 was shown to have two, and only two, copies of ATP3 that encodes the γ‐subunit of H+‐dependent ATP synthase and locates on the right arm of chromosome II. Linkage analyses of the two distinguishably marked copies of ATP3 indicated that the distance between them was about 43 cM. Since YBR030W, an ORF proximal to ATP3 by a distance of 17 kbp, was also found to be duplicated, we marked them with two distinguishable nutritional markers, which were also distinguishable from those used for marking the two copies of ATP3, and achieved four‐point linkage analyses; CEN2 marked with an appropriate nutritional marker gene was included as a reference point. And, the following linkage map was deduced: CEN2– [11 cM]–YBR030Wa– [8 cM]–ATP3a–[47 cM]–ATP3b– [55 cM]–YBR030Wb. From this map, we suspected that a segment spanning at least YBR030W–ATP3 would be inversely duplicated on the right arm of chromosome II. We then carried out chromosome fragmentation analyses, using several laboratory strains including YPH499, and obtained data in accord with our speculation for all strains, although the distance between the two copies of ATP3 varied from 48 kbp to 192 kbp among the strains examined. Copyright