Anaerobic growth of Saccharomyces cerevisiae alleviates the lethal effect of phosphotyrosyl phosphatase activators depletion.

Saccharomyces cerevisiae homologues of phosphotyrosyl phosphatase activator (PTPA) are encoded byRRD1 and RRD2, genes whose combined deletion is synthetic lethal. Previously we have shown that the lethality of rrd1,2delta can be suppressed by increasing the osmolarity of the medium. Here we show that the lethality of rrd1,2delta is also suppressed under oxygen-limited conditions. The absence of respiration per se is not responsible for the suppression since elimination of the mitochondrial genome or a block in heme biosynthesis fail to rescue the rrd1,2delta double mutation.

Functional analysis of RRD1 (YIL153w) and RRD2 (YPL152w), which encode two putative activators of the phosphotyrosyl phosphatase activity of PP2A in Saccharomyces cerevisiae.

In the context of the cooperative project for functional analysis of novel genes uncovered during the systematic sequencing of the Saccharomyces cerevisiae genome, we deleted two paralogous ORFs: YIL153w and YPL152w. Based on the resulting phenotypes, the corresponding genes were named RRD1 and RRD2, respectively. Rrd proteins show significant similarity to the human phosphotyrosyl phosphatase activator (PTPA). Both single mutants, rrd1delta and rrd2delta, were viable. Deletion of RRD1 caused pleiotropic phenotypes under a wide range of conditions, including sensitivity to Ca2+, vanadate, ketoconazole, cycloheximide and Calcofluor white, and resistance to caffeine and rapamycin. The only phenotypes found for rrd2delta - resistance to caffeine and rapamycin - were weaker than the corresponding phenotypes of rrd1delta. The double mutant rrd1,2delta was inviable on rich glucose medium, but could grow in the presence of an osmotic stabilizer. The rrd1,2delta mutant was partially rescued by inactivation of HOG1 or PBS2, suggesting an interaction between the RRD genes and the Hog1p signal transduction pathway. Introduction of slt2delta into the rrd1,2delta background improved the growth of rrd1,2delta on sorbitol-containing medium, indicating that the Rrd proteins also interact with the Slt2p/Mpk1p signaling pathway. Suppression of the lethal phenotype of the rrd1,2delta mutant by overexpression of PPH22 suggested that the products of the RRD genes function positively with catalytic subunits of PP2A. The synthetic lethality was also suppressed by the "viable" allele (SSD1-v1) of the SSD1 gene.

3' Noncoding sequences of the CTA 1 gene enhance expression of the recombinant serine protease inhibitor, CPTI II, in Saccharomyces cerevisiae.

Expression of the gene coding for the recombinant trypsin inhibitor, CPTI II, was enhanced tenfold when yeast transcription terminating sequences were added to the expression cassette of the pJK6 yeast vector. The yield was further increased about 20% in the BJ5464 yeast strain, defective in vacuolar proteases.

Expression of small synthetic genes coding for hEGF, human epidermal growth factor, and CPTI II, serine proteinase inhibitor from Cucurbitacea, cloned in a novel expression/secretion vector in Saccharomyces cerevisiae.

Efficient synthesis of two small eukaryotic polypeptides of human and plant origin was carried out using a novel expression/secretion yeast vector, pYET. The yield was optimized in respect of the yeast strain, expression cassette construction, promoter regulation and culture conditions. Both cloned genes code for biotechnologically important proteins: human epidermal growth factor and a serine proteinase inhibitor from Cucurbitacea.

Genotoxicity assessment of low-molecular weight interferon inducers by the SOS Chromotest.

Tilorone and its aza-analogs, as well as CMA and its butyric analog (CNPA) were investigated as potential genotoxic agents by the SOS Chromotest. The SOS-inducing potency values (SOSIP) were 0.0033 and 0.0009 for SAF and vivakorfen, respectively, after activation with S9 fraction of mouse liver only. In contrast, an SOSIP value for tilorone of 0.0011 was observed in a non-activated assay. The SOSIPs of investigated compounds were low and comparable to the lowest values determined for other genotoxins. CMA and CNPA were not SOS inducers in any test system.

Replication and expression of fragments of phage PM2 cloned in Escherichia coli K-12.

DNA from PM2 phage was cloned, as HindIII fragments and inserted into the pBR322 vector in E. coli cells. It was shown, that replication of recombined plasmids starts from the pBR322 origin. Transcription of recombinant plasmids in E. coli cells, as well as translation in minicells was demonstrated and attributed to pBR322 and/or PM2 DNA sequences.

Cloning of bacteriophage PM2 DNA in Escherichia coli K12.

DNA fragments of phage PM2 restricted with Hin dIII endonuclease was cloned in the vector pBR 322 in an Escherichia coli K12 host. The attempt to clone full length PM2 DNA restricted with PstI endonuclease has been unsuccesful. From six randomly chosen recombinant clones DNA was purified and analysed with EcoRI, PstI and Hin dIII endonucleases. The physical map of three chimeric plasmids was unequivocally established. It was shown, that the whole PM2 genome was cloned, although in separate fragments. However, most of the recombinant clones were instable in the absence of selective pressure.