The Kluyveromyces lactis CPY homologous genes: cloning and characterization of the KlPCL1 gene.

A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.

The response of Ty1 test to genotoxins.

The Ty1 assay is a short-term test for detection of genotoxins based on induction of the transposition of a gene-engineered Ty1 retrotransposon in Saccharomyces cerevisiae cells. Here, we provide evidence that the Ty1 test responds positively in concentration-dependent manner to the carcinogenic genotoxins benz(a)anthracene, benzo(a)pyrene, chenodeoxycholic and taurodeoxycholic free bile acids and to environmental soil samples polluted with carcinogenic substances. The Ty1 test gives negative results with the noncarcinogenic mutagens benz(b)anthracene, benzo(e)pyrene, lithocholic and taurodeoxycholic conjugated bile acids and to soil samples not polluted with carcinogens. Presence or absence of genotoxins in soil samples was evidenced by chemical analysis. Several explanations for the sensitive differential test's response to genotoxins are proposed and discussed. It is concluded that the Ty1 test can complement existing assays in laboratory and environmental studies showing high sensitivity to a wider spectrum of carcinogenic genotoxins.

Enhanced protein export in Saccharomyces cerevisiae nud1 mutants is an active process.

Saccharomyces cerevisiae NUD1 gene codes for a spindle pole body component and nud1 temperature-sensitive mutants arrest at 38 degrees C in late anaphase with a tendency for lysis. We found that addition of 10% sorbitol to the medium complemented the lytic phenotype, and determination of colony-forming units evidenced the viability of nud1 cells for at least 48 hours at 38 degrees C. The protein amount in cell-free medium increased at 38 degrees C, and evidence is presented that intact nud1 cells exported proteins in amounts 10-fold higher compared wild type strains. The observed high amounts of extracellular acid phosphatase, invertase, and bacterial beta-galactosidase suggested the export of secretory proteins. This was evidenced by construction of nudlsec mutants and the observation that interruption of the secretory pathway resulted in absence of protein export at 38 degrees C. Proteins were exported through a cell wall showing increased porosity at 38 degrees C. The extracellular release of Gas1p and the facilitated transformability with plasmid DNA of nud1 cells indicated alternations of their cell walls at 38 degrees C. The export of proteins depends on oxidative phosphorylation as evidenced by disruption of the COX10 gene. Experiments with inhibitors of mitochondrial functions showed that the synthesis of adenosine triphosphate, but not the electron transport along the respiratory chain, has a key role in the export of proteins. The data show that the phenotype of S. cerevisiae nud1 mutants is characterized by enhanced export of secretory proteins and that the passage of proteins through the walls of nud1 cells is an active process.

Genotoxic effect of substituted phenoxyacetic acids.

The potential toxic and mutagenic action of 2,4-dichlorophenoxyacetic acid has been studied in different test systems, and the obtained results range from increased chromosomal damage to no effect at all. We reexamined the effect of this herbicide by simultaneous using three tests based on yeast, transformed hematopoietic, and mouse bone marrow cells. The results obtained demonstrated that 2,4-dichlorophenoxyacetic acid has cytotoxic and mutagenic effects. The positive response of yeast and transformed hematopoietic cells was verified in kinetics and dose-response experiments. The analysis of metaphase chromosomes indicated a statistically proved induction of breaks, deletions, and exchanges after the intraperitoneal administration of 2,4-dichlorophenoxyacetic acid in mice. The study of phenoxyacetic acid and its differently chlorinated derivatives showed that cytotoxicity and mutagenicity are induced by chlorine atoms at position 2 and/or 4 in the benzene ring. The mutagenic effect was abolished by introduction of a third chlorine atom at position 5. Thus 2,4,5-trichlorophenoxyacetic acid was found to have very weak, if any mutagenic effect; however, the herbicide preserved its toxic effect.

NifH and NifM proteins interact as demonstrated by the yeast two-hybrid system.

Biological nitrogen fixation is catalyzed by nitrogenase, a two-component enzyme consisting of the MoFe protein and the Fe protein. Two genes are involved in the formation of active Fe protein: nifH encodes the structural polypeptide, while nifM specifies a stabilizing and activation function by yet unknown mechanisms. Our studies were directed to clarify whether the NifM exerts its function through physical protein-protein interaction with NifH. To accomplish this, we used the yeast two-hybrid system. The simultaneous expression of the GAL4 binding domain-nifH fusion and GAL4 activation domain-nifM fusion resulted in the successful activation of GAL4-responsive HIS3, ADE2, and lacZ reporter genes in the two-hybrid system used. The system was also used to evidence the potential for in vivo NifH and NifM self-association. The results obtained suggest that NifH and NifM form homomers and also associate in between to form higher order complexes, which may be needed to exert the effect of NifM on Fe protein stability and activity.

A new sensitive test based on yeast cells for studying environmental pollution.

Different tests based on yeast cells were developed for determination of mutagenic/carcinogenic action; however, they all showed lower sensitivity compared to bacterial tests, the main reason for this being the limited permeability of yeast cells. We found that general permeability of Saccharomyces cerevisiae cells can be increased by mutation and on this basis we developed a more sensitive test. The aim of this study was to prove the applicability of our test, called D7ts1, in environmental studies. Soil, water and air samples were taken during 1998 from regions in Bulgaria with declared low, average or high pollution levels and investigated for presence of mutagenic/carcinogenic activities in the bacterial test of Ames, the yeast D7 test of Zimmermann and our new D7ts1 test. Results obtained evidenced the following conclusions: (1) the usage of D7ts1 test instead of D7 test permits a clearer measurement of positive samples and detects mutagenic/carcinogenic activities undetectable by D7 test; (2) all samples with positive Ames test were positive in the D7ts1 test; however, some samples, clearly positive in the D7ts1, were negative in the Ames test; therefore, the simultaneous usage of D7ts1 and Ames tests in environmental studies is advantageous because it detects dangers for the human health activities to which bacterial cells do not respond; and (3) regions in Bulgaria declared clean were found to be polluted; particularly troubled are the whole-year positive data in the three tests for air samples from a 'clean' region.

Genotoxic effect of 4-aroyl-1-(2-chloroethyl)-1nitrosohydrazinecarboxamides on Saccharomyces cerevisiae cells.

The study of some 4-aroyl-1-(2-chloroethyl)-1-nitrosohydrazinecarboxamides with a Saccharomyces cerevisiae mutagenicity test of increased sensitivity defined two of them, 4-(4-bromobenzoyl)-1-(2-chloroethyl)-1-nitrosohydrazinecarboxam ide and 4-(4-fluorophenyl)-1-(2-chloroethyl)-1-nitrosohydrazine carboxamide as typical cytostatic agents. At concentrations of 2-5 microg/ml the substances kill up to 60%-70% of cells without having any detectable recombinogenic and mutagenic effects. At the same concentrations, lomustine, well known as a cytostatic reference, demonstrated recombinogenic and mutagenic activity on yeast cells. The advantage of the newly synthesized substances is that, in a certain concentration range, their biological activity is mainly cytotoxic without induction of recombinogenic and mutagenic events in surviving cells.

Enhanced cell permeability increases the sensitivity of a yeast test for mutagens.

ts1 is a mutation which causes a general increase in permeability of Sacharomyces cerevisiae cells in an unspecific manner. The introduction of the ts1 mutation under homozygous conditions into the D7 diploid strain enhanced the sensitivity of the test system described by Zimmermann et al. (1975). The newly constructed strain D7ts1 responded with a four to six times higher frequency compared to the D7 strain for all genetic end-points induced with chemical mutagens (ethyl methanesulfonate, methyl methanesulfonate, hydroxyurea, benzpyrene). The increased sensitivity of D7ts1 is specific only for mutagens active in yeast, since treatment of D7ts1 cells with 5-bromouracil or 5-bromouridine, known to be non-mutagenic in yeast, did not result in the induction of any of the measured genetic alterations. Five out of 14 water samples taken from the environment induced recombinogenic events in D7ts1, whereas all 14 water samples were without effect in the D7 test system. We concluded that D7ts1 cells show a higher sensitivity in the detection of mutagenic or carcinogenic action because of their generally enhanced permeability due to the ts1 mutation.

Evidence that the osmotically fragile yeast S. cerevisiae VY1160 is an actin mutant.

The phenotype of the osmotically dependent S. cerevisiae mutant VY1160 is caused by a single chromosomal mutation, termed srb, with pleiotropic effect. Compared with cells of the parental strain S288C, it was shown that the size and surface structure of the mutant cells are changed. The latter are sensitive to elevated cultivation temperatures as well as to hypotonic pressure and mechanical stress. In these cases, specific plasma membrane alteration were revealed by freeze-fracture electron microscopy. The total actin content is only 88% (21.4 micrograms actin/mg protein) of that of S288C cells. Remarkably, the mutant cells contain only 2.2 micrograms F-actin/mg protein, whereas the S288C cells have 10.3 micrograms F-actin/mg protein. Moreover, the level of reduced glutathione is found to be higher in the mutant cells (23.4 nmole/10(10) cells) than in the parental cells (15.2 nmole/10(10) cells). These results implicate that the srb mutation is localized in the actin gene.

Increased extracellular secretion in fragile mutants of Saccharomyces cerevisiae.

Evidences are presented for a generally increased extracellular secretion capability of the fragile mutants of S. cerevisiae. Proteins secreted in wild type yeasts to the periplasmic space can not be retained by the defective cell wall of the fragile cells and are released into the medium. Changes in the structure of non-mannan components of the cell wall might be the reason for the extracellular release of the periplasmic proteins.

Polyploid fragile strains of Saccharomyces cerevisiae--a novel source of proteins for nutritional purposes.

A series of prototrophic fragile strains of different ploidy (2n, 3n and 4n) has been genetically constructed on the basis of haploid srb1 containing segregants of the fragile Saccharomyces cerevisiae mutant VY 1160. The strains have been characterized by several criteria. In regard to generation time, biomass yield, and nucleic acids content of the cells, the tetraploid srb1 homozygous hybrid is indistinguishable from an industrial strain of S. cerevisiae. However, it is characterized by a higher protein content. Unlike any other laboratory or industrial strains, the original mutant and these hybrids possess an ability for lysis upon suspension in hypotonic solutions. The dependence of the percentage of lysed cells on the growth phase and concentration of osmotic stabilizer in the medium has been investigated. The quantity of proteins in the soluble and insoluble fractions obtained after lysis of these strains by osmotic shock has been determined. These hybrids can be considered as a potential industrial source of proteins for nutritional purposes.

Toyocamycin inhibition of ribosomal ribonucleic acid processing in an osmotic-sensitive adenosine-utilizing Saccharomyces cerevisiae mutant.

An adenosine-utilizing mutant of Saccharomyces cerevisiae (SY 15 ado) is isolated after remutagenesis of an osmotic-sensitive strain, auxotrophic for adenine, with ethyl methanesulfonate. It is shown that the SY15ado mutant can be used to achieve experimental conditions under which cell growth and RNA Synthesis are directly dependent on exogenous adenosine. After starvation for adenosine, toyocamycin is incorporated into pre-rRNA chains of SY15ado cells replacing adenosine residues. The extent of this replacement depends on the concentration of added toyocamycin. Lower doses slow down processing of pre-rRNA into mature rRNA with an accumulation of 27 S and 20 S pre-rRNA. At higher concentrations toyocamycin blocks the last steps of pre-rRNA processing i.e. the conversions 27 S pre-rRNA leads to 25 S rRNA and 20 S pre-rRNA leads to 18 S rRNA. It appears that the main site of toyocamycin action is at the last steps of ribosome formation, while transcription and the early stages of pre-RNA processing are less affected.

Inhibition of ribonuclease II of Escherichia coli by sodium ions, adenosine-5'-triphosphate, and transfer ribonucleic acid.

Ribonuclease II action on polyuridylate is competitively inhibited by transfer ribonucleic acid and noncompetitively inhibited by sodium ions. At low substrate levels, adenosine-5'-triphosphate is also inhibitory.