[Comparative analysis of respiratory activity in the wild type strain of Neurospora crassa and its photoreceptor complex mutants].

Cell respiratory activity of protoplasts obtained from the wild type of Neurospora crassa and photoreceptor complex WCC--white collar 1 (wc-1) and white collar 2 (wc-2)--mutants of Neurospora crassa strains was investigated. Respiration inhibition by KCN in the presence of 25 mM succinate was similar in all strains and did not exceed 83-85% against control. The significant induction of KCN-resistant respiratory pathway occurred under 1% glucose oxidation in wc-1 and wc-2 mutants if compared with the wild type strains. The inhibitors of the main (cytochrome) pathway of electron transfer in mitochondria-1 mM KCN and antimycin A (4 microg/ml)--blocked the respiration rate of the protoplasts from N. crassa wild type by 75%, while the cell respiration of wc-1 and wc-2 strains was suppressed by approximately 50%. The specific inhibitor of alternative oxidase--10 mM salicylhydroxamic acid (SHAM)-in combination with the blockers of mitochondrial electron transfer chain caused the total suppression of respiratory activity of protoplasts in all studied strains. It is supposed that an increase of KCN-resistance in WCC mutants under glucose oxidation is connected with alternative oxidase activation as the result of failure in reception and signal transduction of active oxygen species.

[The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica].

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.

[A comparative study of the components of the antioxidant defense system during growth of the mycelium of a wild-type Neurospora crassa strain and mutants, white collar-1 and white collar-2].

A comparative study of the changes in the components of the antioxidant defense system (ADS), the activity of superoxide dismutase (SOD) and catalase and the level of extractable SH-groups, during the growth of wild-type and mutant (white collar-1 and white colar-2) Neurospora crassa strains was performed. Oxidative stress developing during spore germination and upon the transition to a stationary growth phase was accompanied in all strains by an increase in the level of extractable SH-groups and SOD activity, whereas the total catalase activity decreased during growth. However, in contrast to the wild-type strain, the activity of the catalase in the mutant strains wc-1 and wc-2 slightly increased upon the transition to the stationary phase. In the wc-2 mutant, SOD activity and the level of extractable SH-groups in the exponential growth phase were always lower than in the wild-type and wc-2 strains. The role of wc-1 and wc-2 genes in the level regulation of reactive oxygen species is discussed.

[Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis]. / Izoliatsiia i pervichnaia kharakteristika mutantov po biogenezu peroksisom u metilitrofnykh drozhzhei Hansenula polymorpha.

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.

[Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources]. / Toksichnost' merkaptoétanola dlia mutantnykh linii drozhzhei Pichia methanolica, rastushchikh na razlichnykh istochnikakh ugleroda.

Addition of beta-mercaptoethanol at a concentration of 2-3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeast Pichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. beta-Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of beta-mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.

[Synthesis of polypeptides of various length in isolated mitochondria of rat liver]. / Sintez polipeptidov razlichnoi deliny v izolirovannykh mitokhondriiakh pecheni krysy.

The [35S]methionine labeled mitochondrial translation products were separated by SDS-PAGE electrophoresis in the presence of 8 M urea. The time of the major polypeptide synthesis was determined after fluorography and scanning. It was found that the changes in the time of polypeptide synthesis are not correlated with their lengths. These data are consistent with the hypothesis on the unsteady rate of protein synthesis with a sharp deceleration (pause) of translation during the reading of middle portions of mRNA.

[Incorporation of labeled formate in rat liver mitochondrial proteins and initiation of protein synthesis]. / Vkliuchenie mechenogo formiata v belki mitokhondrii pecheni krysy i initsiatsiia belkovogo sinteza.

During incubation of rat liver mitochondria in vitro labeled formate is incorporated into TCA-insoluble substance during mitochondrial translation. Data from hydrolysis with CNBr (after methionine residues) or with 0.5 N HCl (deformylation of amino acid N-formyl derivatives) suggest that about half of the total protein radioactivity is incorporated in formate groups of N-terminal methionine. Labeling of growing polypeptides with formate (but not with phenylalanine or methionine) oscillates with a period of about 13 min. The potential initiation capacity is unchangeable and exceeds that observed experimentally by one order of magnitude. The data obtained are consistent with the previously proposed hypothesis no synchronization of mitochondrial protein synthesis which cannot be induced by the steps preceding the formation of the first peptide bound.

[Kinetic characteristics of the translation of rat liver mitochondria incubated under various conditions]. / Kineticheskie osobennosti transliatsii v inkubiruemykh pri razlichnykh usloviiakh mitokhondriiakh pecheni krysy.

The mode of distribution of labelled amino acids between the nascent and completed polypeptides during incubation of rat liver mitochondria in vitro was studied. This distribution corresponds to protein synthesis of uneven type with a sharp deceleration (pause) during the translation of the middle part of mRNA. The correspondence manifests itself in the fact that i) regular increment of radioactivity of nascent and completed polypeptides in coupled mitochondria was observed in interval less then ts (time necessary for the synthesis of an average polypeptide), and, ii) serine hydroxamate or norleucine have much less effect on the labelling of total protein as well as on the duration of synthesis of an average polypeptide, as could be expected from their inhibition of unmasked elongation. The duration of an expected pause during translation might exceed 4-fold the time necessary for elongation of the largest part of the polypeptide.

[Interpretation of the low rate of polypeptide synthesis in mitochondria in vitro]. / Interpretatsiia nizkoi skorocti sinteza polipeptidov v mitokhondriiakh in vitro.

The distribution of radioactivity between the growing and finally formed polypeptides in rat liver mitochondria incubated with labelled amino acids was studied. The rate of an "average" polypeptide synthesis in the mitochondrial translation system is much lower than that in bacterial and eukaryotic cytoplasmic protein synthesizing systems. A kinetic study of mitochondrial polypeptide labelling suggests that the rate of ribosomal movement along mRNA in mitochondrial translation systems is variable. It is assumed that the low rate of polypeptide synthesis in mitochondria is due to the delay in ribosome movement along the middle part of mRNA.