[Comparative structural and functional characteristics of different forms of Saccharomyces cerevisiae red pigment and its synthetic analogue].

Structural and functional characteristics of the yeast red pigment (product of polymerization of N1-(beta-D-ribofuranosyl)-5-aminoimadazole), isolated from adel 1 mutant cells of Saccharomyces cerevisiae, its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N1-methyl-5-aminoimadazole in vitro) has been obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. The entire compounds studied were able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in presence of these compounds--they were merged into conglomerates that were more stable and resistant to the effects of ultrasound in comparison with insulin aggregates grown without pigments. We speculate that all these compounds can cause coalescence of fibrils, partially block their loose ends and, thereby, inhibit the attachment of new monomers to growing fibrils.

[Genetic control of metabolism of mutagenic purine base analogs 6-hydroxylaminopurine and 2-amino-6-hydroxylaminopurine in yeast Saccharomyces cerevisiae].

The influence of inactivation of genes, which control biosynthesis of inosine monophosphate (IMP) de novo and the purine utilization and interconversion pathway, on sensitivity of yeast Saccharomyces cerevisiae cells to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA) was studied. It was shown that the manifestation of HAP and AHA mutagenic properties involves the action of enzyme adenine phosphoribosyltransferase encoded in yeast by APT1 gene. A blockade of each stage of IMP biosynthesis, with the exception of the block mediated by inactivation of genes ADE16 and ADE17 leading to the accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), was shown to enhance yeast cell sensitivity to the HAP mutagenic effect; however, it does not affect the sensitivity to AHA. A blockade of conversion of IMP into adenosine monophosphate (AMP) causes hypersensitivity of yeast cells to the mutagenic action of HAP and to the toxic effect of HAP, AHA, and hypoxanthine. It is fully probable that this enhancement of sensitivity to HAP and AHA is conditioned by changes in the pool of purines. This indicates that genes ADE12, ADE13, AAH1, and HAM1 controlling processes of purine utilization and interconversion in yeast make the greatest contribution to the system of protection against the toxic and mutagenic action of the examined analogs. Possible mechanisms of HAP detoxication in bacteria, yeast, and humans are considered.

Multiple antimutagenesis mechanisms affect mutagenic activity and specificity of the base analog 6-N-hydroxylaminopurine in bacteria and yeast.

Base analog 6-N-hydroxylaminopurine is a potent mutagen in variety of prokaryotic and eukaryotic organisms. In the review, we discuss recent results of the studies of HAP mutagenic activity, genetic control and specificity in bacteria and yeast with the emphasis to the mechanisms protecting living cells from mutagenic and toxic effects of this base analog.

[Transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts by a plasmid carrying the ADE2 gene of Saccharomyces cerevisiae]. / Transformatsiia drozhzhei Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus plazmidoi s genom ADE2 Saccharomyces cerevisiae.

Red adenine-dependent mutants of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts have been transformed by the plasmid pYE (ADE2) 2 DNA containing ADE2 gene from Saccharomyces cerevisiae. The analysis of two P. guilliermondii Ade+-transformants has revealed the integration of pYE (ADE2)2 sequence into the recipient strain genome and partial restoration of the deficient function.

[Identification of mutations in the asporogenic yeast Candida tropicalis using intrageneric fusion of protoplasts]. / Identifikatsiia mutatsii u asporogennykh drozhzhei Candida tropicalimetodom mezhrodovogo sliianiia protoplastov.

The possibility of genetic identification of mutations in asporogenic yeast by the technique of intrageneric fusion of yeast protoplasts of Candida tropicals and Saccharomyces cerevisiae has been demonstrated for Candida tropicals strains G5-9 (Ade- Leu-) and G32-4 (Leu-). The mutations to auxotrophy ade- in the strain G5-9 and leu- in G32-4 of Candida tropicals are allelic to ade2 and leu1 mutations in the genes of Saccharomyces cerevisiae yeast. The allelic character of adenine auxotrophy mutation in Candida tropicals and ade2 mutation in Saccharomyces cerevisiae is confirmed by the absence of AIR-carboxylase activity in cellular extract from the strain G5-9.

[A simple method of selecting Saccharomyces cerevisiae ade1 and ade2 mutants]. / Prostoi metod selektsii mutantov ade1 i ade2 drozhzhei Saccharomyces cerevisiae.

A simple technique of selection for spontaneous red mutants of yeast Saccharomyces cerevisiae is proposed. It is based on incubation of yeast cells carrying a deletion in the GCN4 gene on a solid selective medium containing 3-amino-1, 2, 4-triazole (3AT). After incubation for three weeks at 30 degrees C, the 3 AT-resistant mutants ade2 and ade1 were easily visualized due to the pink or red colony color; they were then selected and analyzed by genetic and biochemical methods. The frequency of these colored mutants was approximately 10-9. The method proposed is shown to be suitable for selection and analysis of both spontaneous mutants ade2 and ade1 and those induced by various mutagens. The possible molecular mechanism of 3AT-resistance of red mutants is discussed. This mechanism involves GCN4-independent activation of expression of genes of purine and histidine metabolism. This activation results from purine starvation caused by arrest of purine biosynthesis the ade2 and ade2 mutations.

[Genetic and biochemical study of yeast acid phosphatases. XI. Gene ACP80 controls inorganic phosphate transport]. / Genetiko-biokhimicheskoe izuchenie kislykh fosfataz drozhzhei. Soobshchenie XI. Gen ACP80 kontroliruet transport neorganicheskogo fosfata.

Mutations in the gene ACP80 which simultaneously lead to constitutive synthesis of repressible acid phosphatases, resistance to 1 X 10(-3) M sodium arsenate and to the disturbance of phosphate uptake in the cell were obtained. Mutations suppressible by ochre suppressors in this gene were identified. The conclusion that the gene ACP80 encodes a protein which participates in the phosphate transport into the cell, was drawn. A selective method for isolation of mutations in this gene is proposed.

[Suppression of frameshift mutation as a result of partial inactivation of translation termination factors in Saccharomyces cerevisiae yeast]. / Supressiia mutatsii "sdvig ramki schityvaniia" v rezultate chastichnoi inaktivatsii faktorov terminatsii transliatsii u drozhzhei Saccharomyces cerevisiae.

Special search for frameshift mutations, which are suppressed by the cytoplasmic [PSI] factor and by omnipotent nonsense suppressors (recessive mutations in the SUP35 and SUP45 genes), partially inactivating a translation termination complex, was initiated in the LYS2 gene in the yeast Saccharomyces cerevisiae. Mutations were obtained after exposure to UV light and treatment with a mixture consisting of 1.6- and 1.8-dinitropyrene (DNP). This mixture was shown to induce mutations of the frameshift type with a high frequency. The majority of these mutations were insertions of one A or T, which is in good agreement with the data obtained in studies of DNP-induced mutagenesis in other eukaryotes. Frameshift suppression in yeast was first shown on the example of the mutation obtained in this work (lys2-90), which carried the insertion of an extra T in the sequence of five T. This frameshift suppression was shown to occur in the presence of the [PSI] factor (i.e., due to the prion form of the translation release factor eRF3) and as a result of mutations in genes SUP35 or SUP45, which partially inactivate translation termination factors eRF3 and eRF1, respectively. Alternative mechanisms of programmed translational frameshifting in the course of translation and the possibility of enhancing the effectiveness of such frameshifting in the presence of the [PSI] factor are considered.

[A method of biochemical identification of mutations in red adenine-dependent yeast strains]. / O metode biokhimicheskoi identifikatsii mutatsii u krasnykh adeninzavisimykh shtammov drozhzhei.

A simple biochemical technique is proposed for quantitative estimation of expression of the ADE2 and ADE1 genes, coding for the structure of AIR-carboxylase and SAICAR-synthetase in the yeast Saccharomyces cerevisiae. The technique is based on determining the enzyme specific activities in the yeast crude extracts. The technique was applied to estimate quantitatively the expression of the ADE2 and ADE1 genes in Saccharomyces cerevisiae. The method is available for identification of mutation in the analogous genes of non-saccharomyces yeasts.

[Testing the activity of enzymes responsible for biosynthesis of purine nucleotides AIR-carboxylase and SAICAR-synthetase in human cell extracts]. / Testirovanie aktivnosti fermentov biosinteza purinovykh nukleotidov AIR-karboksilazy i SAIKAR-sintetazy v ékstraktakh kletok cheloveka.

Activities of AIR-carboxylase (ES 4.1.1.21) and SAICAR-synthetase (EC 6.3.2.6) were found in lysates of human erythrocytes, thrombocytes and leukocytes and in homogenate of the stomach biopsy sample. However, these activities were absent in blood plasma and bile. The human erythrocyte enzyme preparation, which had both activities, was isolated and purified about 200 times. The copurification of both activities and properties of the enzyme preparation suggest that two consecutive reactions of purine biosynthesis de novo (from AIR to SAICAR) in human cells are catalyzed by one bifunctional enzyme which is probably encoded by one gene.

[Isolation and properties of phosphoribosyl-aminoimidazole-succinocarboxyamide-synthestase from Saccharomyces cerevisiae yeasts]. / Vydelenie i svoistva fosforibozil-aminoimidazol-suktsinokarboksamid- sintetazy drozhzhei Saccharmomyces cerevisiae.

An isolation procedure for phosphoribosyl succinocarboxamideaminoimidazole synthetase (SAICAR synthetase) (EC 6.3.2.6) has been developed. Pure SAICAR synthetase was found to be a monomeric protein with the apparent molecular weight of 36 kDa. The Michaelis constant for the three substrates of the reaction are 1.6 microM for CAIR, 14 microM for ATP and 960 microM for aspartic acid. The structural analogs of CAIR, 5-aminoimidazole ribotide and 5-aminoimidazole-4-carboxamide ribotide, act as competitive inhibitors of SAICAR synthetase. GTP and 2'-dATP can substitute for ATP in the reaction, while CTP and UTP inhibit the enzyme. No structural analogs of the aspartic acid were found to have affinity for SAICAR synthetase. The optimal reaction conditions for the enzyme were established to be at pH 8.0 and magnesium chloride concentration around 5 mM.

[Substrate specificity of phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR-synthetase) from Saccharomyces cerevisiae yeast]. / Substratnaia spetsifichnost' fosforibozil-aminoimidazol-suktsionokarboksimid-sintetazy (SAIKAR-sintetazy) drozhzhei Saccharomyces cerevisiae.

The substrate specificity of phosphoribosyl-aminoimidazole-succinocarboxamide-synthetase (SAICAR-synthetase, EC 6.3.2.6) of the yeast Saccharomyces cerevisiae towards a set of carboxyaminoimidazole ribotide (CAIR) analogs with modifications in the imidazole ring, ribose and phosphate moieties, as well as aspartic acid analogs has been studied. It was found, in particular, that: i) the presence of double charged phosphate group, 2'- and 3'-hydroxyl groups in the ribose fragment and of an amino group in the imidazole ring of the CAIR molecule is not the absolute requirement for the enzymatic reaction; ii) 3'-carboxy-1.2.4-triazole analog of CAIR is a competitive inhibitor of the enzyme; iii) 2'-deoxy-CAIR is a substrate for both yeast SAICAR-synthetase and its avian liver and human erythrocyte counterparts. A new method designed to determine the SAICAR-synthetase activity with the help of bifunctional enzymes possessing, in addition to the SAICAR-synthetase activity, also a phosphoribosyl-aminoimidazole-carboxylase activity, is proposed; this method is based on the use of 2'-deoxy-CAIR. Some aspartic acid analogs (L-malic acid, beta-threo-oxy-, and beta-threo-fluoro-aspartic acids and alanosine) are substrates for yeast SAICAR-synthetase. The possible involvement of malate as an alternative substrate for the SAICAR-synthetase reaction in vivo is discussed. The results of a comparative analysis of already established primary structures of yeast, bacterial, human, and chicken SAICAR-synthetases are presented.