Structural and functional characteristics of various forms of red pigment of yeast Saccharomyces cerevisiae and its synthetic analog

Structural and functional characteristics of the yeast red pigment (product of polymerization of N¹-(β-D-ribofuranosyl)-5-aminoimidazole), isolated from ade1 mutant cells of Saccharomyces cerevisiae and its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N¹-methyl-5-aminoimidazole in vitro) were 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. All the studied compounds are 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 the presence of these compounds—they are merged into conglomerates more stable and resistant to the effects of ultrasound than are insulin aggregates grown without pigments. We suggest that all these compounds can cause coalescence of fibrils partially blocking the loose ends and, thereby, inhibit attachment of monomers and formation of new fibrils.     

Identification of mutations in the asporogenic yeast Candida tropicalis using intrageneric fusion of protoplasts

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.     

Genetic control of metabolism of mutagenic purine base analogs 6-hydroxylaminopurine and 2-amino-6-hydroxylaminopurine in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We studied the effect of inactivation of genes, which control biosynthesis of inosine monophosphate (IMP) de novo and purine salvage and interconversion pathways, on sensitivity of yeast Saccharomyces cerevisiae to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA). It was shown that the manifestation of HAP and AHA mutagenic properties depends on the action of enzyme adenine phosphoribosyltransferase encoded in yeast by APT1 gene. A blockade of any step 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 block 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 possible that this enhancement of sensitivity to HAP and AHA is due to changes in the pool of purines. We conclude that genes ADE12, ADE13, AAH1, and HAM1 controlling processes of purine salvage and interconversion in yeast, make the greatest contribution to the protection against the toxic and mutagenic action of the examined analogs. Possible mechanisms of HAP detoxication in bacteria, yeast, and humans are discussed.     

Partial Inactivation of Translation Termination Factors Causes Suppression of Frameshift Mutations in the Yeast Saccharomyces cerevisiae

Special search for frameshift mutations, which are suppressed by the cytoplasmic [PSI] factor and by omnipotent nonsense suppressors (recessive mutations in theSUP35and SUP45genes), partially inactivating a translation termination complex, was initiated in theLYS2gene in the yeast Saccharomyces cerevisiae.Mutations were obtained after exposure to UV light and treatment with a mixture 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 was 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 first shown to occur in the presence of the [PSI] factor (i.e., due to the prionization of the translation release factor eRF3) and as a result of mutations in genes SUP35orSUP45, 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.     

Transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts by a plasmid carrying the ADE2 gene of 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.     

Suppression of frameshift mutation as a result of partial inactivation of translation termination factors in Saccharomyces cerevisiae yeast]

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

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.     

Isolation and properties of phosphoribosyl-aminoimidazole-succinocarboxyamide-synthestase from Saccharomyces cerevisiae yeasts

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.