[The phenomenon of predetermination of the cytoplasm upon interaction between alleles of the ADE2 and ADE13 in the yeast Saccharomyces cerevisiae]. / Iavlenie predeterminatsii tsitoplazmy pri vzaimodeistvii allelei genov ADE2 i ADE13 u drozhzhei Saccharomyces cerevisiae.

Our previous data showed that mutation ade13-1, blocking steps 8 and 12 of purine biosynthesis in the yeast Saccharomyces cerevisiae, caused the inability of strains manifesting this activity to grow on the complete nutrient medium with glucose in addition to the loss of adenylosuccinate lyase activity. It was also determined that the ade2-D mutation, inactivating aminoimidasole ribonucleotide carboxylase (the enzyme of step 6), suppressed this phenotypic manifestation of ade13-1; i.e., the ade2-D mutation restores the ability to grow on this medium. When spores of a hybrid that contained both mutations in the heterozygote were germinated on the YEPD medium, almost complete viability of segregants with genotypes ADE2 ADE13 and ade2-D ADE13 and the absence of ADE2 ade13-1 growth were observed. The number of growing segregants ade2-D ade13-1 amounted to approximately half of the possible number. In this work, a decrease in the proportion of segregants with this genotype was shown to occur only when they were obtained as a result of the segregation of hybrids with the normal allele (ADE2) in the heterozygote. The proportion of segregants with genotype ade2-D ade13-1 did not decrease upon segregation of hybrids similar in the genetic background and containing the ade2-D mutation in the homozygote and ade13-1 in the heterozygote. Spores with this genotype formed in the diheterozygous diploid were able to germinate on a medium containing glycerol and to further grow on a medium with glucose. The data suggest that, when a product of the normal ADE2 allele or of another gene, the synthesis of which is stimulated in the presence of this allele, enters spores with genotype ade2-D ade13-1 during meiosis, some of these spores lose their ability to grow on the medium with glucose; i.e., the ADE2 allele can be phenotypically expressed in the spores that did not contain this allele. This phenomenon is similar to the maternal effect known in some species of animals from various systematic groups.

Adenylosuccinate synthetase of the yeast Saccharomyces cerevisiae: purification and properties.

Adenylosuccinate synthetase (AS-synthetase) was purified from the yeast Saccharomyces cerevisiae. The purification procedure included chromatography on DEAE-cellulose, phosphocellulose, and heparin-agarose. The pH and temperature optima for the enzyme activity (7.0 and 35 degreesC, respectively) and also pH and thermostability of AS-synthetase were determined. The native form of the enzyme exists as a dimer. The Km values for IMP, GTP, and L-aspartate are 1.7, 0.16, and 6.7 mM, respectively. ATP cannot be used instead of substrate GTP, whereas 2'-dGTP and dd-GTP are able to substitute for GTP in the reaction. ITP also can be a substrate as an analog of GTP and as an analog of IMP. Two intermediates of purine nucleotide biosynthesis de novo, 5-amino-4-(N-succinocarboxamide)imidazole ribonucleotide (ASCIR) and 5-amino-4-carbamoyl-imidazole ribonucleotide (ACIR), inhibit AS-synthetase. Hydroxylamine and aspartate analogs also inhibit the enzyme. Effective binding requires a four-carbon-atom chain and unsubstituted amino group; the charge of the beta-carboxy group is not necessary. Comparison of primary structures and substrate specificity of yeast ASCIR- and AS-synthetases suggests independent origin of these proteins.

[New phenotypic manifestation of the ad2 mutation in Saccharomyces cerevisiae yeast--the inability to grow on a synthetic medium with glycerol and hypoxanthine]. / Novoe fenotipicheskoe proiavlenie mutatsii ade2 drozhzhei Saccharomyces cerevisiae--nesposobnost' rasti na sinteticheskoi srede s glitserinom i gipoksantinom.

The ADE2 gene of Saccharomyces cerevisiae yeast encodes aminoimidazole ribonucleotide-carboxylase (AIR-carboxylase), an enzyme catalyzing the sixth stage of purine nucleotide biosynthesis. Strains bearing the ade2 mutation are able to grow on a glucose-containing synthetic medium with the addition of adenine or hypoxanthine, which under the action of the cellular phosphoribosyltransferases are converted into adenosine monophosphate and inosine monophosphate, respectively. Our studies showed that ade2 mutants were unable to grow on a synthetic medium with glycerol and hypoxanthine. This newly described feature is not constitutively manifested, because some strains can contain suppressor mutations which restore the ability to grow on a synthetic medium with glycerol and hypoxanthine. The ade4, ade5, ade8, ade6, and ade7 mutations were found to suppress the phenotypic manifestation of the ade2 mutations via inactivation of enzymes catalyzing the first, second, third, fourth and fifth stages of purine biosynthesis, while the ade1 mutation, which inactivates enzyme of the seventh stage, lacks suppressive activity. Strains with single adenine mutations, ade4, ade5, ade8, ade6, ade7, or ade1 grow on glycerol- and hypoxanthine-containing media. Our data suggest that the new property of the ade2 mutations could be associated with the accumulation of the AIR-carbole-ribonucleotide. A mutation resulting in the requirement for serine on the medium with glycerol, but not glucose, is described.

ADE6 gene of Saccharomyces cerevisiae yeast encoding formylglycinamidine-ribonucleotide synthetase. Cloning, sequencing, and analysis.

The ADE6 gene of Saccharomyces cerevisiae yeast encoding the enzyme formylglycinamidine-ribonucleotide (FGAM)-synthetase of de novo synthesis of purine nucleotides was cloned and sequenced. The gene encodes a protein consisting of 1358 amino acids. The flanking regions of 1208 (5') and 728 bp (3') were also sequenced. The nucleotide motif TGACTC inherent to the promotor regions of other purine genes of yeast was located (-276 bp) in the 5'-region of the gene. The amino acid sequence of the yeast FGAM-synthetase was found to contain repeats (Leu430-Ala620 and Pro810-Ile1000). Repeats of similar patterns of conserved amino acids were also detected in the structure of all other FGAM-synthetases. A homology of FGAM-synthetases with certain proteins of viruses from the Herpesviridae family was found.

[Genetic control of metabolism of a mutagenic analog of 6-N-hydroxylaminopurine bases in Saccharomyces cerevisiae yeasts]. / Izuchenie geneticheskogo kontrolia metabolizma mutagennogo analoga osnovanii 6-N-gidroksilaminopurina u drozhzhei Saccharomyces cerevisiae.

Yeasts were shown to utilize 6-substituted adenine analogues as a purine source via the reutilization pathway leading to the formation of inosine monophosphate (IMP). This occurs because the ade12 strains with blocked conversion of IMP into adenosine monophosphate (AMP) cannot grow on media containing the above analogues as a sole purine source. Haploid strains with the double mutation ham1 ade2 or ham1 ade5 were also incapable of growing on a medium with 6-N-hydroxylaminopurine (HAP) as a sole purine source. However, in this case, this inability was caused by the occurrence of recessive lethal mutations rather than by a defect in purine reutilization. Yeast adenine aminohydrolase (AAH) can deaminate HAP to hypoxanthine. Adenine aminohydrolase (AAH) was uniformly active both in strains with a mutation in the HAMI gene and in strains wild-type with respect to this trait.

[Glycine amide ribonucleotide synthetase (EC 6.3.4.13)--is aminoimidazole ribonucleotide synthetase (EC 6.3.3.1) from Saccharomyces cerevisiae]. / Glitsinamidribonukleotid-sintetaza (KF 6.3.4.13)--aminoimidazolribonukleotid-sintetaza (KF 6.3.3.1) Saccharomyces cerevisiae.

The bifunctional enzyme GAR-synthetase-AIR-synthetase (E2-E5) of the yeast Saccharomyces cerevisiae has been studied. The yeast strain with overproduction of E2-E5 has been obtained. The enzyme from this strain, E2-E5, has been purified and characterized. The protein is a dimer composed of two subunits with M(r) of 87 kDa. The pH and temperature optima, pH stability and thermostability for E2 and E5 have been determined. The kinetic constants for E2 and E5 have been estimated. E2 and E5 are active only in the presence of Mg2+. E5 is a K(+)-dependent enzyme as is E5 from other sources. AMP is a competitive (to ATP) inhibitor for E5; hence, in yeast cells the purine nucleotide biosynthesis de novo is regulated at the first and fifth steps. Partial chymotryptic digestion of the purified protein gives rise to two fragments with M(r) of about 40 and 46 kDa; and E2 activity remains, while that of E5 disappears in the process.

[Mutation of ade13-1 of the yeast Saccharomyces cerevisiae leads to the absence of growth on a complete medium with glucose and epistatically interacts with mutations in other genes for purine biosynthesis]. / Mutatsiia ade13-1 drozhzhei Saccharomyces cerevisiae privodit k otsutstviiu rosta na polnoi srede s gliukozoi épistaticheski vzaimodeistvuet s mutatsiiami v drugikh genakh purinovogo biosinteza.

Saccharomyces cerevisiae gene ADE13 encodes enzyme adenylosuccinate lyase catalyzing steps 8 and 12 of the de novo purine pathway. The mutation ade13-1 induced by ethyl methanesulfonate leads to a complete loss of enzymatic activity. Stocks with this mutation cannot grow on complete organic media with glucose or fructose as a carbon source, but they can grow on media with glycerol or ethanol. The inhibiting effect of glucose is confirmed by the lack of growth on the medium with glycerol instead of glucose. Three independently obtained ade2 mutations and mutations ade1-p3, ade4, ade5, and ade5,7-33 suppress growth inhibition by glucose; i.e., they are epistatic with respect to this phenotypical expression of mutation ade13-1. The activity of adenylsuccinate lyase is not reconstituted. Mutations ade1-263, ade6, ade7-23, and ade8-21 do not manifest an epistatic effect. It was suggested that purine synthesis genes participate in the control of glycolysis or in the transduction of signal about the carbon source in the medium. The properties of ade mutations found by us were previously unknown. The results of our study contribute to the knowledge about the function of purine synthesis genes in yeasts.

A new diagnostic technique for adenylosuccinate lyase deficiency.

A convenient and simple method of diagnosing adenylosuccinate lyase (ASL) deficiency is described. This method consists of (1) isolation of SAICA riboside and S-Ado with a cation exchange resin; (2) measurement of the UV absorbance of the ammonia eluate at 270 and 250 nm; (3) calculation of the A270/A250 ratio. If the value of this ratio is less than 0.45, the patient has a normal level of ASL activity. If the value of this ratio is greater than 0.70, the patient has ASL deficiency.

[Saccharomyces cerevisiae ADE12 gene, coding for adenylosuccinate synthetase (EC 6.3.4.4). Cloning, sequencing, expression, and superproduction]. / Gen ADE12 drozhzhei Saccharomyces cerevisiae, kodiruiushchii adenilosuktsii-sintetazu (KF 6.3.4.4). Klonirovanie, sekvenirovanie, izuchenie ékspressii, superproduktsiia.

The cloning and sequencing of Saccharomyces cerevisiae ADE12 gene encoding the structure of adenylsuccinate synthetase (ASS) are reported for the first time. Comparative analysis of all known ASS sequences was carried out. Regulation of ADE12 gene expression by exogenic adenine was carried out. The properties of ASS which was isolated and purified from overproducing yeast strain were examined.

[An enzyme study of purine nucleotide biosynthesis in the plerocercoids of cestodes in the fam. Ligulidae]. / Izuchenie fermentov biosinteza purinovykh nukleotidov u plevrotserkoidov tsestod sem. Ligulidae.

By means of spectrophotometric method there was determined the activity of three enzymes of biosynthesis of purine nucleotides: amino imidazole ribonucleotide-carboxylase (AIR-carboxylase, EC 4.1.1.21), an enzyme of biosynthesis of purine nucleotides de novo in plerocercoids of Schistocephalus pungitii and Digramma interrupta; inosine monophosphate-dehydrogenase (IMPh-dehydrogenase, EC 1.2.1.14), an enzyme of salvage path, and adenylosuccinate lyase (EC 4.3.2.2), an enzyme taking part both in biosynthesis de novo and salvage in plerocercoids of Schistocephalus pungitii. The activity of AIR-carboxylase was not determined. Specific activities of adenylosuccinate lyase and IMPh-dehydrogenase amount to (1.3 +/- 0.3) x 10(-3) and (1.2 +/- 0.4) x 10(-3) mumole/min.mg protein, respectively. The activity of the three enzymes was determined in the liver of ten-spined stickleback, a host of S. pungitii plerocercoids. The question of metabolic dependence of Ligulidae plerocercoids on hosts to provide for purine bases is discussed.

[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.

[The role of histidine in regulating the synthesis of purine nucleotides in Escherichia coli cells]. / Rol' gistidina v reguliatsii sinteza purinovykh nukleotidov v kletkakh Escherichia coli.

Coordination of GTP and 5-aminoimidazole-4-carboxamide riboside 5'-phosphate pools changes was studied. The CTP pool is an important component of Escherichia coli metabolism, while AICAR 5'-phosphate being one of alarmones controls the synthesis of GTP. Main attention was paid to histidine, the biosynthesis of which is connected with formation of purine nucleotides. The expression of the histidine operon and biosynthesis of histidine are shown to change the AICAR pool and help the formation of the GTP pool. The ribosomal antibiotics streptomycin and chloramphenicol may cause the temporary deficiency of GTP eliminated by the increase of alarmone AICAR pool. The latter event is concluded to cause the increase in GTP pool independent of the means of AICAR accumulation (C1-pholatedependent restriction of metabolization or, vice versa, the stimulation in the histidine biosynthesis pathway).

[A method of diagnosing congenital adenylosuccinase enzymopathy]. / Metod diagnostiki vrozhdennoi adenilosuktsinaznoi énzimopatii.

The authors present a simple and convenient method for the diagnosis of a recently described condition, congenital adenyl succinase enzymopathy, that is characterized, among other signs, by the appearance in patients' urine of succinylaminoimidazole carboxamide riboside (SAICRs) and succine adenosine (SA), dephosphorylated (nucleoside) forms of two adenylsuccinase substrates. Both the nucleosides may be sorbed by ion exchanger when the urine is let pass through H+ cationite and then eluated by solved ammonia (earlier this was known only in respect of SAICRs). The new feature of the procedure is detection of the afore-said nucleosides in ammonia eluate from its UV spectrum. The value of the ratio between ammonia eluate absorption in 270 and 250 nm (A270/A250) was found a reliable criterion of both SAICRs and SA absence in the urine and therefore of a normal adenyl succinase level (A270/A250 ratio is 0.25-0.45 in this case) and of their presence in the urine and therefore of their presence of adenyl succinase enzymopathy (A270/A250 ratio, above 0.70, typically 0.90-1.0).

[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.

[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.

[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.