The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control.

The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation. The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5'-noncoding region and 292-bp 3'-noncoding region. The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues. The promoter of the ADE1 gene contains a copy of the 5'-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control. Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions. An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein. One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.

[Development of a system of intragenic mapping for molecular genetic analysis of mutations in the gene LYS2 of Saccharomyces yeasts]. / Razrabotka sistemy vnutrigennogo kartirovaniia dlia molekuliarno- geneticheskogo analiza mutatsii v gene LYS2 u drozhzhei sakharomitsetov.

The collection of overlapping lys2 deletions (five in the chromosomal and seven in the plasmid LYS2 gene) is constructed in this work. The deletions overlap the whole coding region of the gene and provide the system for intragenic recombinational mapping of lys2 mutations in one of 14 controlled regions. A portion of these regions can be correlated with the regions on the physical map of LYS2. Mutations in two regions can be easily cloned. The system constructed gives the possibility for the study of intragenic and molecular specificity of mutagenesis.

Precise excision of bacterial transposon Tn5 in yeast.

We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a lambda::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle episomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion. All insertions inactivated the LYS2 gene and were able to revert with low (about 10(-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.

[Cloning of the gene of Saccharomyces cerevisiae yeasts that determines resistance to the toxic action of cadmium ions]. / Klonirovanie gena drozhzhei Saccharomyces cerevisiae, opredeliaiushchego rezistentnost' k toksicheskomu deistviiu ionov kadmiia.

A gene conferring resistance to cadmium in Saccharomyces cerevisiae was isolated from a yeast gene library created on the basis of the pL3 vector. The phenotype of resistance is only expressed in the yeast cells with cloned DNA inserted into a multicopy plasmid. Integration of the plasmid into chromosome or introduction of the centromeric region into the plasmid decreases the level of cadmium resistance. The cloned Sau3A I fragment of the yeast chromosome is 3.5 kbp in size. Restriction analysis and subcloning experiments showed the gene to be located within 1.6 kbp of the XhoI-Sau3A I fragment of DNA. Instability was observed in the vicinity of the XhoI-Sau3A I fragment of the yeast DNA in Escherichia coli.

[Site of integration of IS1-element responsible for expression of the yeast ADE1 gene in Escherichia coli]. / Issledovanie mesta integratsii IS1-élementa, obespechivaiushchego ékspressiiu gena ADE1 drozhzhei v kishechnoi palochke.

Cloned yeast ADE1 gene is expressed in Escherichia coli, due to integration of the IS1 bacterial element into the non-coding 5' region. Primary structure analysis of the integration site of IS1 element demonstrated that this site is situated in a region flanked by two inverted repeats of yeast DNA which are homologous to the right end of the IS1 element. The region up-stream (5') to the left inverted repeat is AT-rich.

[Cloning of the ADE2 gene of Saccharomyces cerevisiae and localization of the ARS-sequence]. / Klonirovanie ADE2-gena Saccharomyces cerevisiae i lokalizatsiia ARS-posledovatel'nosti.

Mutational changes in ADE2 result in the accumulation of red pigment in cells, which serves as an indicator for the selection of mutants. This easily detectable phenotype of red-coloured colonies can account for the wide use of ade2 mutants in yeast genetics. ADE2 gene was cloned in a shuttle vector by complementing the ade2 mutation in the yeast. It was shown that the 2.2 kbp HindIII fragment of yeast DNA contains structural sequences of the ADE2 gene as well as the ARS sequence. Deletion analysis of the 5' end of the ADE2 gene showed the ARS sequence to be situated at the distal end of the 1 kbp HindIII fragment. Removal of the ARS sequence does not influence ADE2 gene complementation ability. Transformants containing the ADE2 gene comprised in their plasmids form white colonies. Loss of the plasmids results in colour change of colonies.

[Nucleotide sequence of the ADE 1 gene of the yeast Saccharomyces cerevisiae]. / Nukleotidnaia posledovatel'nost' gena ADE 1 drozhzhei Saccharomyces cerevisiae.

The yeast ADE 1 gene has been cloned and sequenced. The primary structure deduced from the nucleotide sequence demonstrated that phosphoribosylaminoimidazole-succinocarboxamide synthetase is a protein with molecular weight of 34 500 D.

[Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae]. / Klonirovanie i fizicheskoe kartirovanie gena ADE1 drozhzhei Saccharomyces cerevisiae.

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.

[Ontogenetic switchover in Bacillus subtilis. II. The dynamics of the stationary phase processes during growth under conditions of catabolite repression]. / Izuchenie ontogeneticheskogo perekliucheniia u Bacillus subtilis. Soobshchenie II.Dinamika protsessov statsionarnoi fazy pri roste v usloviiakh katabolitnoi repressii.

To reveal the pecularities of the growth under the conditions of catabolite repression (medium 2) of Bacillus subtilis and the mutants obtained, the investigations of dynamics of the following processes were carried out: alteration of the pH of the culture exhaustion of glucose in the medium, appearance of the activity of both aconitase in the cells and extracellular metal- and serine proteases in the supernatant, and the appearance of the thermoresistant spores. The following features were observed during the growth under the conditions of catabolite repression: 1. Bacillus subtilis WB 746 and cgs mutants: the death of the main part of the culture after the Iogarithmic phase of growth (LPG), the presence of the secondary LPG of the survived cells which have the increasing activity of aconitase, the appearance and sharp increase in the extracellular serine protease activity 6 hours before thermoresistant spore formation. In the case of cgs mutants the activity of metal proteases appears and increases during the secondary LPG; 2. In the culture of cgl mutants the pH is lowered to 5.1 at the end of the LPG and after the glucose exhaustion the death of almost all the culture follows; 3. cgr mutants: a comparatively high activity of aconitase in the cells is found by the time of the early LPG, and at the end of the LPG the activity of both metal- and serine proteases appear in the supernatant of the culture and the secondary induction of the serine protease activity 6 hours before thermoresistant spore formation is observed. The serine protease activity found in the supernatant before and after the secondary induction of the enzyme belongs to the identical protein. During the stationary phase of the growth of cgr mutants, the high rate of 3H-uridine incorporation into the RNA molecules which have the electrophoretic mobility of mRNA was observed. The sporulation of Bac. subtilis strains under investigation, except cgl mutants, occurs when the culture has reached the definite state: the alkaline pH, the presence of the aconitase activity in the cells and the induced activity of serine protease.