[Formation of ARS-independent miniplasmids upon transformation of yeast Pichia methanolica with DNA molecules containing "transforming" and "nontransforming" genes]. / Formirovanie ARS-nezavisimykh miniplazmid pri transformatsii drozhzhei Pichia methanolica molekulami DNK s "transformiruiushchimi" i "netransformiruiushchimi" genami.

In the study of transforming DNA molecules in cells of the methylotrophic yeast Pichia methanolica, it was found that the efficiency of transformation and the possibility of autonomous replication of hybrid plasmids and miniplasmids consisting of only transforming gene (Trg) sequences do not depend on the presence of ARS sequences with the canonical structure, such as 2 microns DNA in the yeast Saccharomyces cerevisiae. Data supported a suggestion about the existence of the relationship between gene transforming activity, its replicative ability, and transcription. The efficiency of vector activity of Trg is not sufficient to provide the transformation of other sequences of transforming DNA (tDNA). The loss of nonselected sequences of tDNA observed upon transformation is the result of rearrangements and deletions and is associated with the activity of cellular systems involved in nucleic acid metabolism. Miniplasmids similar in structure were shown to differ in their stability in independent transformants. The rate of loss of autonomously replicating plasmids strongly varied (from 1 to 99%) under nonselective conditions and nearly did not depend on plasmid structure.

[Mechanism of transformation in Pichia methanolica yeast: transforming and nontransforming genes]. / Mekhanizmy transformatsii drozhzhei Pichia methanolica: transformiruiushchie i netransformiruiushchie geny.

Two types of genes were found in the study of transformation in yeast Pichia methanolica: transforming (Trg) and nontransforming (Ntg) genes. Transforming genes (P-ADE7,4 and S-LEU2), as linear DNA molecules, can transform competent cells with high efficiency inversely proportional to the molecule size. Nontransforming genes (P-ADE5 and H-LEU2) transform P. methanolica cells at an extremely low rate even when they are combined with transforming genes. The analysis showed that linear DNA molecules with Trg and Ntg can be either rearranged and integrated in random sites of the recipient genome or form circular plasmids, which are capable of autonomous replication irrespective of the presence of specific replicative elements.

Cloning, sequencing, and functional analysis of H-OLE1 gene encoding delta9-fatty acid desaturase in Hansenula polymorpha.

H-OLE1, a gene encoding delta9-fatty acid desaturase (FAD) in Hansenula polymorpha strain CBS 1976, was isolated by hybridization based upon its homology with the P-OLE1 gene cloned earlier from a related species, Pichia angusta IFO 1475. The sequence of the H-OLE1 gene revealed high structural conservation with delta9-FADs from various organisms. A putative 451-amino acid polypeptide encoded by the gene, like all other delta9-FADs, contained two domains: an N-terminal catalytic domain containing three conserved histidine clusters, and a C-terminal cytochrome b5-like domain which has been suggested to be involved in electron transport in desaturation reactions. The whole H-OLE1 gene complemented a H. polymorpha fad1 mutation leading to a defect in delta9-FAD. However, the unsaturated fatty acid requirement that the Saccharomyces cerevisiae ole1 mutant displays was complemented by only the open reading frame of H-OLE1 driven by S. cerevisiae glyceroaldehyde-3-phosphate dehydrogenase promoter, but not by the intact H-OLE1, suggesting that the H. polymorpha delta9-FAD was compatible with the desaturation system of S. cerevisiae whereas the promoter of the H-OLE1 gene had no activity in heterologous cells. It was shown by Northern hybridization that transcription of the H-OLE1 gene in H. polymorpha was slightly repressed by exogenous delta9-unsaturated fatty acid. An H. polymorpha disruption mutant (deltaH-OLE1) was created by transformation of an fad1/FAD1 diploid with disrupted H-OLE1::S-LEU2 linear DNA. It was shown by genetic and molecular analyses that input DNA was integrated in several copies into the chromosomal target to replace the mutated fad1 allele. Gas chromatography analysis showed identical fatty acid compositions in cells of both fad1 and deltaHOLE1 disruption mutants.

Constitutive biosynthesis and localization of alcohol oxidase in the ethanol-insensitive catabolite repression mutant ecr1 of the yeast Pichia methanolica.

The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization. Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates. No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media. Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm3)during isopicnic centrifugation of the organelles in sucrose density gradients. The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P. methanolica may not require any regulatory signals induced by methanol.

[Genetic control of purine biosynthesis in Pichia methanolica yeast. The ADE5 (PUR4) gene, controlling 5'-phosphoribosylformyl glycineamide synthetase]. / Geneticheskii kontrol' biosinteza purinov u drozhzhei Pichi methanolica. Gen ADE5 (PUR4), kontroliruiushchii 5'-fosforibozilformilglitsinamidsintetazu.

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system "from red to white," it was shown that the PUR4 gene encoding 5'-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeast Saccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe (the ade3 gene), and Pichia methanolica (the ADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5sU alleles (mutations with a high reversion frequency ranging from 0.2 x 10(-6) to 2 x 10(-6)) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for the P. methanolica ade5 mutation and probably is the structural P-ADE5 gene, was cloned from a genomic library of P. methanolica by the ade6 mutation complementation in the recipient S. cerevisiae strain.

[Genetic control of purine biosynthesis in the yeast Pichia methanolica. "Roman's effect" in red adenine-dependent pur6 and pur7 mutants]. / [Geneticheskii kontrol' biosinteza purinov u drozhzhei Pichia methanolica. "Effekt Romana" u krasnykh adeninzavisimykh mutantov pur6 i pur7]

Most ade1 and ade2 mutants of the yeast Pichia methanolica generate white and pink secondary colonies (SCs) on the surface of red colonies in complete medium. The formation of SCs was shown to be caused by "Roman's effect" described in Saccharomyces cerevisiae. This effect is known to result from the preferential growth in a colony of spontaneous mutations for genes controlling the first five steps of purine biosynthesis. It has been found that the ADE3, ADE5, ADE4, 7, and ADE8 loci correspond to these genes. In studies on twelve red adenine-dependent mutants, the expression of Roman's effect was shown to vary in different cultures, dependent on the original mutant ade1 and ade2 alleles. It was assumed that spontaneous mutability of each gene of purine biosynthesis ADE ade does not depend on mutations in other genes of this biosynthetic pathway. On the basis of this assumption and data from genetic analysis of mutants, it is concluded that the ade1 and ade2 mutant alleles do not determine the mutability level but affect the level of selective advantage of double mutants. This conclusion was used to estimate the lowest frequencies of spontaneous mutability of early purine genes in P. methanolica (with respect to the identified mutants). As calculated for one red mutant, frequencies of spontaneous mutations in the most mutable ADE5 gene and in the least mutable ADE8 gene of this group were 2.5 x x 10(-8) and 0.4 x 10(-8), respectively.

[Development of a method for vector transformation of the methyltrophic yeast Pichia methanolica]. / Razrabotka metoda vektornoi transformatsii metilotrofnykh drozhzhei Pichia methanolica.

A method for transformation of the methylotrophic yeast Pichia methanolica (formerly P. pinus MH4) was developed. Mutants leu1 were shown to be transformed with different efficiency using 2.2-10.7-kb linear and circular DNA molecules containing the LEU2 gene of the yeast Saccharomyces cerevisiae, which complemented the leu1 mutation of the recipient. Efficiency of transformation with short molecules was higher than with long molecules. Transformation with linear DNA was more efficient than with circular DNA of equal size. Transformants contained both replication-unstable and integration forms in different proportions. Significant rearrangements in the episome and integration forms of transforming DNA were found in transformants obtained using the 10.7-kb circular YEp13 plasmid. Integration of linear DNA was not accompanied by rearrangements in DNA molecules. Certain clones isolated after transformation with linear DNA contained autonomously replicating circular vector molecules formed as a result of a ligase reaction in vivo. The LEU2 gene of S. cerevisiae contains an unknown sequence acting as the ARS replicon in cells of both P. methanolica and of another species of the methylotrophic yeasts, Hansenula polymorpha.

Divergence and conservation of SUP2 (SUP35) gene of yeast Pichia pinus and Saccharomyces cerevisiae.

SUP2 (SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part and a region homologous to EF-1 alpha. The comparison of amino acid sequences of the Sup2 proteins reveals high conservation (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae were found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.

[Comparative analysis of the structure of SUP2 genes in Pichia pinus and Saccharomyces cerevisiae]. / Sravnitel'nyi analiz struktury genov SUP2 drozhzhei Pichia pinus i Saccharomyces cerevisiae.

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. A SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of temperature-sensitive sup2 mutation of S. cerevisiae. Nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa exceeding the SUP2 protein of S. cerevisiae for 6 kDa. The SUP2 gene product of P. pinus is similar to the Sup2 protein of S. cerevisiae by its structure and includes a highly conservative (76%) C-terminal region homologus to EF-1 alpha and a lowly conservative N-terminal region. The relation between the evolutionary conservativity of different regions of the Sup2 protein and their functional significance is discussed.

[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.

["Illegal" transformation of red adenine-dependent mutants of the yeast Pichia pinus by the pYE(ADE2)2 plasmid]. / "Nezakonnaia" transformatsiia krasnykh adeninzavisimykh mutantov drozhzhei Pichia pinus plazmidoi pYE(ADE2)2.

The red adenine-dependent mutants ade1 of the yeast Pichia pinus blocked in the VI step of adenine biosynthesis (lack of AIR-carboxylase) and ade2 mutants blocked in the VII step of adenine biosynthesis (lack of SAIKAR-synthase) were transformed with the plasmid pYE(ADE2)2 containing ADE2 gene of Saccharomyces cerevisiae encoding AIR-carboxylase. The appearance of white Ade+ clones with the frequency 2-7.10(-8) (which is ten-fold higher than reversion frequency) was only observed in the case of ade2 transformation. Genetic analysis points to connection of the "illegitimate" transformants' appearance with the change in the mutant ade2 locus or in a locus closely linked to the former. Ade+ phenotype was stable during 20 generations of mitotic budding. Southern blotting assay of transformant chromosomal DNA indicates that reconstitution of ade2 defective gene is related with its "correction", owing to integration of pYE(ADE2)2 sequence in the vicinity of the mutant locus.

[Expression of human leukocyte interferon A gene in Saccharomyces cerevisiae under the control of regulatory yeast elements PHO5, GAL1 and GAL10]. / Ekspressiia gena leikotsitarnogo interferona A cheloveka v drozhzhakh Saccharomyces cerevisiae pod kontrolem reguliatornykh élementov drozhzhevykh genov PHO5, GAL1 i GAL10.

A chromosomal gene for human leucocyte interferon A is expressed in Saccharomyces cerevisiae yeasts due to interaction of 5'-nontranslating region of the cloned interferon gene with the regulatory elements of yeast genes PHO5, GAL1 and GAL10. Regulated systhesis of interferon was obtained in all cases. The level of interferon genes expression in case using GAL1 and GAL10 genes regulatory elements (5 X 10(5) and 5 X 10(6) u X l-1) correlated with the distances to their promoters. The highest yield of interferon (10(8) u X l-1) was obtained when the PHO5 gene regulatory elements were used.

[Expression of human leukocyte interferon type A in Saccharomyces cerevisiae under the control of regulatory elements of the yeast gene URA3]. / Ekspressiia gena leikotsitarnogo interferona A cheloveka v drozhzhakh Saccharomyces cerevisiae pod kontrolem reguliatornykh élementov drozhzhevogo gena URA3.

Expression of a chromosomal gene for human leucocyte interferon A was obtained due to interaction of 5'-nontranslating region of a cloned interferon gene with the regulatory sequences from UNA3 yeast gene. The sequence of 5'-nontranslating region from interferon gene essential for its expression in yeast is localized within 130 b p. from the initiating codon. Due to increasing of plasmid stability and copy number a 60-fold increase. in interferon yield was obtained in yeast transformants reaching the level of 6 X 10(7) u X 1(-1). The data are presented supposing the existence of functional polycistronic mRNA in yeast.

[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.

[A new vector for cloning of genes and replicons in yeasts]. / Novyi vektor dlia klonirovaniia genov i replikonov v drozhzhakh.

A novel Escherichia coli-Saccharomyces cerevisiae shuttle vector lambda MAN78 has been constructed. The vector contains phage lambda 47.1 DNA, Sacch. cerevisiae chromosomal segment with TRP1 gene and the yeast ARS1 replicator. This vector may be propagated as a phage and, similar to parental lambda 47.1, allows direct selection of large DNA inserts (15-24 kbp) in E. coli. lambda MAN78 can efficiently transform LiCl-treated yeast cells (3-5 X 10(3) transformants per 1 microgram DNA). Replication of hybrid molecules in E. coli cells does not influence the ability of the molecules to transform yeast cells and replicate in the cells.

Genetic control of cell type and complex organization of the mating type locus in the yeast Pichia pinus.

Genetic mechanisms of switching the mating type locus MAT1 in the homothallic yeast Pichia pinus were studied. By analysis of mutations affecting MAT1 complex structural and functional organization of this locus was shown. The existence of two functional regions in MAT1 is postulated. Region I controls mating ability of haploid cells, determines the neutrality of heterozygous cells and regulates the work of Region II. Region II controls meiosis and/or sporulation in the cultures heterozygous for Region I as well as controls switching MAT1 ⇋ MAT1α in haploid cells.

[Comparative study of the protein makeup in diploid and haploid forms of Saccharomyces and Pichia]. / Sravnitel'noe izuchenie sostava belkov u diploidnykh i gaploidnykh form Saccharomyces i Pichia.

The rates of growth, biomass accumulation, and electrophoretic spectra of mobile cytoplasmic proteins were studied with nonisogenous haploid and diploid cultures of Saccharomyces cerevisiae and Pichia guilliermondii as well as with isogenous haploid-diploid pairs of Saccharomyces cerevisiae and Pichia pinus. On a mineral medium with glucose, differences in these parameters in various yeast strains were found to be due to the genotype of a strain rather than to ploidy: nonisogenous haploid and diploid cultures displayed considerable and random variability of these properties while no differences were found in isogenous haploid-diploid pairs. Studies on solubility of protein fractions in various solvents made it possible to reveal differences connected with ploidy, namely: both in nonisogenous and isogenous haploid-diploid systems, the content of the water-soluble fraction decreased in diploid cultures.