[Using adaptive mutagenesis system for identification of early genes encoding de novo purine biosynthesis in methylotrophic yeast Pichia methanolica MH4].

A collection of spontaneous "Roman's mutants" (1654 mutants) for early genes of purine biosynthesis PUR1-PUR5 was obtained from 16 parental ade1(pur6) and ade2(pur7) strains of the methylotrophic yeast Pichia methanolica. Two genes, bifunctiional ADE7,4(PUR2,5) and ADES(PUR4), were identified earlier. For identification of the two remaining early genes (ADE3 and ADE8), a novel approach was used: a comparison of spectra of spontaneous Roman's mutants and relative sizes of genes (with regard to the length of polypeptides in amino acid residues). Significant correlation between relative sizes of genes and a proportion of mutants in the spectrum was shown in yeast Saccharomyces cerevisiae (according to analysis of data from the literature). Data on average polypeptide product lengths of genes encoding the first five steps of purine biosynthesis in 22 species of lower eucaryotes were obtained using the PubMed database. A correlation between the spectrum of spontaneous Roman's mutants and relative gene sizes was also shown experimentally in P. methanolica yeast. On the strength of this evidence, the gene ADE3 was identified as PUR1 and gene ADE8 as PUR3. Spontaneous mutagenesis producing Roman's mutants most probably is the result of transcription activity of early genes, which essentially agrees with experimental data on the correlation between the mutant spectrum and gene sizes. It was supposed that the adaptive mutagenesis, i.e., an enhancement of early genes transcription activity in response to stress, the accumulation of toxic metabolites (AIR or CAIR) as a result of the block of 6-7 stages of purine biosynthesis underlies the Roman's effect.

[The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica].

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.

[Structural, functional and genetic aspects of peroxisome biogenesis].

Intracellular organelles, peroxisomes, occur in cells of most eukaryotic species. Human severe congenital disorders are associated with defective assembly and functioning of peroxisomes, which partly explains the attention of researchers paid to peroxisome biogenesis. It has been shown that peroxisomes are involved in the realization of eukaryotic developmental programs (in particular, neuroblast differentiation and postembryonic development). Cytobiochemical and electron-microscopic studies of mutations involving peroxisomes showed that the primary role in peroxisome biogenesis is played by synthesis of proteins (peroxins) and their transport and incorporation into peroxisome membranes. More than 30 peroxin-encoding genes have been examined. These genes are synthesized on free polysomes and transported into peroxisomes by means of specific signaling peptides, PTS1, PTS2, and PTS3. The import of matrix proteins depends on at least two shuttle receptor proteins, Pex5p and Pex7p. Some proteins regulating peroxisome proliferation in cells have been identified.

[Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis]. / Izoliatsiia i pervichnaia kharakteristika mutantov po biogenezu peroksisom u metilitrofnykh drozhzhei Hansenula polymorpha.

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.

[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.

["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.

[Ploidy and liquid-holding recovery in yeasts sensitive to radiation and nitrous acid]. / Ploidnost' i vosstanovlenie v bufere zhiznesposobnosti drozhzhei, chuvstvitel'nykh k radiatsii i azotistoi kislote

The effects of genome ploidy and posttreatment incubation on inactivation by nitrous acid (NA) were studied in normal, radio- and nitrous acid-sensitive strains of yeast. In normal yeast cells the increase of ploidy (haploid to triploid) resulted in "the protective effect", i.e. haploid cells were the most sensitive, triploid -- the most resistant. This "protective effect" is absent in polyploid yeast homozygous for the xrs1-5 (rad 54) mutation; in this case the NA-sensitivity rises with the increase of ploidy, i.e. haploid cells are the most resistant ones. The effect of liquid holding (LH) after the NA treatment depends on the genetic background and ploidy of treated cells. Posttreatment incubation in buffer has practically no effect on the survival of wild-type Berkeley's yeast strains (1n, 2n, 3n). The highly homozygous haploid strains from Zakharov' collection, both wild type and xrs1-5 mutant, exhibit no LH-recovery too. However the death of wild-type cells drastically rises in LH-condition as the ploidy increases. 24 hours incubation in buffer results in at least a ten-fold decrease in survival of wild type 2n, 3n, 4n cells. The loss of viability is proportional to the time of incubation, but the cell titer being constant. The strains (2n, 3n, 4n) homozygous for the xrs1-5 mutation (rad 54) show considerable LH-recovery. It is supposed that the xrs1-5 mutation results in the derepression of the prereplicative pathway of LH-recovery which eliminates the NA-INDUCED DAMAGE OF DNA.