[Molecular Polymorphism of ß-Galactosidase LAC4 Genes in Dairy and Natural Strains of Kluyveromyces Yeasts].

The ability to ferment lactose is a characteristic peculiarity of dairy Kluyveromyces lactis yeasts; the vast majority of other yeast species are not able to assimilate this disaccharide. Molecular polymorphism of LAC4 genes encoding ß-galactosidase controlling lactose fermentation is not well studied, and the published data concern only a single strain (K. lactis var. lactis NRRL Y-1140) isolated from cream in the United States. We studied ß-galactosidase genes in lactose-fermenting К lactis strains isolated from dairy products and natural sources in different regions of the world using molecular karyotyping, Southern hybridization, and sequencing. It was established that the ability to ferment lactose in К. lactis var. lactis dairy yeasts is controlled by at least three polymeric LAC loci with different chromosomal localization: LAC1 (chromosome III), LAC2 (II), and LAC3 (IV). Most of the strains we studied had the LAC2 locus. A comparative analysis of ß-galactosidases of the Kluyveromyces genus yeasts and these enzymes from other yeasts was conducted for the first time. Phylogenetic analysis detected significant differences between the LAC4 proteins of yeasts of the Kluyveromyces genus (K. lactis, К. marxianus, К. aestuarii, К. nonfermentans, К. wickerhamii), Scheffersomyces stipitis, Sugiyamaella lignohabitans, and Debaryomyces hansenii. A correlation between ß-galactosidase sequences and ecological origin (dairy products and natural sources) of Kluyveromyces strains was found. The group of dairy strains is heterogeneous and includes К. lactis var. lactis and К. marxianus yeasts (99.80-100% similarity), which indicates a common origin of their LAC4 genes. Phylogenetic analysis of ß-galactosidases indicates a close genetic relationship of dairy and hospital strains of К. lactis var. lactis and К. marxianus. Clinical isolates are able to ferment lactose and appear to originate from the dairy yeasts.

[Taxonomic Genetics of Methylotrophic Yeast Genus Komagataella: New Biological Species K. kurtzmanii].

Genetic hybridization analysis revealed that industrially important species Komagataella kurtzmanii has re productive postzygotic isolation from K. pastoris, K. populi, K. phaffii, K. pseudopastoris, and K. ulmi. Therefore, it represents a new biological species of the genus Komagataella. The genetic data are in perfect agree ment with the molecular taxonomy of the genus Komagataella.

New family of pectinase genes PGU1b-PGU3b of the pectinolytic yeast Saccharomyces bayanus var. uvarum.

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.

[Molecular polymorphism of IB-fructosidase SUC genes in the yeast].

SUC gees encoding bea-fructosidase has been investigated in the yeast genus Saccharomyces. We have determined nucleotide sequences of subtelomeric SUC3, SUCS, SUC7, SUC8, SUC9, S UCO10 genes of S. cerevisiae and SUCa gene o:f S. arboricola. Comparisns of nucleotid sequences of all known SUCgenes revealed predominance of transitions C-->T in the third codon position, Which are silent. The amilioacids sequences of p-fructosidases studied have identity of 88-100%.4'Most divergent are SUCa (S.'arboricola) and SUCb (S. bayanus), having amino acid identity.with the other SUCproteins less than 92%. It wasdetermined that accumulation of the polymeric SUC genes takes place in industrial populations of S. cerevisiae, while the other Saccharomyces species (S. arboricola, S.:bayanus, S. cariocanuis, kudriavzevii, S. mikatae and S. paradoxus) each harbor only one SUCgene. Subtelomeric repeats of pructosidase SUCgenes cobetald appear in the genome of S. cerevisiae under the effect of selection in the course of their domestication.

[Comparative genetics of yeast Saccharomyces cerevisiae: chromosomal translocations carrying the SUC2 marker].

Three different translocations involving chromosome IX have been detected in natural Saccharomyces cerevisiae strains using pulsed-field gel electrophoresis with intact chromosomal DNA and their hybridization with the SUC2 probe. Hybrids of these strains with genetic lines having normal molecular karyotype were shown to have back dislocation of at least marker SUC2 due to crossingover. The significance of the detected translocations is discussed.

[Comparative genetics of yeasts: a novel beta-fructosidase gene SUC8 in Saccharomyces cerevisiae].

Molecular and genetic analyses revealed that the distillers race XII, which is an ancestor of Saccharomyces cerevisiae Peterhof and Gatchina genetic lines, has three polymeric beta-fructosidase genes: SUC2, SUC5, and SUC8. The latter gene located on the X chromosome was identitied in this work for the first time. The presence of the single SUC2 gene in yeasts used in the international project on sequencing of the S. cerevisiae genome is discussed.

[Taxonomic genetics of Zygowilliopsis yeasts].

Genetic hybridization analysis was conducted with 16 natural Zygowilliopsis strains isolated in different geographical regions and maintained in collections under species names Z. californica, Hansenula dimennae, and Pichia populi. Genetic relatedness was determined on the basis of mating, viability of hybrid progeny, and meiotic recombination of markers. Four new biological species are recognized in the former monotypic genus Zygowilliopsis. Species Z. californica and Zygowilliopsis sp. 3 probably include divergent geographical populations. It is necessary to reconsider the species composition of the genus Zygowilliopsis and generic assignment of P. populi yeasts. Genetic and molecular identifications of the Zygowilliopsis species are in perfect agreement.

[Sonographic characteristics of non-traumatic focal hourglass-like nerve constriction]. / Sonograficheskie kharakteristiki netravmaticheskoi fokal'noi konstriktsii nerva po tipu 'pesochnykh chasov'.

AIM: To describe the sonographic phenomenon of the focal 'hourglass-like constriction' of the peripheral nerves (FCPN). MATERIAL AND METHODS: The authors described 7 patients meeting the criteria for the diagnosis of neuralgic amyotrophy with unilateral FCPN identified with ultrasound in 4 cases and detected intraoperatively in 3 cases (preliminary ultrasound was not performed). The US scanner Sonoscape Pro mode gray scale in the transverse and longitudinal scanning, linear probe 8-15 MHz and Logiq9 scanner with elastography were used. RESULTS: FCPN was detected in the single nerve in 4 patients, in two nerves in 2 patients and in 3 nerves in one patient. Among all the nerves involved in the pathological process, the radial nerve and its branches were affected in 73% (8 nerves); the ulnar nerve was involved in 18% (2 nerves) and the musculo-cutaneous nerve in 9%. The length of the constriction of the peripheral nerve did not exceed 1.7 mm. The deformation coefficient (DC) of constriction area was 3.8 to .,9; the change in the elasticity in the form of an increase of DC to 5.9 when compared to the intact portion of the nerve and a decrease in echogenicity were observed in one patient. CONCLUSION: High-resolution ultrasound of the nerve can be an informative method for the diagnosis of idiopathic non-traumatic FCPN mononeuropathy.

[The use of hybridization in breeding of eukaryotic microorganisms].

The article deals with the genetic bases of breeding of eukaryotic microorganisms. Using the data on the Saccharomyces yeasts, application of different genetic approaches and methods to breeding is discussed, including interstrain, interlinear, and distant interspecific hybridization, as well as heterosis, polyploidy, cytoduction, and meiotic recombination.

Genetic variation of the repeated MAL loci in natural populations of Saccharomyces cerevisiae and Saccharomyces paradoxus.

In Saccharomyces cerevisiae, the gene functions required to ferment the disaccharide maltose are encoded by the MAL loci. Any one of five highly sequence homologous MAL loci identified in various S. cerevisiae strains (called MAL1, 2, 3, 4 and 6) is sufficient to ferment maltose. Each is a complex of three genes encoding maltose permease, maltase and a transcription activator. This family of loci maps to telomere-linked positions on different chromosomes and most natural strains contain more than one MAL locus. A number of naturally occurring, mutant alleles of MAL1 and MAL3 have been characterized which lack one or more of the gene functions encoded by the fully functional MAL loci. Loss of these gene functions appears to have resulted from mutation and/or rearrangement within the locus. Studies to date concentrated on the standard maltose fermenting strains of S. cerevisiae available from the Berkeley Yeast Stock Center collection. In this report we extend our genetic analysis of the MAL loci to a number of maltose fermenting and nonfermenting natural strains of S. cerevisiae and Saccharomyces paradoxus. No new MAL loci were discovered but several new mutant alleles of MAL1 were identified. The evolution of this gene family is discussed.

The chromosome end in yeast: its mosaic nature and influence on recombinational dynamics.

Yeast chromosome ends are composed of several different repeated elements. Among six clones of chromosome ends from two strains of Saccharomyces cerevisiae, at least seven different repeated sequence families were found. These included the previously identified Y' and X elements. Some families are highly variable in copy number and location between strains of S. cerevisiae, while other elements appear constant in copy number and location. Three repeated sequence elements are specific to S. cerevisiae and are not found in its evolutionarily close relative, Saccharomyces paradoxus. Two other repeated sequences are found in both S. cerevisiae and S. paradoxus. None of those described here is found (by low stringency DNA hybridization) in the next closest species, Saccharomyces bayanus. The loosely characterized X element is now more precisely defined. X is a composite of at least four small (ca. 45-140 bp) sequences found at some, but not all, ends. There is also a potential "core" X element of approximately 560 bp which may be found at all ends. Distal to X, only one of six clones had (TG1-3)n telomere sequence at the junction between X and Y'. The presence of these internal (TG1-3)n sequences correlates with the ability of a single Y' to expand into a tandem array of Y's by unequal sister chromatid exchange. The presence of shared repeated elements proximal to the X region can override the strong preference of Y's to recombine ectopically with other Y's of the same size class. The chromosome ends in yeast are evolutionarily dynamic in terms of subtelomeric repeat structure and variability.

[Comparative molecular-genetic analysis of the beta-fructosidases in yeast Saccharomyces].

To infer the molecular evolution of polymeric beta-fructosidase SUC genes of the yeast Saccharomyces, we have cloned and sequenced a new SUC gene from S. cariocanus and determined the sequence similarity of beta-fructosidases within the genus Saccharomyces. The proteins of Saccharomyces cerevisiae and its five sibling species (S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, S. paradoxus) have high degree of identity - 90-97%. The invertase of S. bayanus is the most divergent among the proteins studied. The data obtained indicated that the yeast invertases are highly conservative. In the coding regions of the SUC genes the pyrimidine transitions were the most abundant event due to silent changes mainly in the third codon position. There is only one, probably, non-telomeric SUC gene in each of the Saccharomyces species. In S. cerevisiae, S. bayanus, S. kudriavzevii, S. mikatae and S. paradoxus the SUC gene have been mapped on chromosome IX, whereas in S. cariocanus this gene is located in chromosone XV, in the position of translocation.

[Molecular polymorphism of viral dsRNA of yeast Saccharomyces paradoxus].

An analysis of 53 strains of yeast Saccharomyces paradoxus (YSP) of different geographic origins enabled us, for the first time, to find viral double-stranded RNA (L and M fractions) in YSP and to study natural polymorphism. As in the cultured Scerevisiae, the size of L dsRNA was constant (4.5 kb). The size of minor M dsRNA varied from 1.5 to 2.4 k.b. In YSP, we determined 7 types of M dsRNA (M1-M7), which were not connected with the source of isolation or geographic origin of the host strains.

[Genetic differentiation of the sherry yeasts Saccharomyces cerevisiae].

Molecular genetic study of the yeast Saccharomyces cerevisiae isolated at various stages of sherry making (young wine, solera, and criadera) in various winemaking regions of Spain demonstrated that sherry yeasts diverged from the primary winemaking yeasts according to several physiological and molecular markers. All sherry strains independently of the place and time of their isolation carry a 24-bp deletion in the ITS 1 region of ribosomal DNA, whereas the yeasts of the primary winemaking lack this deletion. Molecular karyotypes of the sherry yeast from different populations were found very similar.

[Invertase Overproduction May Provide for Inulin Fermentation by Selection Strains of Saccharomyces cerevisiae].

In some recent publications, the ability of selection strains of Saccharomyces cerevisiae to ferment inulin was attributed to inulinase activity. The review summarizes the literature data indicating that overproduction of invertase, an enzyme common to S. cerevisiae, may be responsible for this phenomenon.

[Towards Reinstatement of the Yeast Genus Zygowilliopsis Kudriavzev (1960)].

Experimental data on genetic molecular classification and identification of the yeast genus Zygowilliopsis are summarized. The genus is represented by at least five biological species and three varieties: Z. californica: var. californica, var. dimennae, and var. fukushimae. Biogeography, ecology and killer activity of Z. californica yeasts is considered. Heterogeneity of the taxonomic genus Barnettozyma Kurtzman et al. (2008) and the necessity for its revision are discussed.

[Hybrid Sterility of the Yeast Schizosaccharomyces pombe: Genetic Genus and Many Species in statu nascendi?].

A phenomenon of ascospore death was observed in a number of Schizosaccharomyces pombe interstrain hybrids. Meiotic recombination of the control parental auxotrophic markers was, however, observed in a random ascospore analysis. Genetic and molecular biological data indicated existence of at least geographical divergence of the genomes in Sch. pombe populations. Classification of the genus, species, and varieties of these yeasts is discussed.

A new family of polymorphic metallothionein-encoding genes MTH1 (CUP1) and MTH2 in Saccharomyces cerevisiae.

By pulsed-field gel electrophoresis of chromosomal DNA and hybridization with a cloned MTH1 (CUP1) gene, we determined the locations of metallothionein-encoding gene sequences on chromosomes in monosporic cultures of 76 natural strains of Saccharomyces cerevisiae. Most of the strains (68) exhibited a previously known location for the MTH sequence on chromosome (chr.) VIII. Seven strains (resistant or sensitive to Cu2+) showed a MTH sequence in a new locus, MTH2, on chr. XVI. One strain carried an MTH locus on both chromosomes VIII and XVI. Restriction fragment and Southern blot analyses showed that the two MTH loci were very closely related. The strains displayed heterogeneity in the size and structure of their MTH2 locus. The length of the repeat unit of MTH2 varied: a 1.9-kb or 1.7-kb unit was found, instead of the 2.0-kb unit of the MTH1 locus. The most resistant strain (resistant to 1.2 mM CuSO4) contained a 0.9-kb repeat unit in addition to those of 1.9 kb and 1.7 kb. All three sensitive (to over 0.3 mM CuSO4) strains with an mth2 locus had a repeat unit of 1.9 kb or 1.7 kb, suggesting the presence of at least two copies of the MTH2 gene, with one always being in the junction area outside of the repeat unit. A monogenic tetrad segregation of 2:2 was usually found in crosses of resistant MTH2 and sensitive mth2 strains. Hybrids between strains with different MTH loci in all combinations showed low ascospore viability, suggesting that the complete lack of an MTH locus may lead to the death of segregants on YPD medium. The MTH1 and MTH2 loci were exchangeable. Strains with a high level of Cu2+ resistance were also resistant to Cd2+. However, these two properties did not cosegregate in heterozygotic hybrids.

Genetic characterization of the nonconventional yeast Hansenula anomala.

We describe genetic, molecular and taxonomic characteristics of the yeast Hansenula anomala. Pulsed-field gel electrophoresis of chromosomal DNAs from 19 H. anomala strains and related species indicated that H. anomala had a clearly different karyotype. Chromosome length polymorphism of the H. anomala strains was independent of their geographic origin and source of isolation. The strains were classified into four groups of similar karyotypes and one strain showed a unique profile. The sizes of chromosomes ranged from 850 to 3500 kb in different strains. The haploid chromosome number of H. anomala is at least nine. We have found RAPD primers discriminating at both the species and strain levels. All the primers tested except the M13 core sequence generated unique patterns with most strains. The results indicate the usefulness of PCR analysis with primer M13 for identification of the H. anomala species. Screening of the CBS (Utrecht) collection strains of H. anomala showed that they are rather difficult objects for genetic hybridization analysis. The strains have low fertility, viz. very poor sporulation, low mating type activities and, as a rule, nonviable ascospores. The majority of the hybrids obtained are polyploid, probably tetraploid, as judged by the segregation of control auxotrophic markers. Nevertheless, some monosporic cultures of the strains studied, including the biocontrol yeast J121, formed diploid hybrids with regular meiotic segregation of control auxotrophic markers. As a rule, H. anomala isolates are homothallic, showing delayed self-diploidization. Rare stable heterothallic strains of H. anomala also occur.