Gene, 1992 May 1, 114(1), 67 - 73 Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2; Sakai Y et al.; Alcohol oxidase (AOD) is the first key enzyme for methanol metabolism in methylotrophic yeasts . AOD activity is strictly regulated by carbon source . The AOD1 gene was cloned from a gene library of the asporogenous formaldehyde-producing methylotrophic yeast, Candida boidinii S2 . The complete nucleotide sequence of the gene and its 5'- and 3'-flanking regions (4174 bp) were determined . To identify the conserved and divergent sequences of the AOD1 gene and its 5'-flanking sequences among different species of methylotrophic yeasts, the AOD-encoding genes from C . boidinii S2 (AOD1), Hansenula polymorpha (MOX) and Pichia pastoris (AOX1 and AOX2) were compared . In addition to conserved amino acid sequences, several DNA segments in the G+C-rich region of 5'-flanking sequences were also found to be conserved . Northern analysis showed that the AOD1 gene transcript was induced by methanol, but was not detected when cells were grown on ethanol or glucose . Thus, as in ascosporogenous methylotrophic yeasts, AOD1 gene expression in C . boidinii appears to be controlled at the RNA level. Ukr Biokhim Zh, 1992 May-Jun, 64(3), 96 - 100 {Cellular microbiosensors for methanol and ethanol determination on the basis of pH-sensitive field transistors}; Korpan IaI et al.; Cellular sensors for methanol and ethanol determination were developed using immobilized mutant cells of methylotrophic yeasts Hansenula polymorpha and Pichia pinus (able to extrude protons in the presence of alcohol) and pH-sensitive field effect transistors (pH-SFETs) . The intact cells of yeasts were immobilized in Ca-alginate gel to obtain a biomembrane . The minimal detectable response was obtained to approximately 0.5 mM of methanol and ethanol, a linear dependence of biosensor's response on the logarithmic alcohol concentration was observed in the range from 5 to 100 mM for both types of alcohol . The prospects for application of biosensors to determine alcohols in the analytical practice are discussed. J Biotechnol, 1992 May, 23(3), 303 - 14 On-line biomass monitoring by capacitance measurement; Fehrenbach R et al.; An in-situ, steam-sterilizable capacitance probe was used to follow the biomass concentration on-line, in bioreactors from 20 to 2000 l total volume . Microbial cultures of Saccharomyces cerevisiae, Pichia pastoris and Streptomyces virginiae were grown in batch and fed-batch culture in both defined and complex media in order to demonstrate the wide dynamic operating range of the instrument . A linear correlation was found between the on-line capacitance measurement and the off-line measurements (optical density, OD620; packed mycelial volume, PMV; biomass concentration X, and colony forming units, CFU ml-1) for biomass concentrations (dry cell weight) up to 30 g l-1 (St . virginiae), 106 g l-1 (S . cerevisiae) and 89 g l-1 (P . pastoris) . The on-line capacitance measurement was slightly influenced by variations in agitation speed and strong extraneous radio frequencies . A specific capacitance constant (Cs) was defined for all microbial cells which was dependent on cell viability and cell size . The Cs was easy to calculate using the on-line capacitance measurement and an off-line estimation of biomass concentration . The Biomass Monitor proved suitable for precise on-line monitoring of both homogeneous (uni-cellular) and heterogeneous (mycelial) cultures in bioreactors. Appl Environ Microbiol, 1992 Mar, 58(3), 990 - 7 Geographic distribution and genetics of killer phenotypes for the yeast Pichia kluyveri across the United States; Starmer WT et al.; Representative strains (n = 61) of the yeast Pichia kluyveri from across the United States were studied for their ability to kill 71 other strains (representing 25 species) of yeast . This survey showed killing activity in 69% of the P . kluyveri strains tested . More extensive analysis of killer activity of 197 P . kluyveri strains against strains of five tester species showed comparable activity (67% of strains tested) . This activity was shown to be equally variable within localities, within regions, and across the continent . The genetic basis of the variability was ascertained by tetrad analysis and is most likely due to alleles segregating at three epistatic loci . Evidence for the idea that killer toxins have a role in excluding other yeasts from particular habitats is discussed. J Basic Microbiol, 1992, 32(5), 331 - 8 Mitotically unstable polyploids in the yeast Pichia guilliermondii; Klinner U et al.; Attempts to obtain triploids or tetraploids of P . guilliermondii by sexual hybridization led to mitotically stable hybrids . However, their DNA content per cell was not higher than in diploids . The results of random spore analysis demonstrate that these hybrids were in fact aneuploids which obviously suffered drastic chromosome losses immediately after mating . This phenomenon could have been caused either by aneuploidy already present in the parental strains or it might have been due to a general inability of P . guilliermondii to maintain a polyploid genome. Biol Cell, 1992, 75(1), 19 - 23 Ultrastructural immunodetection of a Pichia anomala killer toxin: a preliminary study; Cailliez JC et al.; A monoclonal antibody (mAb KT4), produced against a Pichia anomala killer toxin, was used to study the secretion process of toxin producing cells . The indirect immunofluorescence assay, performed with large concentrations of mAb KT4, showed a homogeneous distribution of the epitope at the cell surface of the P anomala cells . When increasing dilutions of mAb KT4 were employed, a 'punctuated' labeling appeared on the yeast's cell wall which suggested a heterogeneous secretion of the killer toxin . Similar labeling was also observed by immunodetection on live yeast cells held in buffered suspension . These results confirmed that 'punctuated' labeling was not an artefact due to a distortion of the cell's shape by having been dried on glass slides . Indirect immunodetection was performed in electron microscopy on ultra-thin sections of cells embedded in Araldite resin . The labeling thus obtained showed both the presence of the epitope in the cytoplasm and its sensitivity to strong glutaraldehyde fixation . Indirect immunodetection, performed on ultra-thin frozen sections, showed a cytoplasmic and cell wall labelling . However, the amount of gold particles observed in the cell wall was too low to confirm the heterogeneous killer toxin secretion observed in immunofluorescence . In this case, killer cells were fixed with a low concentration of glutaraldehyde which preserved the structure of the epitope complementary with mAb KT4. Gene, 1991 Dec 20, 109(1), 89 - 97 Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis; Amore R et al.; The XYL1 gene of the yeast Pichia stipitis has been isolated from a genomic library using a specific cDNA probe, and its nucleotide (nt) sequence has been determined . In the 5' noncoding region of the P . stipitis XYL1 gene a TATAAA element (known to be necessary for transcription initiation in most yeast genes) is found at nt -81, and two CCAAT recognition motifs (often referred to as the CCAAT box) are present at nt -146 and -106 . The XYL1 encodes a polypeptide of 35,927 Da that constitutes a NADH/NADPH-dependent xylose reductase (XR) . The enzyme is part of the xylose-xylulose pathway that is absent or only weakly expressed in Saccharomyces cerevisiae . Extensive homology is found to the N terminus of the XR of Pachysolen tannophilus and Candida shehatae . None of the known cofactor binding domains found in many NAD-dependent dehydrogenases are present in the protein . Transformants of S . cerevisiae containing XYL1 of P . stipitis synthesize an active XR . Fusion of XYL1 with the prokaryotic tac promoter leads to a gene that can be expressed in S . cerevisiae and Escherichia coli. J Biol Chem, 1991 Dec 5, 266(34), 22807 - 17 Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris; Trimble RB et al.; Saccharomyces SUC2 invertase, secreted by the methylotrophic yeast Pichia pastoris and purified to homogeneity from the growth medium by DE-52 chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a diffuse ladder of species at 85-90 kDa, while the secreted Saccharomyces form migrated as a broad band from 100 to 150 kDa . Endo-beta-N-acetylglucosaminidase H released the Pichia invertase carbohydrate generating a 60-kDa protein with residual Asn-linked GlcNAcs and oligosaccharides separated on Bio-Gel P-4 into Man8-11GlcNAc . Nearly 75% of the oligosaccharides were equally distributed between Man8,9GlcNAc, while 17% were Man10GlcNAc and 8% were Man11GlcNAc . Oligosaccharide pools were analyzed for homogeneity by high-pH anion-exchange chromatography, and structures were assigned using 500 MHz one- and two-dimensional 1H NMR spectroscopy . Pichia Man8GlcNAc was the same isomer as found in Saccharomyces, which arises by removing the alpha 1,2-linked terminal mannose from the middle arm of the lipid-oligosaccharide Man9GlcNAc (Byrd, J . C., Tarentino, A . L., Maley, F., Atkinson, P . H., and Trimble, R . B . (1982) J . Biol . Chem . 257, 14657-14666) . The Man9GlcNAc pool was 5% lipid-oligosaccharide precursor and 95% Man8GlcNAc isomer with a terminal alpha 1,6-linked mannose on the lower-arm alpha 1,3-core-linked residue (Hernandez, L . M., Ballou, L., Alvarado, E., Gillece-Castro, B . L., Burlingame, A . L., and Ballou, C . E . (1989) J . Biol . Chem . 264, 11849-11856) . An alpha 1,2-linked mannose on the new alpha 1,6-linked branch in Man9GlcNAc provided 80% of the Man10GlcNAc, which is the structure on Saccharomyces invertase (Trimble, R . B., and Atkinson, P . H . (1986) J . Biol . Chem . 261, 9815-9824) . A minor Man10GlcNAc (12%) and the principal Man11GlcNAc (82%) were the major Man9,10GlcNAc with novel alpha 1,2-linked mannoses on the preexisting alpha 1,2-linked termini . Although Pichia glycans did not have terminal alpha 1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes. Vaccine, 1991 Dec, 9(12), 901 - 6 Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties; Romanos MA et al.; Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens . We expressed the immunogenic outer membrane protein pertactin (P69) from B . pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris . In high-density fermentations, engineered P . pastoris yielded greater than 3 g of the protein per litre of culture . Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines. Biotechnology (N Y), 1991 Nov, 9(11), 1090 - 5 Xylitol production by recombinant Saccharomyces cerevisiae; Hallborn J et al.; We obtained efficient conversion of xylose to xylitol by transforming Saccharomyces cerevisiae with the gene encoding the xylose reductase (XR) of Pichia stipitis CBS 6054 . Comparison of the chromosomal and cDNA copies of the XYL1 gene showed that the genomic XYL1 contains no introns, and an XR monomer of 318 amino acids (35,985 D) is encoded by an open reading frame of 954 bp . The amino acid sequence of the P . stipitis XR is similar to several aldose reductases, suggesting that P . stipitis XR is part of the aldoketo reductase superfamily . S . cerevisiae transformed with the XYL1 gene gave over 95% conversion of xylose into xylitol, a yield not obtainable with natural xylose utilizing yeasts. FEBS Lett, 1991 Oct 21, 291(2), 299 - 302 Overexpression of alcohol oxidase in Pichia pastoris; de Hoop MJ et al.; The protein import capacity of peroxisomes in methylotrophic yeasts was studied using Pichia pastoris containing one or two extra copies of the gene encoding the peroxisomal protein alcohol oxidase . The alcohol oxidase overproduced in this strain was only partially imported and assembled into the active, octameric form of the protein . The excess remained in the cytosol as protein aggregates composed of monomers . These results are discussed in view of the possible application of peroxisomes as storage compartments for heterologous proteins. Curr Opin Biotechnol, 1991 Oct, 2(5), 742 - 5 Gene expression in yeast: Pichia pastoris; Vedvick TS; Recent studies have shown the versatility and utility of the Pichia pastoris expression system . Improvements in strains have boosted the yield of proteins and peptides to the commercially feasible range . The Pichia pastoris expression system will soon be used to manufacture proteins for human clinical trials. Gene, 1991 Sep 15, 105(2), 205 - 12 Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies; Clare JJ et al.; We have constructed a synthetic secretion cassette encoding the alpha-factor prepro leader peptide from Saccharomyces cerevisiae fused to mouse epidermal growth factor (mEGF) . This was used to compare the secretion of mEGF, a 53-amino acid polypeptide, in S . cerevisiae and Pichia pastoris . In both yeasts the leader sequence was accurately and efficiently cleaved showing that the S . cerevisiae-derived alpha-factor prepro region is correctly recognised and processed in P . pastoris . Of the total mEGF produced, over 90% was exported to the culture supernatant, although the final level of accumulation was dependent on the composition of the growth medium . With P . pastoris there was instability of the protein in minimal medium (yeast nitrogen base), probably caused by extracellular proteases . This was overcome by adding 1% Casamino acids and buffering the medium to pH 6.0 . To increase the level of secreted mEGF we have developed a method for rapidly screening large numbers of P . pastoris transformants for the presence of many copies of a foreign gene . Using this procedure we isolated a strain containing 19 integrated copies of the mEGF gene which secreted 450 micrograms/ml of mEGF in high-density fermentations . Characterisation of the yeast-derived mEGF showed the presence of truncated forms, mEGF1-51 and mEGF1-52, as was found with S . cerevisiae-secreted human EGF {George-Nascimento et al., Biochemistry 27 (1988) 797-802} . In addition, the full-length protein, mEGF1-53, was secreted by P . pastoris. J Cell Biol, 1991 Sep, 114(5), 893 - 904 Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes; Keller GA et al.; Peroxisomes, glyoxysomes, glycosomes, and hydrogenosomes have each been classified as microbodies, i.e., subcellular organelles with an electron-dense matrix that is bound by a single membrane . We investigated whether these organelles might share a common evolutionary origin by asking if targeting signals used for translocation of proteins into these microbodies are related . A peroxisomal targeting signal (PTS) consisting of the COOH-terminal tripeptide serine-lysine-leucine-COOH has been identified in a number of peroxisomal proteins (Gould, S.J., G.-A . Keller, N . Hosken, J . Wilkinson, and S . Subramani . 1989 . J . Cell Biol . 108:1657-1664) . Antibodies raised to a peptide ending in this sequence (SKL-COOH) recognize a number of peroxisomal proteins . Immunocryoelectron microscopy experiments using this anti-SKL antibody revealed the presence of proteins containing the PTS within glyoxysomes of cells from Pichia pastoris, germinating castor bean seeds, and Neurospora crassa, as well as within the glycosomes of Trypanosoma brucei . Western blot analysis of purified organelle fractions revealed the presence of many proteins containing this PTS in both glyoxysomes and glycosomes . These results indicate that at least one of the signals, and therefore the mechanism, for protein translocation into peroxisomes, glyoxysomes, and glycosomes has been conserved, lending support to a common evolutionary origin for these microbodies . Hydrogenosomes, the fourth type of microbody, did not contain proteins that cross-reacted with the anti-PTS antibody, suggesting that this organelle is unrelated to microbodies. Antonie Van Leeuwenhoek, 1991 Jul, 60(1), 21 - 30 Phylogenetic relationships among species of Williopsis and Saturnospora gen . nov . as determined from partial rRNA sequences; Liu ZW et al.; Phylogenetic relationships among those yeast species that form saturn-shaped ascospores and which are assigned to the genera Williopsis and Pichia were estimated from their extent of nucleotide sequence divergence in three regions of ribosomal RNA . The Pichia species (P . dispora, P . saitoi, P . zaruensis and P . sp . nov.) are a closely clustered group only distantly related to Williopsis, and it is proposed that they be reassigned to Saturnospora gen . nov . The extent of divergence among Williopsis species (W . californica, W . mucosa, W . pratensis, W . saturnus and W . sp . nov.) is greater than that previously observed within other ascomycetous yeast genera. Antonie Van Leeuwenhoek, 1991 Jul, 60(1), 13 - 9 DNA relatedness among saturn-spored yeasts assigned to the genera Williopsis and Pichia; Kurtzman CP; Saturn-spored species assigned to the genera Williopsis and Pichia were compared from extent of nuclear DNA complementarity . Of the Pichia spp., four were recognized as distinct taxa: P . dispora, P . saitoi, P . zaruensis and Pichia sp . nov . Among Williopsis spp., the following were accepted: W . californica, W . mucosa comb . nov., W . pratensis, W . saturnus var . saturnus, W . saturnus var . mrakii comb . nov., W . saturnus var . sargentensis comb . nov., W . saturnus var . subsufficiens comb . nov . and Williopsis sp . nov . The new Pichia and Williopsis species are described elsewhere . Moderate (36-68%) DNA relatedness was detected between the former Pichia sargentensis and varieties of W . saturnus again demonstrating that nitrate assimilation is not a reliable criterion for separating yeast species. Mikrobiologiia, 1991 Jul-Aug, 60(4), 686 - 92 {Competition of isogenic haploid and diploid cells of the yeast Saccharomyces cerevisiae and Pichia pinus growing in mixed populations}; Naidkhardt Kh et al.; During "quasi-continuous" cultivation in rich and minimal media diploid yeast cells of Saccharomyces cerevisiae completely displace isogenic haploid ones . When Pichia pinus are cultivated in the minimal medium, the diploids also have an advantage over isogenic haploids . The results are discussed within the framework of the hypothesis of fixation of diploid phase in the course of biological evolution. J Immunol, 1991 Jun 15, 146(12), 4180 - 6 Modification of lymphocyte migration by mannans and phosphomannans . Different carbohydrate structures control entry of lymphocytes into spleen and lymph nodes; Weston SA et al.; Previous in vitro studies suggest that recognition of phosphomannosyl structures by lymphocytes plays a central role in the binding of lymphocytes to high endothelial venules . However, the physiologic relevance of phosphomannosyl recognition in in vivo lymphocyte migration has not been established . This paper describes experiments that examined this question . It was demonstrated that the phosphomannan monoester core (PPME) from Pichia holstii, a potent inhibitor of peripheral node high endothelial venule interactions in vitro, was a very effective inhibitor of in vivo lymphocyte migration, as little as 39 micrograms/mouse significantly inhibiting popliteal lymph node entry . Furthermore, PPME exhibited a similar hierarchy of inhibition in vivo as previously reported in vitro, most effectively inhibiting entry of lymphocytes into popliteal lymph node, somewhat less effectively inhibiting mesenteric lymph node entry and being a relatively poor inhibitor of Peyer's patch entry . Additionally, PPME inhibited splenic entry of lymphocytes, and inhibition of lymphoid organ entry was accompanied by a substantial leukocytosis . Two additional mannose-containing compounds were found to modify lymphocyte migration, namely a well defined mannose containing pentasaccharide (PENT) with terminal mannose-6-phosphate (M6P) and an unphosphorylated yeast mannan . Both PENT and mannan induced leukocytosis and were particularly effective at inhibiting splenic entry of lymphocytes . In fact, detailed dose-response curves indicated that mannan was a much more potent inhibitor of splenic entry than PPME or PENT, whereas in lymph nodes PPME was the most effective inhibitor . Pretreatment of lymphocytes before injection with either PPME or mannan demonstrated that PPME could act at the lymphocyte level, whereas mannan probably acted at some other site . Collectively, these data suggest that different carbohydrate structures are involved in the entry of lymphocytes into different lymphoid organs, with mannose recognition playing an important role in splenic entry and recognition of M6P-like structures controlling lymph node entry . In contrast, it was found that mannose-and M6P-containing structures, unlike sulfated polysaccharides such as fucoidan, did not affect the subsequent positioning of lymphocytes within lymphoid organs. Biotechnology (N Y), 1991 May, 9(5), 455 - 60 High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene; Clare JJ et al.; We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus . In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture . The purified protein was as effective as native fragment C in immunizing mice . In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia . The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts) . The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette . Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome . These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations . Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event . We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia. Antonie Van Leeuwenhoek, 1991 Apr, 59(3), 139 - 45 Differential toxinogenesis in the genus Pichia detected by an anti-yeast killer toxin monoclonal antibody; Polonelli L et al.; The differential toxinogenesis of 25 isolates belonging to species of the potential yeast killer genus Pichia that were previously classified in the genus Hansenula was comparatively demonstrated by two serologic techniques (indirect immunofluorescence and double immunodiffusion) by using a monoclonal antibody against a yeast killer toxin produced by a selected strain of Pichia anomala (UCSC 25F) . The killer phenotypes of the Pichia isolates were evaluated by their ability to kill each other . The results, although of insufficient taxonomic value for a reliable separation of either species or genera, attest to the genomic heterogeneity for the killer character in the genus Pichia as well as the presumptive dual killer/sensitive identity for each single isolate. J Clin Microbiol, 1991 Apr, 29(4), 718 - 22 Evaluation of the Baxter-MicroScan 4-hour enzyme-based yeast identification system; Land GA et al.; A new 4-h Yeast Identification Panel (YIP; Baxter-MicroScan, W . Sacramento, Calif.) was compared with the API 20C Yeast Identification System (Analytab Products, Inc., Plainview, N.Y.) in the identification of recent clinical yeast isolates . The YIP had a 94% correlation (288 of 306) in identifying 22 species within the genera Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces, and Torulopsis . Correlation dropped to 65% for those species within the genera of slower growing yeasts, i.e., Blastoschizomyces spp., Crpytococcus spp., Geotrichum spp., Hyphopichia spp., Phaeococcomyces spp., Prototheca spp., and Trichosporon spp . Overall correlation with the API 20C was 92% (365 of 401) for those taxa included in the data base and 85% (373 of 437) for all yeasts encountered in the study . There were 36 (8.2%) discrepant identifications, which were due in part to the limited data base . Expansion of the data base plus the easy inoculation, reading, and rapid results of the YIP should make it an excellent method for yeast identification. J Ind Microbiol, 1991 Apr, 7(3), 197 - 201 High-level secretion of biologically active aprotinin from the yeast Pichia pastoris; Vedvick T et al.; A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site . Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion . P . pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein . A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin . The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay . Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg . Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor . The N-terminal extension was variably 11 or 4 amino acids . Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension. Zentralbl Mikrobiol, 1991, 146(3), 173 - 9 {Comparative studies of the growth behavior of wild strains and fusion hybrids of Pichia guilliermondii in discontinuous and continuous culture}; Prahl T et al.; The influence of several temperatures on the wild strain Pichia guilliermondii Pg 0799 and a selected fusion product fp 3/9 was studied in batch- and continuous cultures . Growth and substrate utilization of both strains were compared by determination of specific growth rates, yield coefficients and biomass production . The results showed, that strain fp 3/9 has in general a higher optimum of temperature for biomass production. Antonie Van Leeuwenhoek, 1991 Jan, 59(1), 9 - 13 Regulation of chorismate mutase activity of various yeast species by aromatic amino acids; Bode R et al.; The regulatory properties of chorismate mutase, its cellular localization and isoenzyme pattern were investigated in 23 yeast species . All yeasts contained only a single form of the enzyme, which is localized exclusively in the cytosol . The enzyme activity from all sources was activated 3-(Rhodotorula aurantiaca) to 185-fold (Candida maltosa) by tryptophan . The tryptophan concentration, which was necessary to obtain half maximum velocity was determined to be between 2 (Pichia guilliermondii) and 95 microM (Yarrowia lipolytica) . Ten yeast species possessed an enzyme that was inhibited by both phenylalanine and tyrosine . The chorismate mutase from four strains was inhibited only by tyrosine and the enzyme from two species was inhibited by phenylalanine alone . The enzyme inhibition by phenylalanine and tyrosine was completely reversed by tryptophan . Six enzyme sources were not inhibited and the Y . lipolytica chorismate mutase was slightly activated by both amino acids. J Med Vet Mycol, 1991, 29(4), 235 - 42 'Antibiobodies': antibiotic-like anti-idiotypic antibodies; Polonelli L et al.; Pathogenic micro-organisms such as Candida albicans may be susceptible to the activity of antimicrobial products like yeast killer toxins due to the presence of specific cell wall receptors for these agents . Anti-idiotypic antibodies (anti-Ids) were produced that competed for these receptors with the yeast killer toxin of a strain of Pichia anomala . We report here that affinity chromatography purified anti-Ids may kill C . albicans cells in vitro which are susceptible to the activity of the yeast killer toxin, as well as P . anomala killer cells which are obviously immune to their own toxin despite possessing specific cell wall receptors which can be detected by indirect immunofluorescence with anti-Ids . We propose that these conceptually new antimicrobial immunoglobulins acting as antibiotics be called 'antibiobodies'. Appl Biochem Biotechnol, 1991 Spring, 28-29, 369 - 75 Genetic transformation of xylose-fermenting yeast Pichia stipitis . Scientific note; Ho NW et al.; A plasmid-mediated transformation system has been developed for the xylose-fermenting yeast Pichia stipitis . We found that plasmid vectors containing the Saccharomyces cerevisiae 2 mu replicon and the kanamycin resistance gene (KmR) could be introduced into the Pichia cells and maintained as extrachromosomal elements . Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of KmR . Plasmids identical to those used for transformation can be recovered from the Pichia transformants . Protocols for transformation of P . stipitis by the CaCl2-polyethylene glycol-protoplast process or by direct electroporation of intact Pichia cells have both been developed. Appl Biochem Biotechnol, 1991 Spring, 28-29, 327 - 40 Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae; Takuma S et al.; A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis . DNA sequence analysis showed that the gene consists of 951 bp . The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P stipitis were inducible . S . cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose . Since xylose uptake and accumulation of xylitol by S . cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited. Appl Biochem Biotechnol, 1991 Spring, 28-29, 131 - 44 Ethanolic fermentation of pentoses in lignocellulose hydrolysates; Hahn-Hagerdal B et al.; In the fermentation of lignocellulose hydrolysates to ethanol, two major problems are encountered: the fermentation of the pentose sugar xylose, and the presence of microbial inhibitors . Xylose can be directly fermented with yeasts, such as Pachysolen tannophilus, Candida shehatae, and Pichia stipis, or by isomerization of xylose to xylulose with the enzyme glucose (xylose) isomerase (XI; EC 5.3.1.5), and subsequent fermentation with bakers' yeast, Saccharomyces cerevisiae . The direct fermentation requires low, carefully controlled oxygenation, as well as the removal of inhibitors . Also, the xylose-fermenting yeasts have a limited ethanol tolerance . The combined isomerization and fermentation with XI and S . cerevisiae gives yields and productivities comparable to those obtained in hexose fermentations without oxygenation and removal of inhibitors . However, the enzyme is not very stable in a lignocellulose hydrolysate, and S . cerevisiae has a poorly developed pentose phosphate shunt . Different strategies involving strain adaptation, and protein and genetic engineering adopted to overcome these different obstacles, are discussed. Genetica, 1991, 85(1), 35 - 44 Structure comparison and evolutionary relations between elongation factors EF-Tu (EF-1 alpha) and SUP 2 proteins; Samsonova MG et al.; On the basis of high homology and structural similarity, three genes, SUP2 Saccharomyces cerevisiae, SUP2 Pichia pinus and GST1 Homo sapiens, might be considered as members of one family named SUP2 . Comparison of the primary structure of SUP2 proteins and elongation factors EF-Tu(EF-1 alpha) from 19 different species was performed . It was found that SUP2 proteins bear more homology to eukaryotic elongation factor than to procaryotic EF-Tu, though the degree of sequence conservation in SUP2 proteins is smaller than in EF-1 alpha factors . The extensive phylogenetic analysis of SUP2 and EF-Tu(EF-1 alpha) genes was performed by means of 3 methods, 2 phenetic and one cladystic (maximal parsimony) . The data support the close relation of SUP2 genes to other elongation factor genes. Bioprocess Technol, 1991, 12, 57 - 83 Physical and chemical cell disruption for the recovery of intracellular proteins; Hopkins TR; There are many ways to disrupt microorganisms and plant and animal tissue . Selecting the best cell disruption method depends on the factors listed in Table 6 . The kind or type of cells is an important consideration . For example, some disruption methods which work well for animal tissue do not work at all for microorganisms . A guideline for the suitabiity of a given disruption method for some cell types is given in Table 7 . The ratings in this table are not incontestable and, as mentioned earlier, combinations of methods can sometimes produce satisfactory results whereas one method alone fails . The disruptibility of cells can be influenced by their growth and storage history . For microorganisms, cells in log phase growth tend to produce thinner cell walls which are more easy to disrupt . This and other conditions which can influence microbial cell disruptiability are listed in Table 8 . The cell disruption method selected will depend on its capability to process samples of a certain size or to be able to process multiple samples in a reasonable period of time . Other considerations are the availability, cost, and general utility of the disruption equipment . Thus, in a research environment the purchase of an expensive cell disrupter which processes a wide variety of cell types may be more easy to justify than a specialized disrupter . And if the long-term goal is to scale up, the choice of disruption methods narrow considerably . Indeed, several of the most successful laboratory cell disruption methods have no possibility of being scaled up . Despite possible scale-up difficulties, in the case of many bioactive recombinant products expressed at high levels in microorganisms, this concern may be irrelevant . Few of these products are likely to be manufactured in really large amounts and current laboratory scale or pilot plant scale production equipment may be entirely adequate . For instance, active human TNF (tissue necrosis factor) can be expressed in Pichia pastoris yeast at levels of 100 g/kg of yeast (dry weight) . At this level of expression, only a few kilograms of r-DNA yeast needs be disrupted to meet the worldwide demand for this research material . Finally, the operating and energy requirements which affect the economics of the disruption process (batch versus continuous, disruption yield, cell fragment size, effect of added enzymes on downstream separation, etc.) are important considerations in the selection of production equipment. Curr Genet, 1990 Dec, 18(6), 493 - 500 Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant; Kotter P et al.; A P . stipitis cDNA library in lambda gt11 was screened using antisera against P . stipitis xylose reductase and xylitol dehydrogenase, respectively . The resulting cDNA clones served as probes for screening a P . stipitis genomic library . The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined . The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa . The XYL2 gene is actively expressed in S . cerevisiae transformants . S . cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source . In contrast to aerobic glucose metabolism, S . cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively. Mycopathologia, 1990 Nov, 112(2), 119 - 24 Oral yeasts in patients with cancer of the mouth, before and during radiotherapy; Paula CR et al.; The yeasts of patients with oral cancer has been studied before and during Xr-therapy . Gram and PAS smears revealed an increase of yeast-like structures, during treatment, from 56% to 66% of the cases . Before radiotherapy oral yeasts were isolated from 56% of the patients with cancer represented by Candida albicans (30%); C . tropicalis (12%); C . glabrata and C . krusei (4%), besides six other different species (2%) . During radiotherapy yeasts were isolated in 72% of the cases, as follow: C . albicans (36%); C . tropicalis (16%); Rhodotorula rubra (6%); C . kefyr; C . krusei and Pichia farinosa (4%), besides other nine species (2%) . C . albicans serotype A represented 93% of the isolated samples, before treatment and 88.8% during Xr-therapy. Yeast, 1990 Nov-Dec, 6(6), 461 - 72 Divergence and conservation of SUP2 (SUP35) gene of yeast Pichia pinus and Saccharomyces cerevisiae; Kushnirov VV et al.; 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. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 17 - 22 Factors influencing the utilisation of L-malate by yeasts; Rodriguez SB et al.; The utilisation of L-malate and the effect of glucose concentration on malate utilisation under semi-anaerobic conditions were investigated in three yeasts unable to grow on malate as sole carbon source (Saccharomyces cerevisiae, Schizosaccharomyces malidevorans, Zygosaccharomyces bailii) and two yeasts able to utilise the TCA cycle intermediate as sole carbon source (Pichia stipitis and Pachysolen tannophilus) . Utilisation of malate by both Schiz . malidevorans and Z . bailii was reduced at high and low levels of glucose . In the absence of glucose, P . stipitis and Pa . tannophilus utilised malate rapidly; however, their utilisation was drastically reduced in the presence of glucose, suggesting that malate utilisation is under catabolite repression. Mol Biol (Mosk), 1990 Jul-Aug, 24(4), 1024 - 36 {Comparative analysis of the structure of SUP2 genes in Pichia pinus and Saccharomyces cerevisiae}; Kushnirov VV et al.; 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. J Gen Microbiol, 1990 Jul, 136 ( Pt 7), 1279 - 84 Acetolysis and 1H NMR studies on mannans isolated from very flocculent and weakly flocculent cells of Pichia pastoris IFP 206; Mbawala A et al.; Growth of the yeast Pichia pastoris IFP 206 in methanol- and glucose-containing media led respectively to very and weakly flocculent cells . Mannans from both kinds of cells were extracted and compared . Chemical analysis and molecular mass estimation showed some differences between the mannans from very and weakly flocculent cells, especially in quantitative amino acid content . 1H NMR analysis showed that both kinds of mannan contained alpha-1,2 and beta-1,2 linkages . Two acetolysis conditions, combined with 1H NMR analysis, revealed that mannans from both kinds of cells were composed of mannose, mannobiose, mannotriose, mannotetraose and mannopentaose side-chains with the following respective structures: Man; Man alpha 1---2Man; Man alpha 1----2Man alpha 1----2Man; Man beta 1----2Man alpha 1----2Man; Man beta 1----2Man beta 1----2Man alpha 1----2Man; Man alpha 1----2Man beta 1----2Man beta 1----2Man alpha 1----2Man . Additionally the beta-1,2 linkages of the non-reducing terminal residues of the mannotetraose were shown to be acetolysis-labile . The mannans from very flocculent cells were richer in mannopentaose than the mannans from weakly flocculent cells . According to these results, the extended conformations in the branching moieties of the mannan could be the basis of the higher degree of flocculation of the methanol-grown cells. Antimicrob Agents Chemother, 1990 Jul, 34(7), 1331 - 5 Involvement of a cell wall receptor in the mode of action of an anti-Candida toxin of Pichia anomala; Sawant AD et al.; Hanes-Woolf, Dixon, and Hill plots of growth rates of Candida albicans RC1 grown in various concentrations of glucose and a Pichia anomala WC65 toxin suggested the presence of toxin-binding sites . Indirect immunofluorescence microscopy with antitoxin antibodies demonstrated binding of the toxin to the cell wall . Resistance to the toxin of a mutant Saccharomyces cerevisiae deficient in cell wall beta-1-6-D-glucan suggests that the glucan either served as the receptor or influenced the number or composition of the receptor . Immunofluorescence that appeared to be associated with the cell membrane of toxin-treated spheroplasts of C . albicans was also observed . Spheroplasts of the resistant mutant of S . cerevisiae were sensitive to the toxin. Mycopathologia, 1990 Jun, 110(3), 169 - 75 Production of yeast killer toxin in experimentally infected animals; Polonelli L et al.; The ability of a killer yeast (Pichia anomala, UCSC 25F) to produce toxin in vivo was demonstrated, for the first time, in tissues of normal and immunosuppressed experimentally infected mice by means of a fluorescent antibody technique and a killer toxin specific monoclonal antibody . The possible significance of the findings is discussed. Antonie Van Leeuwenhoek, 1990 May, 57(4), 215 - 22 Candida shehatae--genetic diversity and phylogenetic relationships with other xylose-fermenting yeasts; Kurtzman CP; The xylose-fermenting yeasts Candida shehatae and Pichia stipitis were compared from extent of nuclear DNA complementarity and ribosomal RNA sequence similarity . Low levels of DNA relatedness confirmed that the two taxa are distinct biological species, but the similarity of rRNA sequences suggests that they only recently diverged . C . shehatae is comprised of three genetically divergent (ca . 50% DNA relatedness) subgroups that were accorded varietal status: C . shehatae var . shehatae, var . lignosa and var . insectosa . Estimates of phylogenetic distance from rRNA sequence similarity show C . shehatae and P . stipitis to be more closely related to Pachysolen tannophilus than to Saccharomyces cerevisiae, and that all of these budding yeasts are well separated from Schizosaccharomyces pombe. Genetika, 1990 Apr, 26(4), 614 - 20 {Cloning of the RIB1 gene coding for the enzyme of the first stage of flavinogenesis in the yeast Pichia guilliermondi, GTP cyclohydrolase, in Escherichia coli cells}; Zakal'skii AE et al.; The RIB1 gene encoding the enzyme of the first stage of the yeast Pichia guillermondii-GTP-cyclohydrolase- was cloned on pFL38 shuttle vector as the Sau3A fragment of chromosomal DNA of about 9 kb . EcoRI fragment of 4 kb with RIB1 gene was subcloned from the pFRI hybrid plasmid obtained into the pUC18 plasmid and then shortened to give 2.9 kb via deletion in SalGI site . The plasmid constructed was designated pR1 . Activity of GTP-cyclohydrolase was 80-100-fold higher in extracts of transformants than in the prototroph strain, which evidence of effective expression of the yeast gene within recombinant plasmids in the cells of this species of bacteria . The enzyme isolated from transformants has molecular mass 179 kDa, is inhibited by PAD and adenyl-nucleotides, which is characteristic of GTP-cyclohydrolase of P . guilliermondii but not of Escherichia coli. J Protein Chem, 1990 Feb, 9(1), 83 - 6 Crystallization of alcohol oxidase from Pichia pastoris . Secondary structure predictions indicate a domain with the eightfold beta/alpha-barrel fold; Tykarska E et al.; Alcohol oxidase from Pichia pastoris has been crystallized from polyethylene glycol 4000 solutions . The crystals are tetragonal, a = 228 A, c = 456 A space group P4(1)2(1)2 . The crystals scatter only to about 6 A resolution; their poor crystallinity may have some physiological function . Secondary structure predictions suggest that the C-terminal part of the molecule, residues 311-664, has the folding of an eightfold beta/alpha-barrel (TIM barrel) . This would indicate common ancestry with four other flavoenzymes: canavalin, glycolate oxidase, flavocytochrome b, and trimethylamine dehydrogenase. Mikrobiol Zh, 1990 Jan-Feb, 52(1), 61 - 4 {The effect of Pichia guilliermondii yeasts on the body of laboratory animals}; Kotliar AN et al.; Conditionally pathogenic species Pichia guilliermondii which, being parenterally introduced to white mice, induced pathological changes in viscera and rarely death, has been revealed among species of genus Pichia previously considered as a nonpathogenic genus . The strains differ considerably between themselves in virulence . The latter does not depend on the yeast sex and the source of strain isolation. Arch Immunol Ther Exp (Warsz), 1990, 38(5-6), 379 - 85 Epidemiology of Candida infection . III . A proposal for a new typing system for Candida albicans strains; Budak A; A proposal of a new typing system for C . albicans strains in this paper is presented . Our system was elaborated on the basis of computer analysis of the results received using various already existing methods (killer system, resistogram, API system and Odd's and Abbott's method) for typing of 350 C . albicans strains . The strains originated from various anatomical and geographical sources . For the new system we have chosen three characters: sensitivity to two killer strains (Pichia sp . STUMM 1035, Hansenula sp . STUMM 1034) and urea assimilation test . The computer-aided program, used as an analytical tool, permitted the selection of 8 types among the strains . The majority of tested isolates belonged to types 6 and 7 . We observed differences in the distribution of types according to the geographical and anatomical origin of the strains . The criteria adopted in our system can be useful in typing of C . albicans for epidemiological studies. Arch Immunol Ther Exp (Warsz), 1990, 38(5-6), 359 - 67 Epidemiology of Candida infection . I . Use of killer system for typing of Candida albicans strains; Budak A; A killer system was used for epidemiological differentiating of 350 Candida albicans strains isolated from human sources of different anatomical and geographical origin . By using 9 killer strains from genera: Hansenula, Pichia, Torulopsis, Saccharomyces it was possible to differentiate 87 killer types . Our findings showed differences in the occurrence of the types according to the origin of the strains . The killer system proves to be valid for the differentiation of strains within the Candida species for epidemiological purposes. Antonie Van Leeuwenhoek, 1989 Nov, 56(4), 337 - 47 Regulation of the lysine biosynthesis in Pichia guilliermondii; Schmidt H et al.; The regulatory properties of four enzymes (homocitrate synthase, alpha-aminoadipate reductase, saccharopine reductase, saccharopine dehydrogenase) involved in the lysine biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory patterns found in other yeast species . The first enzyme of the pathway, homocitrate synthase, is feedback-inhibited by L-lysine . Some other amino acids (alpha-aminoadipate, glutamate, tryptophan, leucine) and lysine analogues are also inhibitors of one or more enzymes . It is shown that only the synthesis of homocitrate synthase is weakly repressed by L-lysine. Mol Gen Genet, 1989 Oct, 219(1-2), 320 - 3 Use of site-specific recombination to regenerate selectable markers; Cregg JM et al.; A method which allows the repeated use of a single selectable marker in DNA transformations was demonstrated . This marker regeneration method employed portions of the Saccharomyces cerevisiae 2 microns circle plasmid: the inverted repeat sequences (FRTs), and the FLP gene whose product, a site-specific recombinase, catalyzes recombination events between FRTs . When FRTs were oriented as direct repeats and integrated into the genome of the yeast Pichia pastoris, FLP-mediated recombination resulted in the efficient and precise deletion of DNA located between the repeats . In the example described, the S . cerevisiae ARG4 gene, placed between a set of FRTs and integrated into Pichia in a prior transformation, was deleted by FLP, thereby regenerating an arginine-requiring phenotype in the P . pastoris strain. Mikrobiologiia, 1989 Sep-Oct, 58(5), 769 - 77 {Relative competitiveness of haploid and diploid yeast cells growing in a mixed population}; Glazunov AV et al.; Saccharomyces cerevisiae was grown in a rich medium under the conditions of "quasi-continuous" cultivation and, after 200-300 generations, its diploid cells almost completely displaced haploid cells from the original mixed "haploid-diploid" population where the ratio between diploid and haploid strains was either 1:1 or 1:100 . The cultivation at 40 degrees C did not change the relative competitive ability of haploids and diploids . When cells were cultivated in a rich medium at 6 degrees C or in a minimal medium at 30 degrees C, none of the strains showed an advantage over others for about 200 generations . Haploid cells had an advantage over diploid cells during "quasi-continuous" growth in the minimal medium at 30 degrees C . When the temperature was elevated to 40 degrees C, diploid cells displaced haploid cells from the mixed population . No advantage was found for diploid or haploid cells grown in a medium with an elevated KCl content (1.5 M) . Haploid cells had an advantage over diploid cells when Pichia pinus was cultivated in a minimal medium . The results are discussed using the hypothesis about the diploid phase being fixed in the course of biological evolution. Indian J Exp Biol, 1989 Aug, 27(8), 742 - 3 Microbial transformation of 13-ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraene-14, 17-dione to its 17-beta hydroxy derivative by Pichia farinosa in pilot plant fermentors; Mehdi I et al.; Parameters for microbial transformation of 13-ethyl-3-methoxy-8, 14-seco-gona-1,3,5 (10), 9(11)-tetraene-14,17-dione to its 17 beta-hydroxy derivative by P . farinosa have been standardised in pilot plant fermentors . The yield of the pure crystalline compound was 80%. J Mol Biol, 1989 Jul 5, 208(1), 211 - 2 Crystallization of alcohol oxidase from Pichia pastoris; Boys CW et al.; Crystals of alcohol oxidase purified from Pichia pastoris were grown in microdialysis buttons in a solution of polyethylene glycol, sodium chloride and sodium azide . The crystals were stratified along the major axis and up to 3 mm in length . X-ray diffraction experiments indicated a space group of P2(1) and unit cell dimensions of a = 157.3 A, b = 171.5 A and c = 231.6 A . Crystals diffract to beyond 2.7 A and are suitable for X-ray structure analysis. Tsitologiia, 1989 Jul, 31(7), 785 - 90 {Regularities of posthyperthermic recovery in haploid and diploid yeasts Saccharomyces cerevisiae and Pichia pinus}; Glazunov AV et al.; Isogenic diploid cells of Saccharomyces cerevisiae are more sensitive to hyperthermic treatment (50 degrees C) than haploid ones, the posthyperthermical recovery efficiency being the same for both the cell types . In contrast to this, thermosensitivity of haploid and diploid cells of Pichia pinus does not practically differ, the posthyperthermic recovery efficiency for both the cell types being also the same . It is shown that diploid cells of P . pinus in the logarithmic phase of their growth are incapable of recovering after hyperthermal treatment, which is largely the reason for their higher sensitivity to such a treatment as compared with cells in the stationary phase of growth. Ukr Biokhim Zh, 1989 Jul-Aug, 61(4), 47 - 54 {Properties of 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5'- phosphate reductase, a enzyme of the second stage of flavinogenesis in Pichia guilliermondii yeasts}; Logvinenko EM et al.; 2,5-Diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been isolated from cells of Pichia guilliermondii and subjected to 20-fold purification by treating extracts with streptomycin sulphate, frationating proteins (NH4)2SO4 at 45-75% of saturation and chromatography on blue sepharose CL-6B . The use of gel filtration through Sephadex G-150 and chromatography on DEAE-cellulose proved to be less effective for the enzyme purification . It has been established that it is 2,5-diamino-4-oxy-6-ribosylaminopyrimidine-5-phosphate but not its dephosphorylated form that is the substrate of the given reductase; Km is equal to 7.10(-5) M . The reaction proceeds in the presence of NADPH or NADH . The enzyme affinity to NADPH (Km = 4.7.10(-5) M) is approximately one order higher than that to NADPH (Km = 5.5.10(-4) M) . The enzyme manifests the optimum of action at pH 7.2 and the temperature of 37 degrees C; the molecular weight is 140 kD . EDTA as well as flavins in the concentration of 1.10(-3) M exert no effect on the reductase activity . The enzyme is labile at 4 degrees C and is inactivated in the frozen state at -15 degrees C . The 2.5-diamino-4-oxy-6-ribosylaminopyrimidine-5'-phosphate reductase has been also revealed in Torulopsis candida, Debaryomyces klockeri, Schwanniomyces occidentalis, Eremothecium ashbyii (flavinogenic species) and Candida utilis . Aspergillus nidulans, Neurospora crassa (nonflavinogenic species) . The synthesis of this enzyme contrary to other enzymes of the riboflavin biosynthesis is not regulated in flavinogenic yeast by iron ions. Eur J Biochem, 1989 Jun 15, 182(2), 333 - 41 Characterization of crystalline formate dehydrogenase from Candida methanolica; Izumi Y et al.; The crystalline formate dehydrogenase from Candida methanolica, which showed the highest specific activity (7.52 U/mg) so far reported, was characterized in detail . The enzyme is a dimer composed of identical subunits, each containing one SH group related to the catalytic activity . The molecular mass of the enzyme is about 82-86 kDa . The Km values were found to be 3.0 mM for formate and 0.11 mM for NAD+ . Even if the enzyme was incubated at pH 6.5-9.5 or at 55 degrees C, the activity remained at 100% . Hg2+, Ni2+, NaCN, NaN3 and p-chloromercuribenzoate strongly inhibited the enzyme activity, while the enzyme showed relatively high resistance to various chelating agents . The amino acid composition and some other physicochemical properties of the enzyme were studied . Immunological studies revealed that formate dehydrogenases of methanol-utilizing yeasts immunologically more or less resemble each other, but differ from those of methanol-utilizing bacteria . Furthermore, yeast formate dehydrogenases can be immunologically classified into three types: (a) the Candida type, (b) the Torulopis/Hansenula/Pichia type and (c) the formaldehyde-resistant yeast type . For simple and large-scale preparation of the enzyme for practical use, treatment of cells of C . methanolica with the commercial cationic detergent, 'Benzalkonium' cation, is useful: the total and specific activities of the enzyme are 1.17-fold and 3.10-fold higher than those of the crude cell-free extract, respectively. Biochem Biophys Res Commun, 1989 May 15, 160(3), 1290 - 5 Glucose-6-phosphate dehydrogenase . Characterization of a reactive lysine residue in the Pichia jadinii enzyme reveals a limited structural variation in a functionally significant segment; Jeffery J et al.; Glucose-6-phosphate dehydrogenase from the yeast Pichia jadinii has a reactive lysine residue in a segment of amino acid sequence Ile-Asp-His-Tyr-Leu-Gly-Lys*-Glu-Met-Val-Lys . This structure differs from that of other characterized glucose-6-phosphate dehydrogenases, but outside yeasts the segment is invariant in known mammalian, insect and bacterial forms . Thus, limited structural variation is now defined within yeasts for a part of the protein otherwise strictly conserved, and for which stringent structural requirements probably relate to enzymic mechanisms. Biochemistry, 1989 May 2, 28(9), 4117 - 25 High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris; Sreekrishna K et al.; Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene . Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively . TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density . TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli . TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column . The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence . Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions . Circular dichroic spectra of TNF resemble those of proteins with high beta structure . TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells . In the criteria examined, TNF derived from P . pastoris closely resembles TNF derived from recombinant E . coli and human HL-60 cells. Rev Infect Dis, 1989 May-Jun, 11(3), 369 - 78 New spectrum of fungal infections in patients with cancer; Anaissie E et al.; We report on 44 cancer patients who had serious infections with unusual fungal pathogens and who were cared for at our cancer center between 1974 and 1986 . Twelve different fungal species accounted for these infections, including Trichosporon beigelii, Fusarium species, Geotrichum candidum, Curvularia species, Drechslera species, Penicillium species (but not Penicillium marneffei), Rhodotorula rubra, Pseudallescheria boydii, Pichia farinosa, Torulopsis pintolopesii, Saccharomyces cerevisiae, and Cunninghamella bertholletiae . Skin lesions were noted in seven patients, and sinusitis occurred in four . Twenty-four patients had disseminated infection, 12 had involvement of a single organ, and eight had fungemia alone . Features that correlated with a poor prognosis were persistent neutropenia and disseminated visceral infection but not fungemia alone . We suggest that unusual fungi have now emerged as significant pathogens in this patient population . Fungal sinusitis, previously caused by Aspergillus species and the phycomycetes, also occurs as a result of some of these newly recognized fungi . A high level of suspicion should be maintained when any of these unusual fungi are cultured from clinical specimens from immunocompromised patients. Yeast, 1989 May-Jun, 5(3), 167 - 77 Structural comparison of the Pichia pastoris alcohol oxidase genes; Koutz P et al.; In methylotrophic yeasts, alcohol oxidase is the first enzyme in the methanol-utilization pathway . The genome of one such yeast, Pichia pastoris, contains two alcohol oxidase genes, AOX1 and AOX2 . Sequence analysis indicated that each gene encodes a similar protein of 663 amino acids . The protein-coding regions of the genes were 92% and 97% homologous at the nucleotide and predicted amino acid sequence levels, respectively . In contrast to homology observed within the protein-coding portions of the AOX genes, no homology was found in either the 5' or 3' non-coding regions . Although alcohol oxidase is found in peroxisomes of P . pastoris, the AOX amino acid sequences did not contain a peptide sequence similar to the peroxisomal transport sequence found at the C-terminus of some peroxisomally located proteins in higher eukaryotes. Yeast, 1989 Apr, 5 Spec No, S351 - 4 Estimation of phylogenetic distances among ascomycetous yeasts from partial sequencing of ribosomal RNA; Kurtzman CP; Extent of taxonomic resolution obtained from nuclear DNA complementarity and ribosomal RNA sequencing is discussed . The phylogenetic relationships of Schizosaccharomyces pombe, Pichia stipitis, Pachysolen tannophilus, and Saccharomyces cerevisiae are compared from partial sequences of 18S and 26S ribosomal RNA. Yeast, 1989 Mar-Apr, 5(2), 107 - 15 Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris; Grinna LS et al.; The secreted glycoproteins of Pichia pastoris contain more than 35% of their N-linked oligosaccharides as structures smaller than Man14GlcNAc2 (Man = mannose; GlcNAc = N-acetylglucosamine) . On heterologous invertase produced in P . pastoris, approximately 85% of the oligosaccharides are in the size range Man8-14GlcNAc2 . The structures appear to contain alpha-linked mannose . In addition, one-third of the structures contain net negative charge and can be radio-labelled in vivo with 32P . The largest oligosaccharides isolated from P . pastoris are significantly shorter than the hypermannosylated structures typical of S . cerevisiae, indicating that the factors which influence the processing of N-linked oligosaccharides in P . pastoris are different from those which influence processing in S . cerevisiae . The smaller N-linked oligosaccharides synthesized by P . pastoris resemble high-mannose oligosaccharides synthesized by animal cells, and this finding increases the utility of P . pastoris as a host for the production of heterologous glycoproteins. Mol Cell Biol, 1989 Mar, 9(3), 1316 - 23 Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris; Cregg JM et al.; In Pichia pastoris, alcohol oxidase (AOX) is the first enzyme in the methanol utilization pathway and is encoded by two genes, AOX1 and AOX2 . The DNA and predicted amino acid sequences of the protein-coding portions of the genes are closely homologous, whereas flanking sequences share no homology . The functional roles of AOX1 and AOX2 in the metabolism of methanol were examined . Studies of strains with disrupted AOX genes revealed that AOX1 was the major source of methanol-oxidizing activity in methanol-grown P . pastoris . The results of two types of experiments each suggested that the difference in AOX activity contributed by the two genes was a consequence of sequences located 5' of the protein-coding portions of the genes . First, the coding portion of AOX2 was able to functionally substitute for that of AOX1 when placed under the control of AOX1 regulatory sequences . Second, when labeled oligonucleotide probes specific for the 5' nontranslated region of each gene were used, it was apparent that the steady-state level of AOX1 mRNA was much higher than that of AOX2 . Except for the difference in the amount of mRNA present, the two genes appeared to be regulated in the same manner . A physiological reason for the existence of AOX2 was sought but was not apparent. J Bacteriol, 1989 Mar, 171(3), 1303 - 8 Comparison of glucose uptake kinetics in different yeasts; Does AL et al.; The kinetics of glucose uptake were investigated in laboratory wild-type strains of Saccharomyces cerevisiae of differing genetic backgrounds, in other species of Saccharomyces, and in other yeasts, both fermentative and respiratory . All yeasts examined displayed more than one uptake system for glucose . Variations in apparent Km values, velocity of uptake, and effects of glucose concentration on carrier activity were observed . The three type strains for the species S . cerevisiae, Saccharomyces bayanus, and Saccharomyces carlsbergensis gave distinctive patterns, and each of the laboratory strains was similar to one or another of the type strains . Other fermentative yeasts (Pichia guillermondi and Pichia strasburgensis) regulated glucose uptake in a manner similar to that of Saccharomyces spp . Such was not true for the respiratory yeasts investigated, Pichia heedi and Yarrowia lipolytica, which did not demonstrate glucose repression of carrier activity; this finding suggests that this mechanism of control of transporter activity may be associated with fermentative ability. FEMS Microbiol Lett, 1989 Feb, 48(3), 323 - 7 Differential fructose effect in Pachysolen tannophilus and Pichia stipitis; Bicho PA et al.; The yeasts Pachysolen tannophilus and Pichia stipitis differed in their ability to utilize D-xylose in the presence of D-fructose . When P . tannophilus was grown aerobically in fructose-xylose mixture, the ketohexose was utilized preferentially over the pentose . However, in P . stipitis cultures, the converse was observed . The effect was associated with the ability of D-fructose to repress the induction of xylose reductase and xylitol dehydrogenase activities in P . tannophilus but not in P . stipitis . Both yeasts grew on D-fructose and fermented it to ethanol when it was supplied as the sole carbon source . The results suggest that there may exist some fundamental difference in the regulation of D-fructose metabolism between P . tannophilus and P . stipitis. Tsitol Genet, 1989 Jan-Feb, 23(1), 61 - 5 {Hybridization and recombination in the yeasts Pichia alni, P . bimundalis, P . finlandica, P . glucozyma, P . henricii, P . hostii, P . minuta var . nonfermentans, P . muscicola}; Naumov GI et al.; A collection of genetic lines in 8 reproductively isolated Pichia species has been created . The above data have permitted realizing intraspecific hybridization and showing normal meiotic segregation of auxotrophic markers. Ukr Biokhim Zh, 1989 Jan-Feb, 61(1), 28 - 32 {Regulation of the activity and synthesis of enzymes participating in the formation of 6,7-dimethyl-8-ribityllumazine, a riboflavin precursor in yeast}; Logvinenko EM et al.; The properties of two flavinogenesis enzymes--synthase of the aliphatic precursor of riboflavin (APR-synthase) and 6.7-dimethyl-8-ribityllumazinesynthase (DMRL-synthase) of Pichia guilliermondii . It is established that DMRL-synthase, uses APR as a substrate which contains, evidently, a phosphate group . The value of Km for APR is equal to 0.7.10(-5) M, for 2.4-dihydroxy-5-amino-6-ribitylaminopyrimidine--1.25.10(-5) M . It is riboflavin but not FAD that inhibits the activity of DMRL-synthase; the value (I)0.5 is equal to 2.10(-5) M . DMRL, riboflavin, flavin mononucleotide and FAD do not affect the APR-synthase activity . In iron-deficient cells of P . guilliermondii, Torulopsis candida, Debaryomyces klockeri and Schwanniomyces occidentalis realizing the oversynthesis of riboflavin there occurs derepression of DMRL-synthase and APR-synthase. Antimicrob Agents Chemother, 1989 Jan, 33(1), 48 - 52 Purification and characterization of the anti-Candida toxin of Pichia anomala WC 65; Sawant AD et al.; Pichia anomala WC 65 secretes a toxin that is inhibitory to a variety of yeasts, including strains of the animal pathogen Candida albicans . The toxin was purified to homogeneity by ultrafiltration, ethanol precipitation, ion-exchange chromatography with a Mono Q column, and gel permeation chromatography with a Superose 12 column . The toxin had a molecular weight of 83,300 as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gradient gels and a molecular weight of 85,290 as determined by gel permeation chromatography . The isoelectric point of the toxin was pH 5.0 . The toxin was stable between pH 2.0 and 5.0 . Chemical analysis of the purified toxin indicated that the toxin was a glycoprotein composed of about 86% protein and 14% carbohydrate . At high concentrations, the toxin showed a tendency to aggregate, with loss of biological activity against C . albicans, Pichia bimundalis, and Saccharomycodes ludwigii . Purified toxin expressed killing activity against C . albicans in contrast to the static activity of the crude toxin. J Basic Microbiol, 1989, 29(2), 99 - 128 Sexual behavior and its pheromonal regulation in ascosporogenous yeasts; Yoshida K et al.; We reviewed our investigations on sexual behaviors and interactions including sexual cell agglutination and pheromone action mainly in non-conventional yeasts, Hansenula anomala, H . wingei, Pichia amethionina, P . heedi, P . opuntiae, Saccharomyces kluyveri, S . globsus, S . exiguus, Saccharomycodes ludwigii . The techniques and genetic models including the cassette model and alpha 1-alpha 2 hypothesis which had been developed largely in S . cerevisiae were applicable to these yeasts in principle . The sexual agglutination was distinctly species-specific while sex pheromones were cross-reactive beyond species' barriers . The successful induction of heterothallic strains from homothallic strains in S . exiguus by mutagenesis enabled to the subsequent biochemical and genetical analysis of sexual behavior in the yeast . The phylogenetic consideration on sex differentiation is also included. Ukr Biokhim Zh, 1989 Jan-Feb, 61(1), 32 - 6 {Preparation and properties of immobilized riboflavin kinase from Pichia guilliermondii}; Kashchenko VE et al.; Riboflavin kinase (ATP: riboflavin-5'-phosphotransferase, EC 2.7.1.26) from n-alkane utilizing Pichia guilliermondii yeast has been immobilized by covalent attachment to CNBr-activated agarose beads . The enzyme activity yield during immobilization reached 71.6% . Immobilized riboflavin kinase showed no significant changes in temperature and pH optima as well as in specificity of the action in relation to synthetic substrate analogues with the substitution of methyl groups at positions 7 and 8 of the isoalloxazine ring . Immobilized riboflavin kinase was stable during FMN synthesis in the continuous-flow packed column enzyme reactor with half-life of 27 days. Eur J Epidemiol, 1988 Dec, 4(4), 415 - 8 Killer systems and pathogenic fungi; Polonelli L et al.; Our own studies on the yeast killer phenomenon have been concentrated on its application for the differentiation of opportunistic pathogenic yeast isolates within the same species and its use as an epidemiological marker in nosocomial infections caused by yeasts . Our most recent investigations have led us to reevaluate the potential uses of this phenomenon, since it is now apparent that other microorganisms, unrelated to yeasts, are susceptible to the effects of these toxins . The yeast killer phenomenon can theoretically be used to study epidemiological aspects of any pathogenic microorganism, especially when other systems are not available . Monoclonal antibodies produced against a crude toxic extract of a killer yeast (Pichia anomala UCSC 25F) active against a large number of microorganisms were used to carry out a serological study on metabolic products of various yeasts with known and unknown genetic determinants of their killer characteristics . The extract itself had demonstrated a therapeutic effect in vivo when applied topically . Anti-idiotypic antibodies against these monoclonal antibodies were raised in rabbits . In vitro, these anti-Ids mimicked the action of the killer toxin used as immunogen in the production of monoclonal antibodies . The perspectives of investigations on yeast killer phenomenon are discussed. FEBS Lett, 1988 Sep 26, 238(1), 74 - 6 Unprecedented lysyloxidase activity of Pichia pastoris benzylamine oxidase; Tur SS et al.; Benzylamine oxidase (EC 1.4.3.6) from the yeast Pichia pastoris is a 106 kDa quinoprotein containing one copper atom per molecule . It has a broad substrate specificity ranging from butylamine to peptidyl lysine in collagen and elastin . The kinetic data obtained using lysine-containing model peptides as substrates indicate an astonishing similarity to mammalian lysyloxidase . This similarity is further supported by the inhibition of both enzymes with beta-aminopropionitrile. J Gen Microbiol, 1988 Jul, 134 ( Pt 7), 1893 - 9 The significance of long-chain fatty acid composition and other phenotypic characteristics in determining relationships among some Pichia and Candida species; Viljoen BC et al.; The long-chain fatty acid compositions of 22 species of Candida were determined, and compared with the fatty acid compositions of 10 species of the genus Pichia that contain coenzyme Q9 . The long-chain fatty acid results were also compared with other phenotypic criteria (i.e . assimilation of carbon sources, coenzyme Q type, G + C content and proton magnetic resonance spectra) in order to establish possible anamorph/teleomorph relations . Close correlations were found between known perfect/imperfect states . The results suggest that C . cacaoi and P . farinosa, and C . maltosa and P . etchellsii, also have anamorph/teleomorph relationships. Appl Environ Microbiol, 1988 May, 54(5), 1099 - 103 Anti-Candida albicans activity of Pichia anomala as determined by a growth rate reduction assay; Sawant AD et al.; Killer toxin activity of Pichia anomala WC65 appeared fungicidal for P . bimundalis WC38 and fungistatic for Candida albicans RC1 . Inhibitory activity against sensitive C . albicans showed a linear relationship between toxin concentrations and the inverse of the reduced growth rates . The plot of toxin concentrations against growth rates was hyperbolic, as is characteristic of saturation kinetics . Sensitivity of C . albicans to the toxin decreased with increased cell age . The measurement of growth rate reduction provided a simple and accurate method for quantitation of toxin. J Clin Microbiol, 1988 Mar, 26(3), 602 - 4 Yeast killer toxin-like anti-idiotypic antibodies; Polonelli L et al.; Anti-idiotypic antibodies (anti-Ids) were raised in a rabbit against a murine monoclonal antibody (MAb) neutralizing the yeast killer toxin produced by a strain of Pichia (Hansenula) anomala . In an immunodiffusion test, the anti-Ids produced in the rabbit recognized the antigen-binding site of the MAb used as the immunogen (KT4) but not that of another heterologous MAb . The absence of any significant cross-reactivity among the anti-Ids raised in a rabbit for a heterologous MAb suggested that the anti-Ids were highly specific for unique variable-region determinants . Furthermore, the P . anomala killer toxin proved to be competing with anti-Ids for the binding site of MAb KT4 . Anti-Ids against the MAb to yeast killer toxin inhibited the growth of Candida albicans, thereby mimicking the effect of the yeast killer toxin . These results suggest that, in some cases, anti-Ids might be useful tools for elucidating structure-function relationships for sensitive cell receptors. Ukr Biokhim Zh, 1988 Jan-Feb, 60(1), 97 - 100 {The nature of methanol-induced acidification of the medium by products of metabolism of methylotrophic yeasts}; Gonchar MV et al.; The washed cells of Hansenula polymorpha and Pichia pinus grown on the medium with methanol rapidly acidify the medium during incubation with the mentioned alcohol or formaldehyde . It is found that proton extrusion is coupled with formate anion efflux . Acidification is proved to be energy-dependent process since it is inhibited by respiration poisons, uncouplers of oxidative phosphorylation, and by ATPase inhibitors. J Basic Microbiol, 1988, 28(4), 265 - 78 High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris; Sreekrishna K et al.; Expression of human tumor necrosis factor-alpha (TNF) and four different TNF analogs has been studied in Pichia pastoris by utilizing the alcohol oxidase gene promoter . TNF expression level in certain transformants accounted for as much as 36% of the soluble protein . TNF expression was stably maintained during high cell density fermentation (100 g dry cell weight/liter) resulting in a TNF production level of 6-10 g/liter . TNF contained in P . pastoris cell lysates was biologically active as determined by its cytotoxic effect on murine L-929 fibroblast cells and the bioactivity was retained for at least 6 months in the lysates stored frozen at -20 degrees C in the presence of protease inhibitor PMSF . TNF expressed in P . pastoris was recognized by monoclonal antibodies prepared against recombinant Escherichia coli derived TNF . TNF purified from P . pastoris has the expected N-terminal amino acid sequence and specific activity of 10(7) units/mg protein . TNF analogs were also expressed at levels comparable to that of native TNF . Three of the four analogs were insoluble when produced in P . pastoris. J Basic Microbiol, 1988, 28(5), 293 - 319 Genetic control of methanol utilization in yeasts; Sibirny AA et al.; Considered are our own data and those found in literature on the properties of yeast mutants impaired in their ability to utilize methanol as sole carbon and energy source; hypotheses about the role of alcohol oxidase and citrate synthase in biogenesis of peroxisomes are proposed . It has been proved that formaldehyde reductase participates in the control of the formaldehyde level in the cell . Properties of mutants defective in the catabolite repression and inactivation of enzymes of methanol metabolism are described . The existence of several autonomous mechanisms of the catabolite repression of alcohol oxidase has been shown . It has been found, that the induction of glyoxysomal enzymes of C2-metabolism is repressed by methanol in the ecr1 mutant of Pichia pinus with the affected repression of alcohol oxidase by ethanol . Data are presented on the regulatory properties of the recently discovered acidification system of the medium induced by methanol . Such acidification occurs due to symport extrusion of protons and formate anions from the cells. J Basic Microbiol, 1988, 28(9-10), 619 - 27 Regulation of metabolic branch points of aromatic amino acid biosynthesis in Pichia guilliermondii; Koll P et al.; The regulatory properties of the enzymes involved in the aromatic amino acid biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory pattern found in other yeast species . 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, anthranilate synthase, chorismate mutase and prephenate dehydrogenase are key regulatory enzymes in P . guilliermondii . Two distinctly regulated isozymes of DAHP synthase, the initial pathway enzyme, which is inhibited by tyrosine or phenylalanine were separated by DEAE-cellulose chromatography and were characterized . Tryptophan is an excellent feedback inhibitor of anthranilate synthase, the first definite step in tryptophan biosynthesis . There are two controlled enzymes within the specific synthesis of phenylalanine and tyrosine, chorismate mutase and prephenate dehydrogenase . Chorismate mutase exhibits a balanced allosteric responsivity to phenylalanine and tyrosine, when these are used as inhibitor; tryptophan acts as an allosteric activator . Tyrosine is an effective inhibitor of prephenate dehydrogenase, whereas the activity of prephenate dehydratase is not affected by any of the aromatic amino acids . The synthesis of the enzymes in the yeast was not repressed by any single exogenous aromatic amino acids, nor by combinations of the same. Biochem Int, 1987 Oct, 15(4), 753 - 9 Relevance of cation-exchange capacity of cell wall in detecting H+-symport in yeasts; Lammert T et al.; Addition of mono- or divalent cations to unbuffered yeast suspensions causes acidification of the medium . In some yeasts (Rhodotorula glutinis and Metschnikowia reukaufii) this represented a mediated H+ extrusion in exchange for cations (mostly K+) . However, in some other yeasts (Pichia humboldtii and Candida albicans) the pH change was very rapid . The rapid acidification of the media by Pichia humboldtii was found to be due to high cation-exchange capacity of its cell wall . This cation-exchange was saturable, exhibited high affinity for divalent cations and was stoichiometric under appropriate experimental conditions . Results suggest that such cation exchange capacity of some yeasts could hamper the measurements related to H+-symport mechanisms. Genetika, 1987 Sep, 23(9), 1699 - 701 {Biochemical functions of RIB5 and RIB6 gene products participating in riboflavin biosynthesis in the yeast Pichia guilliermondii}; Logvinenko EM et al.; The biosynthesis of riboflavin precursor 6,7-dimethyl-8-ribityllumazine was studied in extracts of Pichia guilliermondii yeast mutants of rib5 and rib6 genotypes with impaired synthesis of proteins P1 and P2, respectively . It was shown that synthesis of 6,7-dimethyl-8-ribityllumazine took place in extracts of rib5 mutant (active P1 protein) in the presence of 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and the compound formed from ribose-5-phosphate by extracts of rib6 mutant (active P2 protein) . No lumazine was formed in extracts of rib6 mutant from pyrimidine substrate and ribose-5-phosphate preincubated with extracts of rib5 mutant . Hence, P1 protein (the product of RIB5 gene) participates in the biosynthesis of 6,7-dimethyl-8-ribityllumazine from 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and aliphatic intermediate which is formed from ribose-5-phosphate, under the action of P2 protein (the product of RIB6 gene). Genetika, 1987 Sep, 23(9), 1525 - 34 {"Illegal" transformation of red adenine-dependent mutants of the yeast Pichia pinus by the pYE(ADE2)2 plasmid}; Nestat MA et al.; 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. Biochim Biophys Acta, 1987 Jun 12, 900(1), 139 - 44 Role of de novo protein synthesis in the interconversion of glucose transport systems in the yeast Pichia ohmeri; Verma RS et al.; Glucose-repressed cells of the yeast Pichia ohmeri IGC 2879 transported glucose by facilitated diffusion . Derepression led to the formation of a glucose/proton symport and the simultaneous reduction of the facilitated diffusion capacity by about 70% . Cycloheximide prevented this interconversion indicating its dependence on de novo protein synthesis (proteosynthetic interconversion) . In buffer with 2% glucose the glucose/proton symport suffered irreversible inactivation while the facilitated diffusion system was simultaneously restored . This reverse interconversion process did not require de novo protein synthesis as indicated by its lack of sensitivity to cycloheximide (degradative interconversion) . Thus the glucose/proton symport system appeared to consist of about 70% of the facilitated diffusion proteins turned silent through association with additional protein(s) the latter being sensitive to glucose-induced repression and glucose-induced inactivation. Nucleic Acids Res, 1987 May 11, 15(9), 3859 - 76 Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris; Tschopp JF et al.; Two DNA fragments containing putative control regions regulating the expression of the alcohol oxidase (AOX) and dihydroxy-acetone synthase (DAS) genes from the methylotrophic yeast Pichia pastoris were used in the construction of vectors for the expression of the Escherichia coli lacZ gene . These vectors were transformed into P . pastoris host cells and employed in experiments to measure the control mechanisms employed by each promoter in the production of beta-galactosidase fusion products . Results in P . pastoris suggest that the processes used to regulate the expression of these gene fusions involve both repression/derepression and induction mechanisms . Expression of the AOX-lacZ and DAS-lacZ fusions was examined in Saccharomyces cerevisiae as well . Interestingly, beta-galactosidase was expressed in a regulated manner in the heterologous host. Clin Chem, 1987 Apr, 33(4), 486 - 9 Enzymic ethanol assay: a new colorimetric method based on measurement of hydrogen peroxide; Prencipe L et al.; We describe a new colorimetric method for measuring ethanol in plasma by use of a peroxidase-coupled assay system and alcohol oxidase (EC 1.1.3.13) from Pichia species . Absorptivity is low enough to give useful results without sample dilution . The procedure is also applicable to saliva samples and utilizes only one working reagent . The absorbance of the blue dye that is formed is measured at 600 nm . The standard curve for the method is linear for ethanol concentrations up to 4 g/L . Average analytical recovery of ethanol in human plasma exceeded 99% . Within-run and between-run CVs were less than 2.45% and less than 1.92%, respectively . Results correlate very well with those by gas chromatography (r = 0.9977) . The method is adaptable to automation. Radiobiologiia, 1987 Mar-Apr, 27(2), 189 - 94 {Effect of 2-amino-2-thiazoline on the level of lipid peroxidation products in different yeast species}; Zolotareva LT et al.; The influence of 2-amino-2-thiazoline on lipid peroxidation in yeasts Saccharomyces cerevisiae and Pichia guilliermondii has been studied in vivo and in vitro . In the case the radioresistance of diploid yeasts-saccharomycetes is changed the radioprotector can produce a direct effect on lipid peroxidation . Different radioprotective efficiency of the preparation with regard to different strains of one and the same yeast culture is explained by its different influence on the content of endogenous radioprotective factors exerting a control over the accretion of lipid peroxidation products . The observed differences in the lag-periods of peroxidation in Saccharomyces cerevisiae and Pichia guilliermondii correspond to the level of their natural antioxidant activity of lipids. Ukr Biokhim Zh, 1987 Jan-Feb, 59(1), 80 - 2 {The nature of the pyrimidine substrate of 6,7-dimethyl-8-ribityllumazine synthase participating in riboflavin biosynthesis in yeasts}; Logvinenko EM et al.; 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine-5'-phosphate are studied for their effect on the activity of 6,7-dimethyl-8-ribityllumazine synthase of Pichia guilliermondii yeasts . It is shown that when nonphosphorylated form of pyrimidine and ribose-5-phosphate (donor C-4--a fragment) is used as a substrate, the specific activity of 6,7-dimethyl-8-ribityllumazine synthase is high and Be2+ and F- ions, inhibitors of alkaline phosphatases, do not inhibit it . The value of Km for this pyrimidine is 1.1 X 10(-5) M . Phosphorylated pyrimidine being used as a substrate in the presence of Be2+ and F-, the reaction practically does not proceed . Therefore, only 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine is a pyrimidine substrate of 6,7-dimethyl-8-ribityllumazine synthase of yeast. Gene, 1987, 59(1), 115 - 25 Invertase gene (SUC2) of Saccharomyces cerevisiae as a dominant marker for transformation of Pichia pastoris; Sreekrishna K et al.; A two-step method for the selection of transformants of prototrophic industrial strains of the methylotrophic yeast Pichia pastoris has been developed . This method is based on our observation that P . pastoris cannot use sucrose as the sole carbon source (Suc-) and that introduction of the invertase gene (SUC2) of Saccharomyces cerevisiae renders P . pastoris Suc+ . P . pastoris was transformed with a plasmid which contains the SUC2 gene of S . cerevisiae and an autonomously replicating sequence PARS1 from P . pastoris . The transformants were initially allowed to regenerate on medium containing dextrose and the regenerated cells were pooled and plated on sucrose medium to screen for Suc+ transformants . It was shown that the Suc+ transformants of P . pastoris with the autonomously replicating plasmid were highly unstable with respect to the plasmid maintenance, even when grown on sucrose as the sole carbon and energy source . This high instability was attributed to an efficient cross-feeding by Suc- segregants on glucose and fructose generated due to hydrolysis of sucrose by the invertase enzyme secreted by Suc+ cells . Spontaneous integration of the plasmid DNA resulting in a stable Suc+ phenotype was also observed . However, stable Suc+ transformants were obtained more readily by integration of SUC2 into P . pastoris genome following transformation with a linearized plasmid with the ends homologous to P . pastoris HIS4 locus . All such integrants were completely stable for Suc+ phenotype after 20 generations of growth in a nonselective medium. Mol Gen Mikrobiol Virusol, 1986 Dec, (12), 19 - 23 {Transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts by a plasmid carrying the ADE2 gene of Saccharomyces cerevisiae}; Neistat MA et al.; 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. J Clin Microbiol, 1986 Nov, 24(5), 866 - 9 Reevaluation of the yeast killer phenomenon; Polonelli L et al.; The killer effect of 36 Hansenula, Pichia, Saccharomyces, and Candida species on 26 hyphomycetes isolates, 1 isolate of the achlorophyllous microorganism Prototheca, 4 isolates of the lipophilic yeast Malassezia, 1 isolate of the aerobic actinomycete Nocardia, and 19 isolates of bacteria was studied . The killer phenomenon, which was previously considered to be restricted to yeasts, was found to occur among unrelated microorganisms. Arch Biochem Biophys, 1986 Mar, 245(2), 494 - 503 Acetolysis of Pichia pastoris IFO 0948 strain mannan containing alpha-1,2 and beta-1,2 linkages using acetolysis medium of low sulfuric acid concentration; Kobayashi H et al.; To obtain manno-oligosaccharides containing beta-1,2-linked nonreducing terminal groups from the mannan of Pichia pastoris IFO 0948 strain by acetolysis, an attempt was made to establish the reaction conditions under which cleavage of the alpha-1,6 linkage took place preferentially leaving manno-oligosaccharides composed largely of beta-1,2 linkages . By the action of an ordinary acetolysis medium, a 10/10/1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 13 h or at 25 degrees C for 120 h, the O-acetyl derivative of this mannan gave mannose, mannobiose, mannotriose, and mannopentaose . However, treatment of the same O-acetyl mannan with a 50/50/1 (v/v) acetolysis medium at 40 degrees C for 15 h gave a mannotetraose in addition to mannose, mannobiose, mannotriose, and mannopentaose . Use of a 100/100/1 (v/v) acetolysis medium at 40 degrees C for 36 h gave a more satisfactory result, a mixture of oligosaccharides, from mannose to mannopentaose, which contained more mannotetraose than mannopentaose . Because both mannotetraose and mannopentaose contained alpha-1,2 and beta-1,2 linkages, it was concluded that an acetolysis medium containing a low concentration of sulfuric acid, up to 0.5% (v/v), facilitates the preferential cleavage of the alpha-1,6 linkage, leaving manno-oligosaccharides containing the beta-1,2 linkage which was found to be labile to the action of the 10/10/1 (v/v) acetolysis medium. Yeast, 1986 Mar, 2(1), 53 - 8 Inter- and intra-species crosses between Candida albicans and Candida guilliermondii; Suzuki T et al.; Hybridization was shown to occur both between strains of the imperfect diploid yeast Candida albicans and between C . albicans and the distantly related haploid yeast Candida (Pichia) guilliermondii . Prototrophic hybrids were selected from crosses of multiply marked auxotrophic mutants of the two species . In most cases, mild ultraviolet irradiation of the C . albicans partner was required . Examination of auxotrophic markers in segregants from the hybrids indicated that recombination, rather than heterokaryon formation, had occurred in these crosses . The DNA content of the hybrids varied from diploid or aneuploid (for crosses between C . albicans and C . guilliermondii) to triploid (for C . albicans x C . albicans) . It seems possible that genetic exchange analogous to this may occur in nature. Antonie Van Leeuwenhoek, 1986, 52(5), 437 - 43 Pichia triangularis sp . nov., the teleomorph of Candida polymorpha Ohara et Nonomura, nom . nud; Smith MT et al.; The type strain of Candida polymorpha Ohara et Nonomura, nom . nud . was found to produce hat-shaped ascospores . On the basis of its morphology and physiology, it is considered a new species of the genus Pichia and is described as Pichia triangularis sp . nov. Mol Cell Biol, 1985 Dec, 5(12), 3376 - 85 Pichia pastoris as a host system for transformations; Cregg JM et al.; We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations . The system is based on an auxotrophic mutant host of P . pastoris which is defective in histidinol dehydrogenase . As a selectable marker, we isolated and characterized the P . pastoris HIS4 gene . Plasmid vectors which contained either the P . pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P . pastoris mutant host . DNA transfer was accomplished by a modified version of the spheroplast generation (CaCl2-polyethylene glycol)-fusion procedure developed for S . cerevisiae . In addition, we report the isolation and characterization of P . pastoris DNA fragments with autonomous replication sequence activity . Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 10(5)/micrograms and maintained the plasmids as autonomous elements in P . pastoris cells. Mycopathologia, 1985 May, 90(2), 101 - 6 {Coenzyme Q of various species of the genus Pichia: qualitative and quantitative determinations}; Billon-Grand G; The qualitative determination by paper chromatography and the quantitative determination by high performance liquid chromatography (HPLC) of the Coenzyme Q system have been investigated for in 23 species of the genus Pichia . We have adapted HPLC to the quantitative analysis of the Coenzyme Q system . We have found the presence of 2,3 or 4 ubiquinones in the same Coenzyme Q extract. Mol Cell Biol, 1985 May, 5(5), 1111 - 21 Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris; Ellis SB et al.; The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts . The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes . Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined . The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses . Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription . Finally, DNA subfragments of two of the methanol-responsive genomic clones from P . pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation. Biochem J, 1985 Mar 15, 226(3), 669 - 77 Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis; Verduyn C et al.; Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography . The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose . Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21) . Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme . The activity with NADH was approx . 70% of that with NADPH for the various aldose substrates . NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction . These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol. Genetika, 1985 Mar, 21(3), 368 - 74 {Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts . The detection of a new regulator gene RIB81}; Shavlovskii GM et al.; The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied . The mutants were isolated from a genetic line of Pichia guilliermondii . Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium . An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium . The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class . The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency . Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81 . Thus, riboflavin biosynthesis in P . guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81 . The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes . The hybrids of rib81 mutants with natural nonflavinogenic strain P . guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium . Apparently, the strain NF1453-1 contains an unaltered gene RIB81. Curr Genet, 1985, 9(3), 205 - 9 Transformation of Candida maltosa and Pichia guilliermondii by a plasmid containing Saccharomyces cerevisiae ARG4 DNA; Kunze G et al.; Saccharomyces cerevisiae, Candida maltosa and Pichia guilliermondii have been transformed by the plasmid pYe(ARG4)411, which contains the S . cerevisiae ARG4 gene inserted into pBR322 . In all transformants argininosuccinate lyase as well as beta-lactamase were detected . The ARG+ phenotype of transformants is mitotically unstable . Closed circular pYe(ARG4)411 DNA was detected in transformant DNA preparations by hybridization to pBR322 DNA and by transformation of E . coli to ampicillin resistance. Mycopathologia, 1985 Jan, 89(1), 25 - 34 In vitro determination of phagocytic indices of Candida berkhout species by rat peritoneal macrophages; Salim R et al.; The aims of this investigation were to study and describe the behaviour of 13 different species of Candida, as compared with C . albicans, by means of phagocytosis assays in vitro . Tests were carried out with rat peritoneal macrophages in contact with quantified suspensions of live yeasts . Phagocytic indices, candidacidal activity and filamentation rat were tested microscopically after 3 h incubation at 37 degrees C . The phagocytic indices obtained allowed us to separate the fungi into four groups . Candida albicans and tropicalis belong to Group I; diddensii and shehatae, among others, belong to Group II; sake, krusei, viswanathii, etc., Group III; and C . glaebosa and haploid strains of Pichia ohmeri (C . guilliermondii var . membranaefaciens), Group IV . These data would suggest a possible correlation between pathogenesis and phagocytic indices . There were no evidences of any phagocytes ability to kill yeasts . Candidacidal activity was absent in the species assayed . Yeast lysis may have been observed if our assays would have taken longer than 3 h. Radiobiologiia, 1984 Sep-Oct, 24(5), 654 - 6 {The role of glutathione in natural and modified radioresistance of yeast cells}; Zolotareva LT et al.; A study was made of the dependence of different natural and modified radioresistance upon glutathione content of yeast cells (Saccharomyces cerevisiae and Pichia guilliermondii) . It was shown that glutathione was only involved in the formation of natural radioresistance in Saccharomyces cerevisiae cells . It was also shown that the increase in the radioresistance of yeast cells under the effect of 2-amino-2-thiazoline was accompanied by the increase in the level of total glutathione in them. J Appl Bacteriol, 1984 Apr, 56(2), 331 - 5 A note on the presence of novel DNA species in the spoilage yeasts Zygosaccharomyces bailii and Pichia membranaefaciens; Painting KA et al.; Two novel covalently closed circular DNA species of 5.4 and 6.0 kilobases were detected in strains of Zygosaccharomyces bailii with a rapid small scale isolation procedure . The 5.4 kb species was found in four strains and both species were found in three strains . A novel, covalently-closed circular DNA species of 6.9 kb was detected in four of 12 strains of Pichia membranaefaciens . Plasmid DNA (2 micron) (that is CCC DNA of approximately 6 kb in Saccharomyces cerevisiae) was detected in 38 of 40 strains of Sacch . cerevisiae confirming reports of the widespread distribution of this plasmid. Arch Microbiol, 1984 Apr, 137(4), 357 - 61 Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2; Pfeiffer P et al.; A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain . Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized . The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces . Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified . The pH optimum and the isoelectric point were determined . The killer toxins of S . cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of Mr = 16,000 . The amino acid composition of the toxin type K2 of S . cerevisiae strain 399 was determined and compared with the composition of two other toxins. Antonie Van Leeuwenhoek, 1984, 50(3), 227 - 48 The yeasts of British fresh sausage and minced beef; Dalton HK et al.; Three hundred and eighty three yeasts isolated from samples of unsulphited or sulphited sausages and skinless sausages and minced beef were characterized in detail . Debaryomyces hansenii was the most commonly isolated yeast from most samples followed by Candida zeylanoides and Pichia membranaefaciens . The presence of sulphite in sausages did not appear to affect the numbers and range of yeasts present but did affect their relative proportions . A survey of one factory showed that meat intended for sausage production and equipment harboured the same range of yeasts that are found in the finished products. Antonie Van Leeuwenhoek, 1984, 50(3), 209 - 17 Synonomy of the yeast genera Hansenula and Pichia demonstrated through comparisons of deoxyribonucleic acid relatedness; Kurtzman CP; The relationship between the genera Hansenula and Pichia was examined through comparisons of DNA relatedness among phenotypically similar species . Hansenula minuta and Pichia lindneri showed 75% DNA base sequence complementarity . In other comparisons, H . nonfermentans was found to share nearly 50% of its DNA sequences with both H . minuta and P . lindneri . Because of the high degree of relatedness observed, it is proposed that ability to assimilate nitrate, the sole distinction between Hansenula and Pichia, is of insufficient taxonomic value for the reliable separation of either species or genera . Hat-spored species of Hansenula H . et P . Sydow 1919 are being transferred to Pichia Hansen 1904 . Species of Hansenula and Pichia with Saturn-shaped ascospores will be transferred to the genus Williopsis. Nauchnye Doki Vyss Shkoly Biol Nauki, 1984, (5), 34 - 6 {Effect of 2-amino-2-thiazoline on the sulfhydryl group content of yeasts of various degrees of radiosensitivity}; Zolotareva LT et al.; The influence of 2-amino-2-thiazoline on the level of total sulphydryl groups in the haploid and diploid yeast cells Saccharomyces cerevisiae and Pichia guilliermondii has been studied . The increase of radioresistance of the yeast cells has been shown to be connected with the growing content of the endogenous thiols. Mikrobiologiia, 1983 Sep-Oct, 52(5), 806 - 11 {Conditions and characteristics of the early stages of the sexual interaction of Pichia amethionina var . amethionina and Pichia amethionina var . pachycereana yeast cells}; Vorob'ev AV; Pichia amethionina varieties have different sensitivity to the acidity of the medium: P . amethionina var . amethionina has the optimum pH 3 for agglutination and 5 for conjugation; P . amethionina var . pachycereana has the optimum pH 4-6 for agglutination and 6 for conjugation . The optimum temperature for the both organisms is 24 degrees C . Under the optimum conditions, P . amethionina var . amethionina has