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Gene, 1980 Sep, 10(4), 347 - 56 Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: beta-galactosidase from Kluyveromyces lactis; Dickson RC; Three recombinant DNA vectors carrying the beta-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae . All transformants expressed the beta-galactosidase activity of LAC4 . However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes . Enzyme levels probably reflect gene dosage . LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid . Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded beta-galactosidase synthesized in either S . cerevisiae or in K . lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis . However S . cerevisiae transformed with LAC4 cannot grow on lactose, probably because lactose does not enter the cell. Genetics, 1980 Aug, 95(4), 877 - 90 Mutations affecting synthesis of beta-galactosidase activity in the yeast Kluyveromyces lactis; Sheetz RM et al.; Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac-) were isolated and characterized . All the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9 . Strains bearing mutations in lac3, lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal-) . Since the Gal- and Lac- phenotype co-segregate, they are probably due to a single mutation . Strains bearing mutations in any of the seven complementation groups grow normally on glucose . However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium . Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose . Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate chromosome 2 . The remaining five groups are unlinked . Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.--To further characterize the nature of the Lac- phenotype, the basal and inducible level of beta-galactosidase activity were measured . All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels . Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups . In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells . Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of beta-galactosidase . In summary, these data indicate that several genes are necessary for synthesis of beta-galactosidase activity. J Gen Microbiol, 1980 Apr, 117(2), 449 - 54 Ribosomal RNA genes in Kluyveromyces marxianus; Ojha M; DNA from the yeast Kluyveromyces marxianus was studied for its heterogeneity and the multiplicity of rRNA cistrons . These cistrons banded at a slightly different density from the bulk DNA in preparative CsCl or Hg2+-Cs2SO4 equilibrium density gradients . The reassociation kinetics of the bulk DNA showed that the repetitive fraction represented a small amount of the total cellular DNA (10%) and that the single copy fraction had a complexity of 6.3 X 10(9) daltons . Approximately 2.2% of the DNA hybridized to 3H-labelled rRNA at saturation . On the basis of the above genome size, the multiplicity of the rRNA cistrons was calculated to be about 70 per haploid nucleus. J Cell Sci, 1980 Apr, 42, 329 - 56 Magnesium ions and the control of the cell cycle in yeast; Walker GM et al.; A study has been made of the role of magnesium ions in cell division cycle control in the fission yeast, Schizosaccharomyces pombe, and the budding yeast, Kluyveromyces fraglis . Synchronization of cell division in these organismms can be induced by restoring magnesium to magnesium-exhausted cultures . In S . pombe, a correlation exists between the time taken for cells to enter the first synchronous division and the period of magnesium exhaustion . During short-term incubation in magnesium-deficient media, S . pombe cells are observed to continue growth in length, but they fail to make a cell plate and divide; long-term magnesium deficiency results in the production of aberrant cell forms, and a reduction in viability . Analysis of total cell magnesium in cultures of both S . pombe and K . fragilis, synchronized by various induction and selection procedures, revealed that there is a fairly steady fall in magnesium concentration as cells grow, terminating in a rapid influx of magnesium just before cell division . This leads to the hypothesis that falling magnesium concentration may act as a transducer of cell size, eventually triggering spindle formation and a membrane change which permits rapid uptake of magnesium to a concentration which brings about spindle breakdown . The hypothesis was tested directly using the divalent cation ionophore, A23187, in the absence of calcium ions; the results obtained showed that a short pulse of A23187, very late in the cell cycle, accelerated cells into division and shortened the subsequent cycle . The hypothesis is discussed in relation to current models of cell cycle regulation. Sabouraudia, 1980 Mar, 18(1), 69 - 73 Kluyveromyces fragilis as an opportunistic fungal pathogen in man; Lutwick LI et al.; An immunosuppressed cardiac transplant patient with pulmonary infection due to the yeast Kluyveromyces fragilis is described . Isolation of this fungus from human sources is rarely reported, and previous reports of human infection are unavailable in the modern literature . The organism is poorly pathogenic even in immunocompromised hosts . In vitro susceptibility studies with several strains presented here indicate inhibition by 5-fluorocytosine and miconazole, and borderline susceptibility to amphotericin B . The patient was treated with amphotericin B, and recovered without sequelae. Antonie Van Leeuwenhoek, 1980, 46(2), 177 - 89 Hybridization studies within the genus Kluyveromyces van der Walt emend . van der Walt; Johannsen E; Hybridization studies based on the prototrophic selection technique, involving the use of auxotrophic mutants of strains of all accepted species of the genus Kluyveromyces, are reported . Two main groups of mutally interfertile taxa were established withinthe genus . the first group comprises Kluyveromyces bulgaricus, K . cicerisporus, K . dobzhanskii, K . drosophilarus, K . fragilis, K . lactis; K . marxianus, K . phaseolosporus, K . vanudenii and K . wileenii . The second group consists of K . doazhanskii, K . drosophilarum, K . lactis, K . vanudenii and K . wickerhamii . Hybrids were also detected in crosses involving K . drosophilarum and K . waltii as well as K . marxianus and K . thermotolerans . In terms of the concept of the biological species and in compliance with requirements of the International Code of Botanical Nomenclature, taxa which hybridize with K . marxianus and form fertile recombinants at frequencies observed in intraspecific crosses, are accepted as varieties of K . marxianus. Mol Gen Genet, 1980, 179(2), 273 - 82 Studies of ribosomal proteins of yeast species and their hybrids: gel electrophoresis and immunochemical cross-reactions; Adoutte-Panvier A et al.; The cytoplasmic ribosomal proteins (r-proteins) of seventeen yeast species of the genera Saccharomyces and Kluyveromyces were analyzed by one-dimensional gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate . The electrophoretic patterns of cytoplasmic r-proteins from different species display extensive differences in both the 40S and the 60S subunit . Relatedness of species suggested by r-protein patterns correlates well with that based on DNA/DNA homology (Bicknell and Douglas 1970) . Immunochemical cross-reactions and antibiotic susceptibility tests were also used to compare different species . Analyses of r-proteins from two different interspecific hybrids showed that their ribosomes were hybrid, containing r-proteins from both parents . These findings are discussed in relation to the evolution of yeast species and the regulation of expression of r-proteins in eucaryotes. Mol Gen Genet, 1980, 178(2), 351 - 5 Extrachromosomal oligomycin-resistant mutants of the petite-negative yeast Kluyveromyces lactis . Properties of mitochondrial ATPase and cross-resistance to inhibitors of phosphoryl transfer reactions; Brunner A et al.; The mitochondrial ATPase from oligomycin-resistant mutants which map on different regions of an extrachromosomal DNA (01 and 011 class mutants) showed an increased resistance to oligomycin and venturicidin when assayed in vitro as compared to the sensitive strains . The resistance to oligomycin of the isolated mitochondrial ATPase from 01 class mutants was higher than that of the 011 class mutants . Cross resistance of the oligomycin-resistant mutants to the antibiotics peliomycin and ossamycin, which also inhibit phosphoryl transfer reactions in mitochondria (Walter et al., 1967), was observed, 01 mutants being more resistant to ossamycin than 011 class mutants . At the concentrations of peliomycin studied, no difference in sensitivity among both groups of oligomycin-resistant mutants could be detected . Mitochondrial respiration and isolated mitochondrial ATPase activity are sensitive to venturicidin, suggesting that the previously observed (Brunner et al., 1977) in vivo venturicidin resistance of K . lactis is probably due to an impairment of the influx of the drug at the level of the plasma membrane. Sabouraudia, 1979 Mar, 17(1), 71 - 8 Evaluation of industrial yeasts for pathogenicity; Holzschu DL et al.; Eleven yeasts representative of species of industrial interest were compared with Candida albicans for their potential pathogenicity for untreated and cortisone-treated mice . Only C . tropicalis produced a progressive infection similar to that produced by C . albicans . Candida lipolytica, Torulopsis spp., and Hansenula polymorpha were not recovered from mice 6 days after inoculation . Kluyveromyces fragilis, C . pseudotropicalis, C . utilis, C . guilliermondii and C . maltosa were recovered from mice but did not produce evidence of infection. J Immunol, 1979 Mar, 122(3), 1138 - 45 The immunochemistry of antibodies sharing concanavalin A's anti-mannosyl binding specificity; Reichert CM et al.; Procedures were developed for the synthesis of the disaccharide hapten, p-isothiocyanatophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside, and for its conjugation to hemocyanin . The synthetic carbohydrate: protein antigen was then emulsified in complete Freund's adjuvant and injected into the footpads of New Zealand White rabbits . A population of the resulting anti-conjugate antibodies displayed some binding properties analogous to concanavalin A, the carbohydrate-binding protein of the jack bean . The antisera weakly percipitated mannans from Saccharomyces rouxii, S . cerevisiae, and an alpha-(1 leads to 3)-mannopyranosyl transferase-deficient mutant from Kluyveromyces lactis Y58a . These polysaccharides, possessing side chains containing terminal alpha-(1 leads to 2)-mannobiosyl residues, produce strong percipitation reactions with concanavalin A . In addition, various saccharides were tested for their ability to inhibit the interaction of anti-conjugate antisera with alpha-(1 leads to 2)-mannobiosyl-containing polymers . p-Nitrophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside showed a strong complementarity for the binding sites of both the anti-conjugate antisera and concanavalin A . However, the antibody failed to bind a concanavalin A-reactive mouse fibrosarcoma or to stimulate mitogenesis of human peripheral lymphocytes. Antonie Van Leeuwenhoek, 1979, 45(2), 281 - 91 A comparison of interfertility and in vitro DNA-DNA reassociation as criteria for speciation in the genus Kluyveromyces; van der Walt JP et al.; Hybridization studies based on the use of the prototrophic selection technique were undertaken to compare interfertility and DNA-DNA reassociation as criteria for speciation purposes in the yeast genus Kluyveromyces . Degrees of DNA-DNA reassociation greater than 70% between strains, as reported in the literature, were found to correlate with high recombination frequencies . Degrees of DNA-DNA reassociation less than 20% between strains did, however, not invariably coincide with the absence of interfertility between strains . If interfertility is accepted as criterion for conspecificity, the hitherto reported low degrees (less than 20%) of DNA-DNA reassociation in Kluyveromyces cannot confidently be employed for speciation purposes. Antonie Van Leeuwenhoek, 1979, 45(2), 241 - 52 The Pasteur effect and catabolite repression in an oxidative yeast, Kluyveromyces lactis; Royt PW et al.; The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation . Extracts from these cells contained ATP-sensitive phosphofructokinase activity . Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation . Phosphofructokinase in these cells was ATP-insensitive . The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation . These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K . lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase . Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity . The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts. Antonie Van Leeuwenhoek, 1979, 45(1), 55 - 63 Characterisation and metabolic studies of Saccharomyces cerevisiae and Kluyveromyces fragilis by flow microcalorimetry; Beezer AE et al.; The use of microcalorimetry in the routine identification of microorganisms is critically discussed and assessed . By use of flow microcalorimetric studies on Saccharomyces cerevisiae and Kluyveromyces fragilis the role of physical parameters and that of oxygen tension are discussed . The conclusion reached is that identification of microorganisms by microcalorimetry and subsequent discussion of metabolic events revealed by the thermogram, except under restrictive conditions, is inappropriate . However flow microcalorimetry, in contrast to batch microcalorimetry which has been used in the published material on microorganism identification, may allow characterization of yeasts suitable for particular industrial processes. J Bacteriol, 1979 Jan, 137(1), 51 - 61 Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis; Dickson RC et al.; beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose . When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed . The enzyme has a sedimentation coefficient of 9.6S . This beta-galactosidase and the one from Escherichia coli are not antigenically related . Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent . beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively . The hydrolase and transgalactosylase activities of the enzyme are similar to those of E . coli beta-galactosidase. Cell, 1978 Sep, 15(1), 123 - 30 Molecular cloning and expression in E . coli of a yeast gene coding for beta-galactosidase; Dickson RC et al.; The yeast Kluyveromyces lactis synthesizes a beta-galactosidase (EC 3.2.1.32) which is inducible by lactose . We have isolated the gene that codes for this enzyme using recombinant DNA techniques . K . lactis DNA was partially digested with the restriction endonuclease Eco R1 and joined to Eco R1-digested pBR322 plasmid DNA using DNA ligase . ligase . A lac-mutant of Escherichia coli lacking the structural gene for beta-galactosidase was transformed with ligated DNA . Three lac+ transformants containing recombinant plasmids were selected . Two of the plasmids (pK15 and pK17) contain four Eco R1-K . lactis DNA fragments having molecular weights of 2.2, 1.4, 0.55 and 0.5 x 10(6) daltons . The other plasmid (pK16) lacks the smallest fragment . E . coli carrying any of these plasmids produce beta-galactosidase activity that has a sedimentation coefficient and immunological determinants that are nearly identical to K . lactis beta-galactosidase and distinctly different from E . coli beta-galactosidase . DNA-DNA hybridization studies show that the four Eco R1 fragments in pK15 hybridize to K . lactis but not to E . coli DNA. Nucleic Acids Res, 1978 Jul, 5(7), 2577 - 86 Synthesis of DNA in permeabilized cells of Kluyveromyces lactis; Badaracco G et al.; Kluyveromyces lactis cells permeabilized with nystatin, though no longer viable, were able to incorporate 3H-dATP into DNA . Maximum rate of synthesis was obtained when all four deoxyribonucleoside triphosphates were present . For prolonged incorporation of 3H-dATP into DNA rATP or phosphoenolpyruvate were of absolute requirement . DNA synthesis was inhibited by p-chloromercuribenzoate, N-ethylmaleimide, nalidixate, ethidium bromide and distamycin A . The density of DNA synthesized in permeabilized cells grown on non-fermentable and fermentable carbon sources was analyzed on CsCl gradients in the presence or absence of distamycin A . The DNA synthesized by permeabilized cells previously grown on glycerol was essentially mitochondrial DNA; nuclear DNA (30% of total) was also synthesized by cells previously grown on glucose. Microbios, 1978, 22(88), 73 - 84 Flow microcalorimetric investigation of yeast growth in a complex medium; Beezer AE et al.; The growth of Kluyveromyces fragilis in a complex medium under anaerobic and aerobic growth conditions has been conducted using a flow microcalorimeter . Growth under defined conditions is characterized by an initial exponential rise in heat output rate to a maximum, followed by a decline to a baseline deflection . Inadequate oxygenation can result in a more structured thermogram . The results are used to illustrate the deficiencies of some calorimetric incubations, and observations reported by other authors are critically discussed. Antonie Van Leeuwenhoek, 1978, 44(2), 177 - 82 Different phenotypes for the lactose utilization system in Kluyveromyces and Saccharomyces species; Algeri A et al.; Analysis of the system for the utilization of lactose in some strains of yeasts belonging to the genera Kluyveromyces and Saccharomyces revealed the occurrence of several genetic variants, corresponding to mutants expected on the basis of the model of Jacob and Monod for the lac system in E . coli . These results could be useful in the taxonomic analysis and in the phylogenetic evaluation of the Saccharomycetaceae. Can J Biochem, 1977 Sep, 55(9), 1001 - 6 Purification and partial characterization of an exo-beta-glucanase from the yeast Kluyveromyces aestaurii; Lachance MA et al.; The intracellular-periplasmic exo-1,3-beta-glucanase (EC 3.2.1.58) has been extracted from the yeast Kluyveromyces aestuarii and purified to immunoelectrophoretic homogeneity by ion-exchange and gel-exclusion chromatography . The kinetic constants and activation energies for laminarin, p-nitrophenyl-beta-D-glucoside, and pustulan have been determined, along with the effect of pH . Evidence is presented indicating that the enzyme is composed of a single polypeptide chain, about 24% carbohydrates, and its molecular weight was estimated to be 43 000. Arch Microbiol, 1977 Aug 26, 114(2), 137 - 42 Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis; Hossack JA et al.; Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells . Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S . cerevisiae whether grown at 30 degrees C (generation time 1.1 h) or 20 degrees C (generation time 2.1 h) . Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown . Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.
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