Yeast, 1998 Jan 15, 14(1), 37 - 48 The lysine-rich C-terminal repeats of the centromere-binding factor 5 (Cbf5) of Kluyveromyces lactis are not essential for function; Winkler AA et al.; The gene coding for the centromere-binding factor 5 (CBF5) of Kluyveromyces lactis has been isolated by hybridization of a Saccharomyces cerevisiae CBF5 DNA probe to a K . lactis library . The amino acid sequence of KlCbf5 is highly homologous, 88% identity, to ScCbf5, but also to the rat protein Nap57 (64% identity) . The main difference between both yeast proteins and the rat protein is the presence of a lysine-rich domain with KKE/D repeats in the C-terminal part of the protein . These repeats are thought to be involved in binding of the protein to microtubules . Deletion of the KKE/D domain in KlCbf5 however, has no discernible effect on growth on rich medium, sensitivity to the microtubule-destabilizing drug benomyl or segregation of a reporter plasmid . On the other hand, insertion of two leucine residues adjacent to the KKE domain increases the loss rate of a reporter plasmid . In both yeasts complementation of a lethal CBF5 disruption with the heterologous gene results in a slight increase in benomyl sensitivity . A possible role of CBF5 in chromosome segregation will be discussed. Folia Microbiol (Praha), 1997, 42(4), 319 - 23 Biochemical and molecular-genetic properties of a cytochrome-c-deficient mutant of Kluyveromyces lactis; Hikkel I et al.; We have isolated a respiration-deficient nuclear mutant of the yeast Kluyveromyces lactis that exhibited diminished levels of all cytochromes and did not grow on glycerol and other nonfermentable carbon sources . The mutant named cyc1 was transformed with a K . lactis genomic library and the DNA fragment conferring its wild-type properties was isolated and sequenced . The sequence of the isolated gene showed extensive homology with other eukaryotic cytochrome-c genes . The highest level of homology, based on the deduced amino acid sequences, was observed between the gene products of K . lactis and Hansenula anomala. Mol Gen Genet, 1997 Dec, 257(1), 62 - 70 Characterization of an AP-1-like transcription factor that mediates an oxidative stress response in Kluyveromyces lactis; Billard P et al.; The KlYAP1 gene, encoding the transcription factor Yap1p from Kluyveromyces lactis, was cloned by functional complementation of the cadmium hypersensitivity phenotype of a Saccharomyces cerevisiae strain lacking functional YAP1 and YAP2 genes . The KlYAP1 gene product is 41% identical to Yap1p, the sequence similarity being centered on the bZip domain and extending into the C-terminal portion of both proteins . When expressed in S . cerevisiae, this gene efficiently complements some of the phenotypes associated with both yap1 and yap2 mutations and also mediates AP-1 response element-dependent transcriptional activation in response to H2O2 . Gene disruption experiments in K . lactis indicated that the KlYAP1 gene is involved in both the oxidative and cadmium response pathways . We also demonstrate the existence in K . lactis of inducible protective stress responses to both peroxides and superoxides and investigate the role of the Klyap1p protein in these responses. Yeast, 1997 Dec, 13(15), 1399 - 8 Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis; Gonzalez FJ et al.; The DAO1 gene of Trigonopsis variabilis encoding a D-amino acid oxidase (EC 1.4.3.3) was isolated from genomic clones selected for their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that have been conserved through evolution . The nucleotide sequence of the gene predicts a protein with similarities to human, pig, rabbit, mouse and Fusarium solani D-amino acid oxidases . The open reading frame of the T . variabilis DAO1 gene was interrupted by an intron . The Dao1p sequence displays two regions, one in the N-terminal section--the FAD binding site--and the other near the C-terminal region that contains conserved signatures found in all the D-amino acid oxidases . The three C-terminal amino acids suggest that the enzyme may be located in peroxisomes . Northern blot experiments showed that no transcriptional activation occurred in the presence of D-methionine . The cDNA encoding Dao1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis . Both yeast species are able to synthesize a functional enzyme under the control of the GAL1 promoter . In K . lactis, up to six times more enzyme units per gram of dry weight are produced with a multicopy plasmid in comparison with the wild-type strain of T . variabilis . The yeast expression system we describe may constitute an alternative source for the production of D-amino acid oxidases at industrial level. Appl Environ Microbiol, 1998 Jan, 64(1), 94 - 7 Cloning and characterization of two pyruvate decarboxylase genes from Pichia stipitis CBS 6054; Lu P et al.; In Pichia stipitis, fermentative and pyruvate decarboxylase (PDC) activities increase with diminished oxygen rather than in response to fermentable sugars . To better characterize PDC expression and regulation, two genes for PDC (PsPDC1 and PsPDC2) were cloned and sequenced from P . stipitis CBS 6054 . Aside from Saccharomyces cerevisiae, from which three PDC genes have been characterized, P . stipitis is the only organism from which multiple genes for PDC have been identified and characterized . PsPDC1 and PsPDC2 have diverged almost as far from one another as they have from the next most closely related known yeast gene . PsPDC1 contains an open reading frame of 1,791 nucleotides encoding 597 amino acids . PsPDC2 contains a reading frame of 1,710 nucleotides encoding 570 amino acids . An 81-nucleotide segment in the middle of the beta domain of PsPDC1 codes for a unique segment of 27 amino acids, which may play a role in allosteric regulation . The 5' regions of both P . stipitis genes include two putative TATA elements that make them similar to the PDC genes from S . cerevisiae, Kluyveromyces marxianus, and Hanseniaspora uvarum. Plasmid, 1997, 38(3), 202 - 9 A circular plasmid from the yeast Torulaspora delbrueckii; Blaisonneau J et al.; A new member of the 2-micron family of plasmids, named pTD1, was found in the yeast Torulaspora delbrueckii, a widespread yeast associated with food . Nucleotide sequences revealed the presence of a pair of inverted repeats and three open reading frames, one of which is a homologue of the FLP recombinase gene of 2-micron plasmid . An ARS region was identified, by replication in Saccharomyces cerevisiae and T . delbrueckii, near one of the inverted repeats . By the use of pTD1 derivatives and auxotrophic mutant hosts an efficient host-vector system was established for T . delbrueckii . So far, the 2-micron family of plasmids is restricted to four closely related genera (Q6 group): Saccharomyces, Zygosaccharomyces, Kluyveromyces, and Torulaspora . After a survey of 2500 strains belonging to about 500 species (80 genera) of yeast, no circular plasmids were found in other genera. Biochim Biophys Acta, 1997 Dec 5, 1343(2), 251 - 62 Multiple unfolded states of UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis . Involvement of proline cis-trans isomerization in reactivation; Dutta S et al.; UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis is a dimeric molecule of 75 kDa per subunit with one molecule of cofactor NAD per dimer . It undergoes unfolding and complete dissociation in presence of 8 M urea at pH 7.0 by 10 min . It can be functionally reconstituted almost quantitatively in 2 h by dilution with 20 mM sodium phosphate buffer, pH 7 containing 1 mM extraneous NAD under a second order kinetics {Bhattacharyya, D . (1993) Biochemistry 32, 9726-9734} . Denaturation between 10-60 min inversely affects both the rate and maximum recovery of activity upon refolding . Aggregation of this protein has not been observed under these conditions . The time dependent reaction at the unfolded state is independent of pH between 5.4-10.4 but strongly dependent on temperature of denaturation between 0-20 degrees C . Unfolding at 0 degrees C divides the protein largely into two populations-34% of fast folding species following an apparent first order kinetics and 59% of slow folding species following a second order kinetics of reactivation . A very fast folding species of low abundance 3.5-7.5% depending on temperature of denaturation has been identified, which gets active status within the dead time of mixing . Interaction with the active site directed fluorescence probe 1-anilino 8-naphthalene sulfonic acid (1-ANS) and estimation of bound NAD suggest that the catalytic region of this enzyme is not formed in the long term denatured samples . The whole process of reactivation is catalysed by peptidyl prolyl cis-trans isomerase and thus suggests that one or more proline residues stereochemically control the rate limiting step of reactivation. Biotechnol Appl Biochem, 1997 Dec, 26 ( Pt 3), 189 - 94 Differences in the growth kinetic behaviour of Torulopsis cremoris in batch and continuous cultures; Cristiani-Urbina E et al.; Torulopsis cremoris growth in whey was described satisfactorily by Monod's model . The maximum specific growth rate of T . cremoris obtained in batch culture was approximately 26% higher than that estimated in continuous culture . This rate was higher than that reported for Kluyveromyces fragilis, which is the yeast used in large-scale processes for the biomass production from whey . In batch and continuous cultures, the yeast utilized some other carbon and energy source, different from lactose . In batch cultures, the overall growth yield coefficients exhibited a significant dependence on initial lactose concentration . The single-stage continuous culture is not a convenient system for T . cremoris . The results obtained in this work demonstrate that the kinetic parameters may vary significantly according to the culture type used, and this could have economic repercussions. RNA, 1997 Nov, 3(11), 1337 - 51 Polyadenylation of telomerase RNA in budding yeast; Chapon C et al.; Telomerase RNA is a subunit of a stable ribonucleoprotein particle required for telomere replication . We find that, at steady state, 5-10% of the telomerase RNA in Saccharomyces cerevisiae and Kluyveromyces lactis contains a poly(A) tail of about 80 nt . In S . cerevisiae, the poly(A)+ fraction quickly disappeared when a conditional pap1 or rna15 mutant was shifted to the nonpermissive temperature, indicating that polyadenylation is accomplished by the same machinery that polyadenylates mRNAs . Potential cis-acting polyadenylation elements were identified in the telomerase RNA sequence; when they were mutagenized, the polyadenylation pattern shifted, but was not eliminated . The corresponding mutants displayed wild-type growth . By putting the RNA under the control of an inducible promoter, we were able to show that synthesis of the poly(A)+ RNA precedes that of the poly(A)- fraction . This supports, but does not prove, a model in which all telomerase RNA is first polyadenylated and then rapidly processed to give the stable poly(A)-form . Cell cycle arrest experiments showed an increase in the poly(A)+ form between G1 and S phase, consistent with an induction of telomerase RNA transcription at the time of DNA replication. Eur J Biochem, 1997 Nov 1, 249(3), 762 - 9 The role of subunit VIII in the structural stability of the bc1 complex from Saccharomyces cerevisiae studied using hybrid complexes; Boumans H et al.; The QCR8 genes encoding subunit VIII of the bc1 complex from Kluyveromyces lactis and Schizosaccharomyces pombe partially complement the respiratory-deficient phenotype of a S . cerevisiae QCR8-null mutant . This implies that the heterologous Qcr8 subunits can be imported by S . cerevisiae mitochondria and that they assemble to form a hybrid bc1 complex that is sufficiently active to support growth . In contrast, the QCR8 gene from bovine heart, encoding the 9.5-kDa subunit, is not able to restore respiratory function to the S . cerevisiae null mutant . This lack of functional complementation is directly attributable to the inability of S . cerevisiae mitochondria to import this protein as shown by in vitro assays . However, a hybrid gene encoding the N-terminal 26 residues of S . cerevisiae subunit VIII and the rest of the 9.5-kDa bovine heart homologue, was able to functionally complement the QCR8-null mutant, albeit to a very low extent . Successful import into S . cerevisiae mitochondria was confirmed by in vitro import experiments . Surprisingly, although assembly of these hybrid complexes is reduced to an extent that is proportional to the evolutionary distance of the homologue to S . cerevisiae, the specific activities of the assembled complexes are the same as for the wild-type bc1 complex . After solubilisation of the mitochondrial membranes with the mild detergent dodecyl maltoside, the wild-type enzyme can be inactivated by incubation at increased temperature, independent of protease activity . The rate of inactivation can be significantly increased by the addition of o-phenanthroline {Boumans, H., Grivell, L . A . & Berden, J . A . (1997) J . Biol . Chem . 272, 16753-16760} . The hybrid complexes are much more sensitive to both types of treatment . We conclude that substitution of subunit VIII by a homologous counterpart results in a loosening of the structure of the bc1 complex on the intermembrane space side, resulting in a less stable insertion of the Rieske Fe-S protein in vivo and therefore a lower stability of the assembled enzyme under certain in vitro conditions, but without an effect on catalytic activity. Yeast, 1997 Nov, 13(14), 1309 - 17 Purification and characterization of phosphofructokinase from the yeast Kluyveromyces lactis; Bar J et al.; Phosphofructokinase from Kluyveromyces lactis was purified by 180-fold enrichment, elaborating the following steps: cell disruption, polyethylene glycol precipitation, affinity chromatography, size exclusion chromatography on Sepharose 6B and on Bio-Sil SEC 400 and ion exchange chromatography . The homogeneous enzyme exhibits a molecular mass of 845 +/- 20 kDa as determined by sedimentation equilibrium measurements and a specific activity of 100 units/mg protein . The apparent sedimentation coefficient was found to be s20,c = 20.7 +/- 0.6 S and no significant dependence on the protein concentration was observed in a range from 0.2 to 8 mg protein/ml . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to molecular masses of 119 +/- 5 kDa and 102 +/- 5 kDa, respectively . Thus, the enzyme assembles as octamer composed of two types of subunits . From Western blot analysis applying subunit-specific monoclonal antibodies raised against Saccharomyces cerevisiae phosphofructokinase and from the determination of the N-terminal amino acid sequence, the conclusion was drawn that the 102 kDa-subunit corresponds to the beta-subunit of the S . cerevisiae enzyme . In contrast to bakers' yeast phosphofructokinase, the K . lactis enzyme exhibits no cooperativity with respect to the substrate fructose 6-phosphate . Both activators AMP and fructose 2,6-bisphosphate decrease the Michaelis constant with respect to this substrate . The enzyme from K . lactis is also inhibited by ATP . Fructose 2,6-bisphosphate or AMP diminish the ATP-inhibition . In contrast to the phosphofructokinase from S . cerevisiae, where fructose 2,6-bisphosphate turned out to be more efficient than AMP, both activators exert similar effects on the K . lactis enzyme. J Basic Microbiol, 1997, 37(5), 307 - 12 Physiological and biochemical characterization of intergeneric hybrids of thermotolerant and non-thermotolerant yeasts; Dhamija SS et al.; Kluyveromyces-like intergeneric hybrids of thermotolerant Kluyveromyces marxianus and non-thermotolerant Saccharomyces cerevisiae, produced in a previous study by protoplasmic fusion, have been characterized . On molasses, these strains produced ethanol in excess of 6% (v/v) both at 30 and 45 degrees C as against 3% and 4.2% (v/v) by the former parent at 30 and 45 degrees C, respectively . In hybrids, the increase in ethanol production appeared to be a sequel to increased activities of alcohol dehydrogenase and pyruvate kinase, derived probably from S . cerevisiae parent . Hybrid ADH-isozyme pattern on polyacrylamide gel corroborated the presence of S . cerevisiae ADH in the tested hybrids . Regression analyses indicated a positive correlation between ethanol production and ADH or PK or both (r approximately 0.76-0.84). Eur J Biochem, 1997 Oct 1, 249(1), 248 - 57 Influence of mutations in hexose-transporter genes on glucose repression in Kluyveromyces lactis; Weirich J et al.; The variability of Kluyveromyces lactis strains in sensitivity to glucose is correlated with genetic differences in Kluyveromyces hexose transporter (KHT) genes . The glucose sensitive strain JA6 was shown to contain an additional gene, KHT2, not found in strains that are less sensitive . KHT2 is tandemly arranged with KHT1 which is identical to the low-affinity transporter gene RAG1, except for the C-terminus . Sequence analysis indicated that most of KHT2 had been lost by a recombination event between KHT1 and KHT2 generating the chimeric gene RAG1 . Recombination between KHT1 and KHT2 was also found in mutants of JA6 selected as 2-deoxyglucose resistant colonies . These mutants, like kht1 kht2 double mutants were unable to grow on glucose when respiration was blocked (Rag- phenotype) and glucose repression was strongly reduced . kht1 or kht2 single mutants of JA6 were Rag+ but still an influence of the kht mutations on glucose repression was detectable . Repression was not affected in a Rag- mutant deleted for the phosphoglucose isomerase gene suggesting that the influence of transporter genes on repression is not caused by a reduction of the glycolytic flux . The data rather suggest that sensitivity to glucose repression is dependent on the rate of glucose uptake. J Dairy Sci, 1997 Oct, 80(10), 2264 - 9 Differences in the hydrolysis of lactose and other substrates by beta-D-galactosidase from Kluyveromyces lactis; Kim SH et al.; The hydrolysis of o-nitrophenyl galactopyranoside and lactose by beta-D-galactosidase from Kluyveromyces lactis was enhanced by the addition of Mg2+ and Mn2+, but the rates of activation by each metal on both substrates were not the same . The Co2+, Zn2+, and Ni2+ activated the o-nitrophenyl galactopyranoside-hydrolyzing activity of the enzyme, but these same metals inhibited the lactose-hydrolyzing activity . The addition of Mg2+ and EDTA to the assay buffer increased the hydrolysis of o-nitrophenyl galactopyranoside and lactose at different rates . The responses of o-nitrophenyl galactopyranoside and lactose to the enzyme activity were different as a function of pH . The hydrolyzing activity toward both substrates also was influenced by the concentration of the phosphate in the assay buffer . However, the profile of the enzyme activity toward each substrate was different as a function of concentration . Because the assay of beta-galactosidase using o-nitrophenyl galactopyranoside is fast and convenient, the estimation of lactose-hydrolyzing activity of the enzyme has frequently been made based on the assay of o-nitrophenyl galactopyranoside hydrolysis . As shown in this study, a slight change in the conditions of the assay system and the enzyme application may cause changes in the ability of the enzyme to hydrolyze both lactose and o-nitrophenyl galactopyranoside . The change in o-nitrophenyl galactopyranoside-hydrolyzing activity is not always consistent with that of the lactose-hydrolyzing activity under the given condition, which may cause an inaccurate estimation of the enzyme activity in the enzyme preparation as well as in actual applications of the enzyme. J Photochem Photobiol B, 1997 Sep, 40(2), 132 - 40 Single strand breaks and mutagenesis in yeast induced by photodynamic treatment with chloroaluminum phthalocyanine; Paardekooper M et al.; Photodynamic treatment of the yeast Kluyveromyces marxianus with the sensitizer aluminum phthalocyanine results in loss of clonogenicity . In this paper the effect of this treatment on DNA of this yeast was investigated by searching for single strand breaks and forward mutations . Using the alkaline step elution technique it was found that illumination of the yeast in the presence of aluminum phthalocyanine resulted in an increase in single strand breaks . These could, partially, be repaired by post-incubating illuminated cells in growth medium . At comparable survival levels, photodynamic treatment with aluminum phthalocyanine induced fewer single strand breaks than X-ray treatment . By using a medium containing 5-fluoroorotic acid, mutants in the uracil biosynthetic pathway were selected . Photodynamic treatment resulted in a light dose dependent increase of the mutation frequency . The observed mutagenicity of photodynamic treatment of the yeast with phthalocyanine was lower than the mutagenicity of UVC and X-ray treatment at equal colony forming capacity, indicating that photodynamic treatment is the least mutagenic of those treatments . It is concluded that photodynamic treatment of K . marxianus results in DNA damage . Saccharomyces cerevisiae rad14 and rad52 mutants were used to determine the effect of the nucleotide excision repair and recombinational repair pathways, respectively, on survival after photodynamic treatment . Our data indicate that DNA damage is not the main determinant for cell killing by photodynamic treatment and that the type of damage induced is apparently not subject to RAD14- or RAD52 controlled repair. Arch Biochem Biophys, 1997 Oct 15, 346(2), 294 - 302 Inactivation of the Kluyveromyces lactis H+-ATPase by dicyclohexylcarbodiimide: binding stoichiometry and effect of nucleophiles; Velazquez I et al.; Dicyclohexylcarbodiimide (DCCD) inactivated the plasma membrane H+-ATPase (EC 3.6.1.35) from Kluyveromyces lactis, with a second-order rate constant of 420 M(-1) min(-1) . The inhibition kinetics was apparently complex, due to degradation of DCCD with time . Neither Mg2+ nor Mg-ADP affected the inactivation of the ATPase by DCCD . In contrast, vanadate, a transition state analog of phosphate, partially protected the enzyme with a Kd of 14 microM, indicating a coupling between the DCCD-reactive site and the vanadate-binding site . The incubation of H+-ATPase with 14C-DCCD showed that the incorporation of 1.2 mol of DCCD/mol ATPase leads to complete inactivation . The hydrophobic carbodiimide reacted with the protonated form of the carboxylic group, which displayed a pKa of 7.4, strongly suggesting that the residue is in the hydrophobic environment of the membrane . Benzylamine increased the rate of inactivation by DCCD . In this case, full inactivation of the enzyme was associated with the incorporation of 2.4 mol of DCCD/mol of enzyme, indicating the opening of new reactive sites, resulting from a conformational change induced by benzylamine. Curr Genet, 1997 Oct, 32(4), 267 - 72 Cloning and characterization of KlCOX18, a gene required for activity of cytochrome oxidase in Kluyveromyces lactis; Hikkel I et al.; We describe the isolation and initial characterization of KlCOX18, a gene that is essential for the assembly of a functional cytochrome oxidase in the yeast Kluyveromyces lactis . Cells carrying a recessive nuclear mutation in this gene are respiratory deficient and contain reduced levels of cytochromes a and a3 . The KlCOX18 gene has been cloned by complementation of the respective nuclear mutation, sequenced, and disrupted . KlCOX18 is located on chromosome II and contains an open reading frame of 939 base pairs . The corresponding protein exhibits 70.4% similarity to the Cox18p of Saccharomyces cerevisiae . It contains three possible membrane-spanning domains and a putative amino-terminal mitochondrial import sequence . The strain carrying a null mutation in KlCOX18 does not grow on non-fermentable carbon sources and is deficient in both cytochrome c oxidase and respiratory activity . It is proposed that KlCox18p, like its S . cerevisiae counterpart, provides an important function at a later step of the cytochrome oxidase assembly pathway. Eur J Biochem, 1997 Sep 15, 248(3), 903 - 12 The Kw recombinase, an integrase from Kluyveromyces waltii; Ringrose L et al.; Site-specific recombinases of the integrase family share limited amino-acid-sequence similarity, but use a common reaction mechanism to recombine distinct DNA target sites . Here we report the characterisation of the Kw site-specific recombinase, encoded on the 2 mu-like plasmid pKWS1 from the yeast Kluyveromyces waltii . Using in vitro-translated Kw recombinase, we show that the protein is able to bind and to recombine its putative DNA target site . Recombination is conservative and the Kw target site has a spacer of seven base pairs . We show that Kw recombinase is able to mediate recombination in a mammalian cell line, thus, it has potential for use as a tool for genomic manipulation in heterologous systems. Curr Genet, 1997 Aug, 32(2), 139 - 46 Characterization of the bip gene of Aspergillus awamori encoding a protein with an HDEL retention signal homologous to the mammalian BiP involved in polypeptide secretion; Hijarrubia MJ et al.; A DNA fragment containing an open reading frame of 2016 nucleotides has been cloned from the DNA of Aspergillus awamori by hybridization with a probe internal to the KAR2 (BiP) gene of Saccharomyces cerevisiae . The 73.4-kDa-encoded protein showed very high similarity to the endoplasmic reticulum (ER) lumenal BiP protein of S . cerevisiae, Kluyveromyces lactis, Schizosaccharomyces pombe, and animal and plant cells . The BiP protein contains a polar N-terminal end followed by a 18-amino-acid strongly hydrophobic region corresponding to the leader peptide for transport through the ER membrane . In the C-terminal region the protein ends with the HDEL canonical ER retention signal that targets proteins to the lumen of the ER . The A . awamori bip gene contains three introns as shown by cloning and sequencing the putative intron regions from a cDNA library . The bip gene is transcribed as a monocistronic mRNA of 2.4 kb . Two transcription start sites located 160 and 233 bp upstream of the first translated ATG were identified by primer extension . The promoter region showed no consensus TATA box but it contains CCAAT and CreA boxes known to be involved in both stress and carbon-catabolite regulation of fungal promoters. Nucleic Acids Res, 1997 Sep 15, 25(18), 3657 - 64 Glucose represses the lactose-galactose regulon in Kluyveromyces lactis through a SNF1 and MIG1- dependent pathway that modulates galactokinase (GAL1) gene expression; Dong J et al.; Expression of the lactose-galactose regulon in Kluyveromyces lactis is induced by lactose or galactose and repressed by glucose . Some components of the induction and glucose repression pathways have been identified but many remain unknown . We examined the role of the SNF1 (KlSNF1) and MIG1 (KlMIG1) genes in the induction and repression pathways . Our data show that full induction of the regulon requires SNF1; partial induction occurs in a Klsnf1 -deleted strain, indicating that a KlSNF1 -independent pathway(s) also regulates induction . MIG1 is required for full glucose repression of the regulon, but there must be a KlMIG1 -independent repression pathway also . The KlMig1 protein appears to act downstream of the KlSnf1 protein in the glucose repression pathway . Most importantly, the KlSnf1-KIMig repression pathway operates by modulating KlGAL1 expression . Regulating KlGAL1 expression in this manner enables the cell to switch the regulon off in the presence of glucose . Overall, our data show that, while the Snf1 and Mig1 proteins play similar roles in regulating the galactose regulon in Saccharomyces cerevisiae and K.lactis , the way in which these proteins are integrated into the regulatory circuits are unique to each regulon, as is the degree to which each regulon is controlled by the two proteins. Mol Cell Biol, 1997 Sep, 17(9), 5453 - 60 Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae: involvement of two hexose transporters; Nourani A et al.; In the yeast Saccharomyces cerevisiae, multidrug resistance to unrelated chemicals can result from overexpression of ATP-binding cassette (ABC) transporters such as Pdr5p, Snq2p, and Yor1p . Expression of these genes is under the control of two homologous zinc finger-containing transcription regulators, Pdr1p and Pdr3p . Here, we describe the isolation, by an in vivo screen, of two new Pdr1p-Pdr3p target genes: HXT11 and HXT9 . HXT11 and HXT9, encoding nearly identical proteins, have a high degree of identity to monosaccharide transporters of the major facilitator superfamily (MFS) . In this study, we show that the HXT11 product, which allows glucose uptake in a glucose permease mutant (rag1) strain of Kluyveromyces lactis, is also involved in the pleiotropic drug resistance process . Loss of HXT11 and/or HXT9 confers cycloheximide, sulfomethuron methyl, and 4-NQO (4-nitroquinoline-N-oxide) resistance . Conversely, HXT11 overexpression increases sensitivity to these drugs in the wild-type strain, an effect which is more pronounced in a strain having both PDR1 and PDR3 deleted . These data show that the two putative hexose transporters Hxt11p and Hxt9p are transcriptionally regulated by the transcription factors Pdr1p and Pdr3p, which are known to regulate the production of ABC transporters required for drug resistance in yeast . We thus demonstrate the existence of genetic interactions between genes coding for two classes of transporters (ABC and MFS) to control the multidrug resistance process. Microbiology, 1997 Aug, 143 ( Pt 8), 2615 - 25 The KIPHO5 gene encoding a repressible acid phosphatase in the yeast Kluyveromyces lactis: cloning, sequencing and transcriptional analysis of the gene, and purification and properties of the enzyme; Ferminan E et al.; A secreted phosphate-repressible acid phosphatase from Kluyveromyces lactis has been purified and the N-terminal region and an internal peptide have been sequenced . Using synthetic oligodeoxyribonucleotides based on the sequenced regions, the genomic sequence, KIPHO5, encoding the protein has been isolated . The deduced protein, named KIPho5p, consists of 469 amino acids and has a molecular mass of 52520 Da (in agreement with the data obtained after treatment of the protein with endoglycosidase H) . The purified enzyme shows size heterogeneity, with an apparent molecular mass in the range 90-200 kDa due to the carbohydrate content (10 putative glycosylation sites were identified in the sequence) . A 16 amino acid sequence at the N-terminus is similar to previously identified signal peptides in other fungal secretory proteins . The putative signal peptide is removed during secretion since it is absent in the mature secreted acid phosphatase . The gene can be induced 400-600-fold by phosphate starvation . Consensus signals corresponding to those described for Saccharomyces cerevisiae PHO4- and PHO2-binding sites are found in the 5' region . Northern blot analysis of total cellular RNA indicates that the KIPHO5 gene codes for a 1.8 kb transcript and that its expression is regulated at the transcriptional level . Chromosomal hybridization indicated that the gene is located on chromosome II . The KIPHO5 gene of K . lactis is able to functionally complement a pho5 mutation of Sacch . cerevisiae . Southern blot experiments, using the KIPHO5 gene as probe, show that some K . lactis reference strains lack repressible acid phosphatase, revealing a different gene organization for this kind of multigene family of proteins as compared to Sacch . cerevisiae. Yeast, 1997 Aug, 13(10), 961 - 71 Isolation and characterization of the KlHEM1 gene in Kluyveromyces lactis; Gonzalez-Dominguez M et al.; The KlHEM1 gene from Kluyveromyces lactis encodes a functional 5-aminolevulinate synthase (deltaALA synthase), as confirmed by complementation of a hem1 mutant Saccharomyces cerevisiae strain, homology search, and detection of a 2.3 kb transcript . The gene is highly homologous to the ScHEM1 gene, and the sequence of the promoter region contains a complex combination of putative regulatory signals . Some of them are related to phospholipid biosynthesis, glycolytic metabolism, and regulation by carbon source . Transcription of KlHEM1 increased significantly in response to limited oxygen, and only slightly with the change from repressed (glucose) to derepressed conditions (glycerol) . The deltaALA synthase from K . lactis contains, in the amino-terminal region, two heme-responsive elements that are not present in the protein from Saccharomyces cerevisiae. Yeast, 1997 Aug, 13(10), 945 - 60 Phytopathogenic filamentous (Ashbya, Eremothecium) and dimorphic fungi (Holleya, Nematospora) with needle-shaped ascospores as new members within the Saccharomycetaceae; Prillinger H et al.; Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region) . Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J Industr . Microbiol . 14, 523-530) . In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct . The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation . Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle-shaped ascospores within the family Saccharomycetaceae . After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous . The type species of the genus, K . polysporus is congeneric with the genus Saccharomyces . The data of Cai et al . (1996; Int . J . Syst . Bacteriol . 46, 542-549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K . marxianius, K . dobzhanskii, K . wickerhamii and K . aestuarii, which again can be included in the family Saccharomycetaceae . The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution. Mol Gen Genet, 1997 Jul, 255(3), 341 - 9 Cloning and characterization of the lipoyl-protein ligase gene LIPB from the yeast Kluyveromyces lactis: synergistic respiratory deficiency due to mutations in LIPB and mitochondrial F1-ATPase subunits; Chen XJ; The mgi1-4 and mgi2-1 mutants of the petite-negative yeast Kluyveromyces lactis have mutations in the beta- and alpha-subunits of the mitochondrial F1-ATPase, respectively . The mutants are respiratory competent but can form petites with deletions in mitochondrial DNA . In this study a cryptic nuclear mutation (lipB-1) was identified which, in combination with the mgi alleles, displays a synergistic respiratory-deficient phenotype on glycerol medium . The gene defined by the mutation was cloned and shown to encode a polypeptide of 332 amino acids with an N-terminal sequence characteristic of a mitochondrial targeting signal . The deduced protein shares 27% sequence identity with the product of the Escherichia coli lipB gene, which encodes a lipoyl-protein ligase involved in the attachment of lipoyl groups to lipoate-dependent apoproteins . A K . lactis strain carrying a disrupted lipB allele is severely compromised for growth on glycerol medium . The growth defect cannot be rescued by addition of lipoic acid, but cell growth can be restored on medium containing ethanol plus succinate . In addition, it was observed that lipB mutants of K . lactis, unlike the wild-type, are unable to utilize glycine as sole nitrogen source, indicating that activity of the glycine decarboxylase complex (GDC) is also affected . Taken together, these findings suggest that LIPB is the main determinant of the lipoyl-protein ligase activity required for lipoylation of enzymes such as alpha-ketoacid dehydrogenases and GDC. Yeast, 1997 Jun 30, 13(8), 777 - 81 Cloning and characterization of the KlDIM1 gene from Kluyveromyces lactis encoding the m2(6)A dimethylase of the 18S rRNA; Housen I et al.; The KlDIM1 gene encoding the m2(6)A rRNA dimethylase was cloned from a Kluyveromyces lactis genomic library using a PCR amplicon from the Saccharomyces cerevisiae ScDIM1 gene as probe . The KlDIM1 gene encodes a 320-amino acid protein which shows 81% identity to ScDim1p from S . cerevisiae and 25% identity to ksgAp from Escherichia coli . Complementation of the kasugamycin-resistant ksgA-mutant of E . coli lacking dimethylase activity demonstrates that KlDim1p is the functional homologue of the bacterial enzyme . Multiple alignment of dimethylases from prokaryotes and yeasts shows that the two yeast enzymes display distinctive structural motives including a putative nuclear localization signal. Yeast, 1997 Jun 30, 13(8), 699 - 706 Secretion of mouse alpha-amylase from Kluyveromyces lactis; Tokunaga M et al.; We constructed two mouse alpha-amylase secretion vectors for Kluyveromyces lactis using the well-characterized signal sequence of the pGKL 128 kDa killer precursor protein . Both PHO5 and PGK expression cassettes from Saccharomyces cerevisiae directed the expression of mouse alpha-amylase in YPD medium at a similar level of efficiency . K . lactis transformants secreted glycosylated and non-glycosylated alpha-amylase into the culture medium and both species were enzymatically active . The K . lactis/S . cerevisiae shuttle secretion vector pMI6 was constructed, and K . lactis MD2/1(pMI6) secreted about four-fold more alpha-amylase than S . cerevisiae YNN27 harboring the same plasmid, indicating that K . lactis is an efficient host cell for the secretion and production of recombinant proteins. Yeast, 1997 Jun 15, 13(7), 613 - 20 The linear plasmid pDHL1 from Debaryomyces hansenii encodes a protein highly homologous to the pGKL1-plasmid DNA polymerase; Fukuda K et al.; Both the linear plasmids, pDHL1 (8.4 kb) and pDHL2 (9.2 kb), of Debaryomyces hansenii TK require the presence of a third linear plasmid pDHL3 (15.0 kb) in the same host cell for their replication . A 3.5 kb Bam HI-PstI fragment of pDHL1 strongly hybridized by Southern analysis to the 3.5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of this conserved region in the replication of the two smaller pDHL plasmids . The 4.2 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced . The results showed that the cloned pDHL1 fragment encodes a protein of 1000 amino acid residues, having a strong similarity to the DNA polymerase coded for by ORF1 of the killer plasmid pGKL1 from Kluyveromyces lactis . The catalytic and proof-reading exonuclease domains as well as terminal protein motif were well conserved as in DNA polymerases of pGKL1 and other yeast linear plasmids . Analysis of the cloned fragment further showed that pDHL1 encodes a protein partly similar to the alpha subunit of the K . lactis killer toxin, although killer activity was not known in the DHL system . Analysis of the 5' non-coding region of the two above pDHL1-ORFs reveal the presence of the upstream conserved sequence similar to that found upstream of pGKL1-ORFs . The possible hairpin loop structure was also found just in front of the ATG start codon of the pDHL1-ORFs like pGKL1-ORFs . Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K . lactis plasmids. Nature, 1997 Jun 12, 387(6634), 708 - 13 Molecular evidence for an ancient duplication of the entire yeast genome; Wolfe KH et al.; Gene duplication is an important source of evolutionary novelty . Most duplications are of just a single gene, but Ohno proposed that whole-genome duplication (polyploidy) is an important evolutionary mechanism . Many duplicate genes have been found in Saccharomyces cerevisiae, and these often seem to be phenotypically redundant . Here we show that the arrangement of duplicated genes in the S . cerevisiae genome is consistent with Ohno's hypothesis . We propose a model in which this species is a degenerate tetraploid resulting from a whole-genome duplication that occurred after the divergence of Saccharomyces from Kluyveromyces . Only a small fraction of the genes were subsequently retained in duplicate (most were deleted), and gene order was rearranged by many reciprocal translocations between chromosomes . Protein pairs derived from this duplication event make up 13% of all yeast proteins, and include pairs of transcription factors, protein kinases, myosins, cyclins and pheromones . Tetraploidy may have facilitated the evolution of anaerobic fermentation in Saccharomyces. Biochim Biophys Acta, 1997 Jun 6, 1335(3), 235 - 41 Heterologous Kluyveromyces lactis beta-galactosidase production and release by Saccharomyces cerevisiae osmotic-remedial thermosensitive autolytic mutants; Becerra M et al.; The beta-galactosidase from Kluyveromyces lactis is a high molecular weight protein with commercial interest . A major drawback of its industrial production is the high cost associated with extraction and downstream processing due to its intracellular nature . In this work, the effectiveness of the utilization of Saccharomyces cerevisiae LD1 and LHDP1 strains, osmotic-remedial mutants which lyse at 37 degrees C, for the heterologous production and release into the extracellular medium of this protein has been proved . The highest absolute values of released beta-galactosidase have been obtained with the protease-deficient strain LHDP1 by osmotic shock. Mol Gen Genet, 1997 Jun, 255(1), 9 - 18 Comparative analysis in three fungi reveals structurally and functionally conserved regions in the Mig1 repressor; Cassart JP et al.; The Mig1 repressor is a key effector in glucose repression in the yeast Saccharomyces cerevisiae . To gain further insights into structure-function relationships, we have now cloned the MIG1 homologue from the yeast Kluyveromyces marxianus . The amino acid sequence deduced from KmMIG1 differs significantly from ScMig1p outside the highly conserved zinc fingers . However, 12 discrete conserved motifs could be identified in a multiple alignment that also included the K . lactis Mig1p sequence . We further found that KmMig1p is fully functional when expressed in S . cerevisiae . First, it represses the SUC2 promoter almost as well as ScMig1p . This repression requires the Cyc8 and Tup1 proteins and is dependent on a C-terminal region comprising several conserved leucine-proline repeats . Second, KmMig1p is regulated by glucose in S . cerevisiae, and a KmMig1-VP16 hybrid activator is inhibited by the ScSnf1p kinase in the absence of glucose . This suggests that KmMig1p has retained the ability to interact with several S . cerevisiae proteins, and reinforces the notion that the conserved motifs are functionally important . Finally, we found that the physiological role of Mig1p also is conserved in K . marxianus, since KmMig1p represses INU1, the counterpart of SUC2 in this organism. Curr Genet, 1997 Jun, 31(6), 488 - 93 Mitochondrial ATP synthase subunit 9 is not required for viability of the petite-negative yeast Kluyveromyces lactis; Clark-Walker GD et al.; Specific mutations in nuclear MGI genes encoding the alpha, beta and gamma subunits of the mitochondrial inner membrane F1-ATPase complex allow mitochondrial DNA (mtDNA) to be lost from K . lactis . In the absence of a mutation in any of these three nuclear genes, loss of mtDNA is lethal . These results imply that mtDNA encodes a gene that is essential . Likely candidates for such an essential role are the ATP6, 8 and 9 genes coding for proteins of the ATP synthase-F0 component . The present study removes ATP9 from contention as a vital mitochondrial gene because in a respiratory deficient mutant, Gly- 3 . 9, lacking a nuclear mgi mutation, we have found that a rearrangement in mtDNA has deleted 22 amino acids from the carboxy terminus of the 75 amino-acid subunit-9 protein . Rearrangement in mtDNA has occurred by recombination at a 23-bp repeated sequence in the introns of the ATP9 and large ribosomal RNA (LSU) subunit genes . These two introns, of 394 (ATP9) and 410 (LSU) nucleotides, both belong to group 1. Lett Appl Microbiol, 1997 Jun, 24(6), 455 - 9 Evaluation of the Biolog system for the identification of food and beverage yeasts; Praphailong W et al.; The inconvenience of conventional yeast identification methods has resulted in the development of rapid, commercial systems, mainly for clinical yeast species . The Biolog system (Biolog Inc., Hayward, CA, USA) is a new semi-automated, computer-linked technology for rapid identification of clinical and non-clinical yeasts . The system is based around a microtitre tray and includes assimilation and oxidation tests . This paper evaluates the Biolog system for the identification of 21 species (72 strains) of yeasts of food and wine origin . Species correctly identified included Saccharomyces cerevisiae, Debaryomyces hansenii, Yarrowia lipolytica, Kluyveromyces marxianus, Kloeckera apiculata, Dekkera bruxellensis and Schizosaccharomyces pombe . Zygosaccharomyces bailii and Zygosaccharomyces rouxii were identified correctly 50% of the time and Pichia membranaefaciens 20% of the time. J Biol Chem, 1997 May 9, 272(19), 12462 - 7 Cloning of cutinase transcription factor 1, a transactivating protein containing Cys6Zn2 binuclear cluster DNA-binding motif; Li D et al.; Hydroxy fatty acids from plant cutin were shown previously to induce the expression of the cutinase gene via a palindromic sequence located at -159 base pairs of the cutinase gene in Fusarium solani f . sp . pisi (Nectria hematococca mating type VI) . Of the two overlapping palindromes in this sequence, palindrome 2 was found to be essential for the inducibility of cutinase by hydroxy fatty acids . Screening of a phage expression library with the concatenated palindrome 2 as probe detected a distinct cDNA clone encoding a polypeptide designated cutinase transcription factor 1alpha (CTF1alpha) with a calculated molecular weight of 101,109 . This protein contains a Cys6Zn2 binuclear cluster motif sharing homology to the Cys6Zn2 binuclear cluster DNA-binding domains of transcription factors from Saccharomyces cerevisiae, S . carlsbergensis, Kluyveromyces lactis, Neurospora crassa, Aspergillus nidulans, and A . flavus . CTF1alpha, expressed in Escherichia coli, showed specific binding to the palindrome 2 DNA fragment but not to palindrome 1 or mutant palindrome 2 DNA fragments, suggesting specific binding of CTF1alpha to palindrome 2 . When CTF1alpha was expressed as a fusion protein with the nuclear localization sequence of SV40 in yeast, it transactivated the native cutinase promoter fused to the chloramphenicol acetyl transferase (cat) gene . Mutation of palindrome 2 but not palindrome 1 abolished this transactivation . Thus, CTF1alpha positively acts in vivo by binding selectively to palindrome 2 of the cutinase gene promoter. Biochim Biophys Acta, 1997 Apr 25, 1339(1), 133 - 42 Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis; Bozzi A et al.; The cytosolic and mitochondrial alcohol dehydrogenases from Kluyveromyces lactis (KlADHs) were purified and characterised . Both the N-terminally blocked cytosolic isozymes, KlADH I and KlADH II, were strictly NAD-dependent and exhibited catalytic properties similar to those previously reported for other yeast ADHs . Conversely, the mitochondrial isozymes, KlADH III and KlADH IV, displayed Ala and Asn, respectively, as N-termini and were able to oxidise at an increased rate primary alcohols with aliphatic chains longer than ethanol, such as propanol, butanol, pentanol and hexanol . Interestingly, the mitochondrial KlADHs, at variance with cytosolic isozymes and the majority of ADHs from other sources, were capable of accepting as a cofactor, and in some case almost equally well, either NAD or NADP . Since Asp-223 of horse liver ADH, thought to be responsible for the selection of NAD as coenzyme, is strictly conserved in all the KlADH isozymes, this amino-acid residue should not be considered critical for the coenzyme discrimination with respect to the other residues lining the coenzyme binding pocket of the mitochondrial isozymes . The relatively low specificity of the mitochondrial KlADHs both toward the alcohols and the cofactor could be explained on the basis of an enhanced flexibility of the corresponding catalytic pockets . An involvement of the mitochondrial KlADH isozymes in the physiological reoxidation of the cytosolic NADPH was also hypothesized . Moreover, both cytosolic and KlADH IV isozymes have an additional cysteine, not involved in zinc binding, that could be responsible for the increased activity in the presence of 2-mercaptoethanol. Appl Microbiol Biotechnol, 1997 Apr, 47(4), 329 - 36 Microbial linear plasmids; Meinhardt F et al.; While plasmids were originally considered to be generally circular until almost two decades ago, linear elements were reported to exist as well . They are now known to be common genetic elements in both, pro- and eukaryotes . Two types of linear plasmids exist, the so-called hairpin plasmids with covalently closed ends and those with proteins bound to their 5' termini . Hairpin plasmids are common in human-pathogenic Borrelia spirochetes, in which they are instrumental in escape from the immunological response; cryptic hairpin elements are present in mitochondria of the plant pathogenic fungus Rhizoctonia solani . Plasmids with 5' attached proteins constitute the largest group . In actinomycetous bacteria they are conjugative and usually confer advantageous phenotypes, e.g . formation of antibiotics, degradation of xenobiotics, heavy-metal resistance and growth on hydrogen as the sole energy source . In contrast, the majority of linear plasmids from eukaryotes are cryptic, with only a few exceptions . In some yeasts a killer phenotype may be associated, the most thoroughly investigated elements being those from Kluyveromyces lactis killer strains . In Neurospora spp . and in Podospora anserina, senescence and longevity respectively are correlated with linear plasmids . This review focuses on the biology of linear plasmids, their environmental significance and their use as tools in molecular and applied microbiology. Can J Microbiol, 1997 Apr, 43(4), 328 - 36 The incidence of killer activity and extracellular proteases in tropical yeast communities; Abranches J et al.; The presence of killer and proteolytic yeasts was studied among 944 isolates representing 105 species from tropical yeast communities . We found 13 killer toxin producing species, with Pichia kluyveri being the most frequent . Other killer yeast isolates were Candida apis, Candida bombicola, Candida fructus, Candida krusei, Candida sorbosa, Hanseniaspora uvarum, Issatchenkia occidentalis, Kloeckera apis, Kluyveromyces marxianus, Pichia membranaefaciens, Pichia ohmeri-like, and Sporobolomyces roseus . The communities from which killer yeasts were isolated had strains sensitive to them, and there were interspecific and intraspecific differences in the spectra of their killer activities . Pichia kluyveri had the broadest spectra of activity against sensitive isolates, and it apparently produced different toxins . The coexistence of sensitive and killer yeasts using the same substrate suggests that there is spatial separation in microhabitats or temporal separation in different stages of successions . Basidiomycetous yeasts were more frequently proteolytic than ascomycetous yeasts . Extracellular proteases could be important for the yeasts to have access to more nitrogen nutrients and obtain a better balance with available carbon sources. Int J Syst Bacteriol, 1997 Apr, 47(2), 453 - 60 A phylogenetic analysis of the genus Saccharomyces based on 18S rRNA gene sequences: description of Saccharomyces kunashirensis sp . nov . and Saccharomyces martiniae sp . nov; James SA et al.; A phylogenetic investigation of the ascomycetous yeast genus Saccharomyces was performed by using 18S rRNA gene sequence analysis . Comparative sequence analysis showed that the genus is phylogenetically very heterogeneous . Saccharomyces species were found to be phylogenetically interdispersed with members of other ascomycetous genera (e.g., the genera Kluyveromyces, Torulaspora, and Zygosaccharomyces) . The four species of the Saccharomyces sensu stricto complex (viz., Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus) were found to be phylogenetically closely related to one another, displaying exceptionally high levels of sequence similarity (> or = 99.9%) . These four species formed a natural group that was quite separate from the other Saccharomyces and non-Saccharomyces species examined . Saccharomyces exiguus and its anamorph, Candida holmii, were found to be genealogically almost identical and, along with Saccharomyces barnettii, formed a stable group closely related to, but nevertheless distinct from, Kluyveromyces africanus, Kluyveromyces lodderae, Saccharomyces rosinii, Saccharomyces spencerorum, and Saccharomyces sp . strain CBS 7662T (T = type strain) . Saccharomyces spencerorum and Kluyveromyces lodderae displayed a particularly close genealogical affinity with each other, as did Saccharomyces castellii and Saccharomyces dairensis . Similarly, Saccharomyces servazzii, Saccharomyces unisporus, and Saccharomyces sp . strain CBS 6904 were found to be genotypically highly related and to form a phylogenetically distinct lineage . The recently reinstated species Saccharomyces transvaalensis was found to form a distinct lineage and displayed no specific association with any other Saccharomyces or non-Saccharomyces species . Saccharomyces kluyveri formed a very loose association with a group which included Kluyveromyces thermotolerans, Kluyveromyces waltii, Zygosaccharomyces cidri, and Zygosaccharomyces fermentati . Saccharomyces spp . strain CBS 6334T, on the other hand, displayed no specific association with any of the other Saccharomyces spp . studied, although a neighbor-joining analysis did reveal that this strain exhibited a loose phylogenetic affinity with Kluyveromyces polysporus and Kluyveromyces yarrowii . On the basis of the phylogenetic findings, two new Saccharomyces species, Saccharomyces kunashirensis (with type strain CBS 7662) and Saccharomyces martiniae (with type strain CBS 6334), are described. Arch Microbiol, 1997 Apr, 167(4), 202 - 8 Molecular cloning of the neutral trehalase gene from Kluyveromyces lactis and the distinction between neutral and acid trehalases; Amaral FC et al.; We cloned the Kluyveromyces lactis KlNTH1 gene, which encodes neutral trehalase . It showed 65.2% and 68.5% identity at nucleotide and amino acid sequence level, respectively, with the Saccharomyces cerevisiae NTH1 gene . Multiple alignment of the predicted trehalase protein sequences from yeasts, bacteria, insects, and mammals revealed two major domains of conservation . Only the yeast trehalases displayed in an N-terminal extension two consensus sites for cAMP-dependent protein phosphorylation and a putative Ca2+-binding sequence . Gene disruption of the KlNTH1 gene abolished neutral trehalase activity and clearly revealed a trehalase activity with an acid pH optimum . It also resulted in a high constitutive trehalose level . Expression of the KlNTH1 gene in an S . cerevisiae nth1Delta mutant resulted in rapid activation of the heterologous trehalase upon addition of glucose to cells growing on a nonfermentable carbon source and upon addition of a nitrogen source to cells starved for nitrogen in a glucose-containing medium . In K . lactis, the same responses were observed except that rapid activation by glucose was observed only in early-exponential-phase cells . Inactivation of K . lactis neutral trehalase by alkaline phosphatase and activation by cAMP in cell extracts are consistent with control of the enzyme by cAMP-dependent protein phosphorylation. Biosci Biotechnol Biochem, 1997 Mar, 61(3), 563 - 4 A novel killer yeast effective on Schizosaccharomyces pombe; Kono I et al.; To control the extent of deacidification in wine making, we screened Kluyveromyces strains by their activity to kill the fission yeast Schizosaccharomyces pombe . Among Kluyveromyces IFO strains tested, K . waltii IFO 1666T was shown to have the desired activity . The killer spectrum of this strain was different from those of the other known killer yeasts . The activity was found in the culture medium and was lost by protease treatment . The activity was associated with the precipitate obtained by an increase of ammonium sulfate concentration . The toxin was larger than 10,000 daltons as judged by ultrafiltration. Mol Cell Biol, 1997 Mar, 17(3), 1722 - 30 Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon; Cardinali G et al.; The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown . We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose . We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase . The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer . A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels . The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells . A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells . Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose . We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway. Microbiology, 1997 Feb, 143 ( Pt 2), 321 - 30 3-phosphoglycerate kinase: a glycolytic enzyme protein present in the cell wall of Candida albicans; Alloush HM et al.; We have used a polyclonal antiserum to cell wall proteins of Candida albicans to isolate several clones from a cDNA lambda gt11 expression library . Affinity-purified antibody prepared to the fusion protein of one clone identified a 40 kDa moiety present in cell wall extracts from both morphologies of the organism . Indirect immunofluorescence demonstrated expression of this moiety at the C . albicans cell surface . Sequencing of a pBluescript II genomic clone identified with the cDNA clone revealed an open reading frame for a 417 amino acid protein . The nucleotide sequence showed significant homology with 3-phosphoglycerate kinase (PGK) genes, with 88%, 77% and 76% nucleotide homology with the PGK genes from Candida maltosa, Saccharomyces cerevisiae and Kluyveromyces lactis, respectively . The deduced amino acid sequence was consistent with this identification of the sequence as PGK1 of C . albicans . This finding was confirmed by a positive immunological response of a commercially available purified PGK from S . cerevisiae with the affinity-purified antibody against the fusion protein of the cDNA clone . The presence of PGK in the cell wall was confirmed by two additional methods . Cell wall protein were biotinylated with a derivative that does not permeate the cell membrane to distinguish extracellular from cytosolic proteins . Biotinylated PGK was detected among the biotinylated proteins obtained following streptavidin affinity chromatography . Immunoelectron microscopy revealed that the protein was present at the outer surface of the cell membrane and cell wall as well as expected in the cytoplasm . Northern blot analysis revealed that the gene transcript was present in C . albicans cells growing under different conditions, including different media, temperatures and morphologies . Most of the enzyme activity was found in the cytosol . Low enzymic activity was detected in intact cells but not in culture filtrates . These observations confirmed that PGK is a bona fide cell wall protein of C . albicans. Curr Genet, 1997 Feb, 31(2), 190 - 2 ORF7 of yeast plasmid pGKL2: analysis of gene expression in vivo; Schaffrath R et al.; ORF7 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a putative RNA polymerase subunit of 16 kDa . RNA analysis detected a single, plasmid-dependent ORF7 transcript of 550 nt indicating that the gene is transcribed mono-cistronically . Attempted one-step gene disruption of ORF7 resulted in chromosomal integration of the marker gene rather than the formation of stable recombinant k2ORF7(0) deletion plasmids . Thus, ORF7 appears to be a potential cis-dominant locus the integrity of which is indispensable for plasmid stability . The ORF7 gene product was over-produced as a c-myc-tagged fusion protein in Escherichia coli . Western-blot analysis of total yeast protein extracts using an antibody against this Orf7-c-myc fusion product identified a protein band with an apparent molecular weight of 17 kDa . This protein corresponds in size to the predicted product and is only detectable in plasmid-carrying killer yeasts. J Biol Chem, 1997 Jan 31, 272(5), 2729 - 35 The filamentous fungus Aspergillus niger contains two "differentially regulated" trehalose-6-phosphate synthase-encoding genes, tpsA and tpsB; Wolschek MF et al.; Two genes encoding trehalose-6-phosphate synthase were cloned from Aspergillus niger . tpsA was cloned using the Saccharomyces cerevisiae GGS1/TPS1 gene as a probe . It encodes a 517-amino acid polypeptide with 64-70% similarity to trehalose-6-phosphate synthase of S . cerevisiae, Kluyveromyces lactis, and Schizosaccharomyces pombe . Its transcription occurs constitutively and is enhanced on carbon-derepressing carbon sources, coinciding with the presence of a CreA-binding nucleotide motif in the 5'-noncoding region of tpsA . Disruption of tpsA only weakly reduces growth on glucose, and neither influences the glucose induction of a low affinity glucose permease nor interferes with the catabolite repression of a pectinase; it causes reduced the heat tolerance of conidia . tpsB was cloned by a polymerase chain reaction-based strategy . Its 480 amino acid sequence showed 76.5% identity to tpsA . Its transcription was hardly detectable at ambient temperatures but was enhanced strongly upon heat shock, which agrees with the presence of several copies of a C4T stress-responsive element in its 5'-upstream sequences . Hence the function of yeast GGS1/TPS1 has been split into two differentially regulated genes in A . niger, of which none appears to be involved in glucose sensing. Mol Gen Genet, 1997 Jan 27, 253(4), 469 - 77 The Kluyveromyces lactis equivalent of casein kinase I is required for the transcription of the gene encoding the low-affinity glucose permease; Blaisonneau J et al.; The RAG8 gene of Kluyveromyces lactis, which is one of the genes controlling the expression of the low-affinity carrier gene RAG1, has been cloned by in vivo complementation of the rag8 mutation . The sequence of Rag8p (535 amino acids), deduced from the nucleotide sequence of the cloned RAG8 gene, has been found to share a high degree of identity with the two casein kinases I of Saccharomyces cerevisiae, Yck1p and Yck2p, encoded by YCK1 and YCK2: the proteins are 65-66%, identical overall and show 89-90% identity in the kinase domain . The finding that the RAG8 gene of K . lactis cloned in a centromeric vector was able to complement the growth defect of a yck1 delta yck2(ts) mutant of S . cerevisiae strongly suggested that Rag8p is a casein kinase I . In contrast to the S . cerevisiae homologs, the RAG8 gene of K . lactis seems to be an essential single-copy gene, as shown by Southern blot experiments and the lethality of the rag8 null mutation . Northern blot analysis showed that the transcription of the RAG8 gene was higher on glucose media than in cells grown on a non-fermentable carbon source. Gene, 1997 Jan 15, 184(2), 299 - 306 The P-OLE1 gene of Pichia angusta encodes a delta 9-fatty acid desaturase and complements the ole1 mutation of Saccharomyces cerevisiae; Anamnart S et al.; Three PCR-amplified DNA fragments hybridizing with the OLE1 gene encoding delta 9-fatty acid desaturase of Saccharomyces cerevisiae were obtained using, respectively, genomic DNAs of one strain each of Kluyveromyces thermotolerans, Pichia angusta and Yarrowia lipolytica as templates . A gene designated P-OLE1 was cloned from the above fragment of P . angusta and sequenced . An open reading frame of P-OLE1 encodes a 49.6-kDa protein consisting of 451 amino acid residues, which shows high identity (62%) and similarity (89%) to that deduced from the OLE1 nucleotide sequence . Expression of P-OLE1 driven by the S . cerevisiae GAP promoter or its own promoter complemented the ole1 mutation of S.cerevisiae . Transcription of P-OLE1 in the native host was suggested to be partially repressed by oleic acid in the medium, as was that of OLE1 in S . cerevisiae and a similar gene in Y . lipolytica, but that of a similar gene in K . thermotolerans was not. FEMS Microbiol Lett, 1997 Jan 15, 146(2), 189 - 90 Exponential growth rates of species of the yeast genus Kluyveromyces; Bitzilekis S et al.; Doubling times were measured during exponential growth of 19 strains belonging to 10 of the 17 species of the yeast genus Kluyveromyces . Growth was in shaken aerobic batch culture at 25 degrees C, in a chemically defined medium with D-glucose as sole carbon source . Doubling times were strikingly uniform, being mainly between 2 and 3.5 h. Yeast, 1997 Jan, 13(1), 21 - 9 Galactose-inducible expression systems in Candida maltosa using promoters of newly-isolated GAL1 and GAL10 genes; Park SM et al.; The GAL1 and GAL10 gene cluster encoding the enzymes of galactose utilization was isolated from an asporogenic yeast, Candida maltosa . The structure of the gene cluster in which both genes were divergently transcribed from the central promoter region resembled those of some other yeasts . The expression of both genes was strongly induced by galactose and repressed by glucose in the medium . Galactose-inducible expression vectors in C . maltosa were constructed on low- and high-copy number plasmids using the promoter regions of both genes . With these vectors and the beta-galactosidase gene from Kluyveromyces lactis as a reporter, galactose-inducible expression was confirmed . Homologous overexpression of members of the cytochrome P-450 gene family in C . maltosa was also successful by using a high-copy-number vector under the control of these promoters. Curr Genet, 1997 Jan, 31(1), 15 - 21 Analysis of a transketolase gene from Kluyveromyces lactis reveals that the yeast enzymes are more related to transketolases of prokaryotic origins than to those of higher eukaryotes; Jacoby JJ et al.; The role of the pentose-phosphate pathway in carbohydrate metabolism of the yeast Kluyveromyces lactis, and the evolutionary relationships between the encoding genes, was investigated . For this purpose, we isolated the gene encoding transketolase (KlTKL1) and determined its nucleotide sequence . Surprisingly, comparisons of the deduced amino-acid sequence with those from other organisms revealed that the yeast enzymes are more related to those from prokaryotic sources than to those from higher eukaryotes . Functional analyses showed that KlTKL1 also complemented a Saccharomyces cerevisiae tkl1 tkl2 double mutant for growth in the absence of aromatic amino acids and restored transketolase activity in this mutant . A band detected in these transformants by Western-blot analysis corresponded to a band detected in K . lactis both in a wild-type strain and in a multicopy transformant with elevated transketolase activity. Genetics, 1996 Dec, 144(4), 1445 - 54 The mitochondrial genome integrity gene, MGI1, of Kluyveromyces lactis encodes the beta-subunit of F1-ATPase; Chen XJ et al.; In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast . In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase . The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate . Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the "gain-of-function" phenotype found in mgi1 point mutants . The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations. Rev Invest Clin, 1996 Nov, 48 Suppl, 51 - 61 {Uses of microbial beta-galactosidases to reduce lactose content in milk and dairy products}; Garcia-Garibay M et al.; The commercial sources of microbial beta-galactosidases (lactases) include the yeasts species Kluyveromyces marxianus, Kluyveromyces lactis and Candida kefyr which are used to hydrolyse lactose in milk due to their optimum pH . On the other hand, lactases obtained from the moulds Aspergillus niger and Aspergillus oryzae have an acid optimum pH and therefore are used to hydrolyse lactose in whey to obtain whey syrups to be used as raw materials in the food industry . The lactose intolerance problem has led to many studies concerning lactose hydrolysis by means of these microbial enzymes to obtain milk suitable for people with lactose maldigestion and special diets for ill persons, elderly population and intolerant babies due to secondary deficiency of lactose . Most industries obtain hydrolyzed lactose milk with free enzyme; however, there are some developments of immobilized lactase catalysts which are being used mainly in whey. Curr Microbiol, 1996 Nov, 33(5), 323 - 30 Extranuclear expression of the bacterial xylose isomerase (xylA) and the UDP-glucose dehydrogenase (hasB) genes in yeast with Kluyveromyces lactis linear killer plasmids as vectors; Schrunder J et al.; On the basis of the linear killer plasmid pGKL1 from Kluyveromyces lactis, two new linear hybrid plasmids were constructed . One of these, pRSC126, carried the xylA gene from Streptomyces rubiginosus encoding the xylose isomerase . The other linear hybrid molecule, pRSC128, carried the hasB gene of Streptococcus pyogenes encoding the UDP glucose dehydrogenase . Construction was performed in a way that the putative cytoplasmic promoter element of ORF5 of pGKL2 was fused to the coding region of the heterologous genes . After transformation, in vivo recombination led to the establishment of linear hybrid vectors . Though efficiency of expression was low when compared with bacterial systems, cytoplasmic expression of both genes was clearly demonstrated. Mol Gen Genet, 1996 Oct 28, 252(6), 746 - 50 A vital function for mitochondrial DNA in the petite-negative yeast Kluyveromyces lactis; Clark-Walker GD et al.; Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondrial DNA (mtDNA) from petite-positive yeasts . However, in the petite-negative yeast Kluyveromyces lactis, specific mutations in the nuclear genes MG12 and MG15 encoding the alpha- and gamma-subunits of the mitochondrial F1-ATPase, allow mtDNA to be lost . In this study we show that wild-type K . lactis does not survive in the absence of its mitochondrial genome and that the function of mgi mutations is to suppress lethality caused by loss of mtDNA . Firstly, we find that loss of a multicopy plasmid bearing a mgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA . Secondly, we cloned the K . lactis homologue of the Saccharomyces cerevisiae mitochondrial genome maintenance gene MGM101, and disrupted one of the two copies in a diploid . Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells . By contrast, disruption of MGM101 is not lethal in a haploid mgi strain with a specific mutation in a subunit of the mitochondrial F1-ATPase . These observations suggest that mtDNA in K . lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of Fo. Gene, 1996 Oct 24, 177(1-2), 237 - 41 Sphingolipid synthesis: identification and characterization of mammalian cDNAs encoding the Lcb2 subunit of serine palmitoyltransferase; Nagiec MM et al.; Synthesis of the ceramide portion of sphingolipids in animals has been hypothesized to be tightly regulated thereby controlling the rate of de novo sphingolipid formation . Regulation is predicted to occur at the first and committed biosynthetic step catalyzed by serine palmitoyltransferase (SPT, EC 2.3.1.50) . This hypothesis remains unproven because SPT has been refractory to purification and subsequent characterization . To begin to test this hypothesis we have used a genetic strategy to isolate LCB2 homologs from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe and a cDNA homolog from humans and mice . Identity is supported by overall amino acid sequence similarity between the predicted proteins and the known Saccharomyces cerevisiae Lcb2 protein . In addition, a motif of 56 residues from the human protein functionally substituted for the corresponding region of the S . cerevisiae Lcb2 protein . The 56 residue motif was found to be unique to Lcb2 proteins . Likewise, the base sequence encoding it is unique to the human genome . Finally, a peptide sequence in the motif is known to be part of the catalytic domain of all members of the aminolevulinate synthase superfamily of proteins of which Lcb2 is a member . These data argue that this motif is part of the catalytic domain of SPT and is a signature of Lcb2 proteins . The mammalian LCB2 cDNAs provide valuable reagents for studying the Lcb2 subunit of SPT and for studying how ceramide synthesis is regulated. J Bacteriol, 1996 Oct, 178(20), 5860 - 6 Glucose uptake in Kluyveromyces lactis: role of the HGT1 gene in glucose transport; Billard P et al.; A gene for high-affinity glucose transport, HGT1, has been isolated from the lactose-assimilating yeast Kluyveromyces lactis . Disruption strains showed much-reduced uptake of glucose at low concentrations and growth was particularly affected in low-glucose medium . The HGT1 nucleotide sequence implies that it encodes a typical transmembrane protein with 12 hydrophobic domains and with 26 to 31% amino acid identity with the Hxtp family of glucose transport elements in Saccharomyces cerevisiae . Expression is constitutive (in contrast to RAG1, the major gene for low-affinity glucose uptake in K . lactis) and is controlled by several genes also known to affect expression of RAG1 . These include RAG5 (which codes for the single hexokinase of K . lactis), which is required for HGT1 transcription, and RAG4, which has a negative effect . The double mutant deltahgt1deltarag1 showed further reduced glucose uptake but still grew quite well on 2% glucose and was not completely impaired even on 0.1% glucose. Nature, 1996 Sep 26, 383(6598), 354 - 7 Control of telomere growth by interactions of RAP1 with the most distal telomeric repeats; Krauskopf A et al.; Telomeres, the specialized DNA-protein structures at the ends of eukaryotic chromosomes, are required for chromosomal stability and integrity . Regulation of the overall length of the telomeric DNA repeat tract is likely to be a key requirement for its various biological roles . We have studied telomere length regulation in the yeast Kluyveromyces lactis, which has long (25 base pairs) homogeneous telomeric repeat units that make it highly suitable for telomere studies . In the related Saccharomyces cerevisiae, the DNA-sequence-specific duplex-binding protein RAP1 is a component of the telomeric complex . Here we show that the phenotypic severity of previously described telomerase RNA (ter1) mutations is directly proportional to the loss of RAP1 binding to mutated telomeric repeats . Using a carboxy-terminal-tail mutant of K . lactis RAP1, we also show that, unexpectedly, RAP1 interaction with the most terminal telomeric repeats is crucial for telomere length control. Yeast, 1996 Sep 15, 12(11), 1125 - 33 Isolation of a gene encoding a G protein alpha subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis; Savinon-Tejeda AL et al.; Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0.5 kb (P-1) and 0.8 kb (P-2) . Sequencing showed that these two fragments share high homology with genes coding for the G alpha subunits from different sources . Using the P-1 fragment as a probe we screened a genomic library from K . lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein alpha subunits from Saccharomyces cerevisiae . KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this G alpha subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway . KlGpa2p shares some structural similarities with members of the mammalian G alpha s family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence. Yeast, 1996 Sep 15, 12(11), 1097 - 105 Cloning of Candida albicans SEC14 gene homologue coding for a putative essential function; Monteoliva L et al.; The yeast SEC14 gene product is required for the transport of proteins from the Golgi complex . We have cloned the homologous Candida albicans SEC14 gene (CaSEC14) by functional complementation of a Saccharomyces cerevisiae thermosensitive mutant, sec14ts . Some putative TATA boxes have been identified in CaSEC14 and, contrary to S . cerevisiae SEC14, no introns were found in the Candida homologue . Sequence analysis revealed that CaSec14p is a 301 amino acid protein, 67% identical to S . cerevisiae and Kluyveromyces iactis Sec14p, and 61% identical to the 300 amino-terminal residues of Yarrowia lipolytica Sec14p . Hydrophatic profile analysis of CaSec14p suggests a soluble protein without transmembrane domains as has been described for the S . cerevisiae counterpart . While it was easy to disrupt one allele of SEC14 in C . albicans, repeated attempts to disrupt the second allele were unsuccessful, thus suggesting that the gene could be essential for vegetative growth in C . albicans. Appl Microbiol Biotechnol, 1996 Sep, 46(2), 187 - 90 Fermentation of molasses using a thermotolerant yeast, Kluyveromyces marxianus IMB3: simplex optimisation of media supplements; Gough S et al.; The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var . marxianus was investigated at 45 degrees C . A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v) . The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined . Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration . This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses . The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308-313, 1965) simplex optimisation method . The optimum concentration of the supplements were 0.576 g1(-1) magnesium sulphate, 0.288 g1(-1) potassium dihydrogen phosphate and 0.36% (v/v) linseed oil . Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate. Int J Food Microbiol, 1996 Aug, 31(1-3), 205 - 19 Growth of yeasts in milk and associated changes to milk composition; Roostita R et al.; The growth of several yeast species in milk containing added sodium chloride (0-15%, w/v) at 25 degrees C and 10 degrees C was examined in conjunction with yeast metabolism of milk constituents . Depending on conditions, all yeasts grew to maximum populations of 10(7)-10(8) cfu/ml . Kluyveromyces marxianus gave strong utilisation of lactose and weak metabolism of citrate, protein and fat with the production of ethanol, glycerol, lactic acid and propionic acid . As measured by the production of free amino acids and free fatty acids, Candida lipolytica and Candida catenulata gave strong proteolytic and lipolytic reactions, the specificities of which appeared to be influenced by temperature and the presence of NaCl . These species also metabolised organic acids . Although giving strong growth responses, Debaryomyces hansenii and Saccharomyces cerevisiae did not metabolise lactose and gave only very weak lipolytic and proteolytic reactions . Citrate was metabolised by D . hansenii but not by S . cerevisiae . Both species produced small amounts of ethanol, glycerol and lactic acid. Curr Genet, 1996 Jul 31, 30(2), 145 - 50 Isolation and molecular analysis of the gene for cytochrome c1 from Kluyveromyces lactis; Gbelska Y et al.; By ethyl methanesulphonate mutagenesis of the yeast Kluyveromyces lactis we have isolated five nuclear mutants that were unable to grow on non-fermentable carbon sources . The mutations were found to belong to three complementation groups . After functional complementation of the mutation in one of these mutants we have cloned the structural gene for cytochrome c1, named KlCYT1 . This gene has been assigned to chromosome VI and its nucleotide sequence exhibited 74.3% identity to the homologous gene of S . cerevisiae. J Biotechnol, 1996 Jul 18, 48(1-2), 15 - 24 High-level secretion of human alpha 1-antitrypsin from Saccharomyces cerevisiae using inulinase signal sequence; Kang HA et al.; The use of a proper signal sequence is one of the major determinants for the efficient secretion of heterologous proteins from yeast . The signal sequence derived from inulinase (INU1A) of Kluyveromyces marxianus was evaluated in directing the secretion of a human glycoprotein, alpha 1-antitrypsin (alpha 1-AT), from Saccharomyces cerevisiae . A yeast expression vector for alpha 1-AT was constructed by placing the coding sequence of human alpha 1-AT fused with the INU1A signal sequence downstream of the GAL10 promoter . S . cerevisiae transformants harboring the expression vector secreted about 70% of the total alpha 1-AT synthesized into the culture media . The intracellularly retained form of alpha 1-AT was mostly unglycosylated, whereas the secreted protein had high mannose-type glycosylation . The fed-batch cultivation of the recombinant yeast achieved a high-cell density, leading to the secretion of biologically active alpha 1-AT up to 75 mg l-1 . The secreted protein was purified and subjected to N-terminal sequencing, which confirmed that the secreted alpha 1-AT was processed correctly at the Kex2 cleavage site as expected from the sequence of INU1A signal peptide . The results suggest that the inulinase signal sequence is useful for the high-level secretion of relatively large glycoproteins, such as human alpha 1-AT, from S . cerevisiae. Genes Dev, 1996 Jul 15, 10(14), 1822 - 34 Cap-prevented recombination between terminal telomeric repeat arrays (telomere CPR) maintains telomeres in Kluyveromyces lactis lacking telomerase; McEachern MJ et al.; Deletion of the telomerase RNA gene (TER1) in the yeast Kluyveromyces lactis results in gradual loss of telomeric repeats and progressively declining cell growth capability (growth senescence) . We show that this initial growth senescence is characterized by abnormally large, defectively dividing cells and is delayed when cells initially contain elongated telomeres . However, cells that survive the initial catastrophic senescence emerge relatively frequently, and their subsequent growth without telomerase is surprisingly efficient . Survivors have lengthened telomeres, often much longer than wild type, but that are still subject to gradual shortening . Production of these postsenescence survivors is strongly dependent on the RAD52 gene . We propose that shortened, terminal telomeric repeat tracts become uncapped, promoting recombinational repair between them to regenerate lengthened telomeres in survivors . This process, which we term telomere cap-prevented recombination (CPR) may be a general alternative telomere maintenance pathway in eukaryotes. Antonie Van Leeuwenhoek, 1996 Jul, 70(1), 67 - 78 Analysis of coenzyme Q systems, monosaccharide patterns of purified cell walls, and RAPD-PCR patterns in the genus Kluyveromyces; Molnar O et al.; Analysis of the coenzyme Q system and the monosaccharide pattern of purified cell walls were used for species characterization in the genus Kluyveromyces . All the type strains of the genus possess coenzyme Q-6 and the mannose-glucose ('Saccharomyces type') cell wall sugar pattern . With the help of Random Amplified Polymorphic DNA-Polymerase Chain Reaction analysis 17 species were separated: K . aestuarii, K . africanus, K . Bacillisporus, K . blattae, K . delphensis, K . dobzhanski, K . lactis (anamorph Candida sphaerica), K . lodderae, K . marxianus (syn . K . fragilis, K . bulgaricus, K . cicerisporus, anamorphs Candida macedoniensis, C . pseudotropicalis, C . kefyr), K . phaffii, K . piceae, K . polysporus, K . sinensis, K . thermotolerans (syn . K . veronae, anamorph Candida dattila), K . waltii, K . wickerhamii, K . yarrowii (anamorph Candida tannotolerans) . A strain of K . drosophilarum showed with the type strain of K . lactis only 63% similarity . The strain originally described as the type strain of K . cellobiovorus nom . nud . was excluded from the genus (Q-9), and found to be conspecific with the type strain of Candida intermedia. Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1070 - 5 Phylogenetic relationships of species of the genus Saccharomyces Meyen ex Reess deduced from partial base sequences of 18S and 26S ribosomal RNAs (saccharomycetaceae); Ando S et al.; In order to clarify the phylogenetic relationships among the yeast species classified in the genus Saccharomyces, partial base sequences of 18S and 26S ribosomal RNAs were determined for ten selected strains . The regions determined correspond to positions 1451 through 1618 of the 18S rRNA and positions 493 through 622 and 1611 through 1835 of the 26S rRNA in S . cerevisiae . Analyses of these partial base sequences suggested that the genus Saccharomyces is phylogenetically heterogeneous . Saccharomyces servazzii and S . unisporus showed identical or very similar sequences in all the three regions, and their phylogenetic distance from S . cerevisiae was large enough to introduce a genus independent of Saccharomyces . Saccharomyces kluyveri is also distant from all the other Saccharomyces species examined, and is likely to deserve a new genus . Estimated phylogenetic relationships between Saccharomyces and other genera characterized by the Q-6 system, such as species of Zygosaccharomyces . Torulaspora, Kluyveromyces, Arxiozyma, Pachytichospora, Nadsonia . Hanseniaspora, Kloeckeraspora, and Saccharomycodes, are also discussed. Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1063 - 9 Phylogenetic relationships of species of the genus Kluyveromyces van der Walt (saccharomycetaceae) deduced from the partial sequences of 18S and 26S ribosomal RNAs; Ando S et al.; In order to clarify the phylogenetic relationships of the species classified in the genus Kluyveromyces (Saccharomycetaceae), three partial base sequences of 18S and 26S rRNAs of eighteen strains were determined . The regions determined of the strains corresponded to positions 1451 through 1618 (168 bases) of 18S rRNA and to positions 1611 through 1835 (225 bases) and 493 through 622 (130 bases) of a strain (IFO 2376) of Saccharomyces cerevisiae . The analyses of the partial base sequences suggested that the genus Kluyveromyces is phylogenetically heterogeneous, ranging from the strains that are quite close to the strain of S . cerevisiae to the strains that are distinct enough to be classified in genera separate from the genus Saccharomyces . From our sequence data, we concluded that the extent of the genus Kluyveromyces should be restricted to only one species, K . polysporus, the type species of the genus . Kluyveromyces phaffii was also distinct enough to deserve another genus . Kluyveromyces cellobiovorus was not close to any of the strains of Kluyveromyces species examined, and should be excluded from the genus . Most of the strains of the species examined were fairly close to the strain of S . cerevisiae. Gene, 1996 Jun 12, 172(1), 131 - 6 Low- and high-copy-number shuttle vectors for replication in the budding yeast Kluyveromyces lactis; Chen XJ; Four sets of plasmid vectors for the budding yeast Kluyveromyces lactis (Kl) have been constructed . All plasmids are pUC19-based shuttle vectors having multiple unique sites in their multiple cloning site (MCS) within the bacterial lacZ gene . The first set of vectors contains Klori, the origin of replication for Kl isolated from Kluyveromyces plasmid pKD1, and one of the selectable nutritional markers, URA3, TRP1 or LEU2 . These markers from the yeast, Saccharomyces cerevisiae (Sc), can complement the uraA1, trp1 and leu2 mutations of Kl . The second set of vectors, in addition to Klori, contains the ARS (autonomously replicating sequence) and centromeric sequences of Sc, and are able to replicate in both Sc and Kl . The third group of plasmids is centromeric vectors that are maintained in Kl at low copy number . The last family of vectors was designed for gene overexpression . As they contain the bacterial kanamycin-resistance-encoding gene (kan), plasmid copy number can be amplified to over 100 copies per cell in Kl by growing cells in the presence of the antibiotic G418 (Geneticin) . This type of vector has been used to study the high-copy-lethality phenotype of a truncated version of the Kl MGI2 gene encoding the alpha-subunit of the mitochondrial F1F0-ATP synthase. Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 5963 - 8 Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis; Abeijon C et al.; The mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they lack mannose phosphate and have terminal alpha1-->2-linked N-acetylglucosamine . The biosynthesis of these chains probably occurs in the lumen of the Golgi apparatus, by analogy to S . cerevisiae . The sugar donors, GDP-mannose and UDP-GlcNAc, must first be transported from the cytosol, their site of synthesis, via specific Golgi membrane transporters into the lumen where they are substrates in the biosynthesis of these mannoproteins . A mutant of K . lactis, mnn2-2, that lacks terminal N-acetylglucosamine in its mannan chains in vivo, has recently been characterized and shown to have a specific defect in transport of UDP-GlcNAc into the lumen of Golgi vesicles in vitro . We have now cloned the gene encoding the K . lactis Golgi membrane UDP-GlcNAc transporter by complementation of the mnn2-2 mutation . The mnn2-2 mutant was transformed with a genomic library from wild-type K . lactis in a pKD1-derived vector; transformants were isolated and phenotypic correction was monitored following cell surface labeling with fluorescein isothiocyanate conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine, and a fluorescent activated cell sorter . A 2.4-kb DNA fragment was found to restore the wild-type lectin binding phenotype . Upon loss of the plasmid containing this fragment, reversion to the mutant phenotype occurred . The above fragment contained an open reading frame for a multitransmembrane spanning protein of 328 amino acids . The protein contains a leucine zipper motif and has high homology to predicted proteins from S . cerevisiae and C . elegans . In an assay in vitro, Golgi vesicles isolated from the transformant had regained their ability to transport UDP-GlcNAc . Taken together, the above results strongly suggest that the cloned gene encodes the Golgi UDP-GlcNAc transporter of K . lactis. Curr Genet, 1996 Jun, 30(1), 89 - 92 Isolation and characterization of mutants as an approach to a transformation system in Kluyveromyces marxianus; Basabe L et al.; A method to obtain K . marxianus mutants has been developed . Different auxotrophic mutants were isolated by nystatin and snail-enzyme enrichment procedures using an incubation time of 2 h before adding the antibiotic or the enzyme respectively . All his mutants analyzed by complementation tests turned out to belong to the same complementation group . Some of them were transformed and complemented by the S . cerevisiae HIS3 gene . These non-reverting his3 mutants contain no heterologous sequence, which is essential to make them acceptable for application in the food industry. Curr Genet, 1996 Jun, 30(1), 19 - 28 The nuclear Kluyveromyces lactis MRF1 gene encodes a mitochondrial class I peptide chain release factor that is important for cell viability; Pel HJ et al.; We report the isolation and characterization of the Kluyveromyces lactis MRF1 gene encoding mitochondrial peptide chain release factor mRF-1 . Over-expression of the KlMRF1 gene has a strong antisuppressive effect in a Saccharomyces cerevisiae mitochondrial nonsense suppressor strain . Inactivation of KlMRF1 results in a dual phenotype: most cells die after about 10-13 generations, while a small number of cells exceed this limit . We propose that the lethality is related to a loss of mitochondrial genome integrity . Surviving Klmrf1 cells are able to grow slowly on the non-fermentable substrate glycerol, indicating the existence of a second mitochondrial release factor activity . Our previous comparative analysis of class I release factors is refined by the incorporation of KlmRF-1 and ten recently identified prokaryotic release factor sequences.Keywords Kluyveromyces lactis middle dot Mitochondrial release factor middle dot MRF1 middle dot Peptide chain termination FEBS Lett, 1996 May 27, 387(1), 7 - 10 Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose by Kluyveromyces lactis rag2 mutants; Gonzalez Siso MI et al.; Kluyveromyces lactis mutants defective in the glycolytic enzyme phosphoglucose isomerase are able to grow in glucose media and to produce ethanol, but they depend on a functional respiratory chain and do not grow in glucose-antimycin media . We postulate that this is due to the necessity of reoxidizing, in the mitochondria, the NADPH produced by the pentose phosphate pathway, which may be highly active in these mutants in order to bypass the blockade in the phosphoglucose isomerase step . This oxidation would be mediated by a cytoplasmic-side mitochondrial NAD(P)H dehydrogenase that would pass the electrons to ubiquinone . Data supporting this hypothesis are provided. Gene, 1996 May 24, 171(1), 113 - 7 Cloning of the gene encoding the mitochondrial adenine nucleotide carrier of Schizosaccharomyces pombe by functional complementation in Saccharomyces cerevisiae; Couzin N et al.; We describe the isolation and sequencing of both cDNA and genomic clones encoding the mitochondrial ADP/ATP carrier (Anc) of Schizosaccharomyces pombe (Sp) . The cDNA clone was isolated from a cDNA library of this fission yeast by complementation of a Saccharomyces cerevisiae (Sc) strain defective in adenine nucleotide carrier . The predicted amino acid (aa) sequence (322 aa) shared similarity with the known Anc sequences . It is more closely related to Neurospora crassa (Nc) Anc than to ScAnc1, 2, or 3 or Kluyveromyces lactis (Kl) Anc . Hybridization experiments with ordered libraries of Sp genomic DNA led to the physical mapping (chromosome II, NotI-B region) and the isolation of the Sp ANC1 gene . We also conclude that a single-copy gene encodes the Sp Anc. Nucleic Acids Res, 1996 May 15, 24(10), 1879 - 86 Kluyveromyces lactis killer system: analysis of cytoplasmic promoters of the linear plasmids; Schickel J et al.; All of the 14 genes encoded by the cytoplasmic linear killer plasmids of Kluyveromyces lactis are preceded by upstream conserved sequences (UCSs), cis-acting elements involved in plasmid gene transcription . Using the bacterial glucose-dehydrogenase gene as a reporter, expression driven by seven cytoplasmic promoters was determined . The level of expression ranged from 0.5 to 6 nkat . The highest activity was displayed by UCS 6 of pGKL2 whereas the lowest level was obtained with UCS2 of pGKL2, all other values were in between . Sequences located 5' upstream the UCSs do not influence expression . As exemplified for UCS5 and UCS10, deletion led to an almost complete loss of expression. Antonie Van Leeuwenhoek, 1996 May, 69(4), 357 - 61 Location of the beta-galactosidase of the yeast Kluyveromyces marxianus var . marxianus ATCC 10022; Bacci Junior M et al.; During the growth of Kluyveromyces marxianus var . marxianus ATCC 10022 on lactose, peaks of glucose, but not beta-galactosidase activity, were detected in culture medium . Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-beta-D-galactopyranoside (ONPG), indicating that beta-galactosidase is physically associated with cells . ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells . Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that beta-galactosidase is not located in the periplasm . In addition, the energy inhibitors fluoride or arsenate, as well as the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells . These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage . The taxonomic and physiologic implications of the exclusive intracellular location of beta-galactosidase of K . marxianus var . marxianus ATCC 10022 are discussed. Mol Microbiol, 1996 May, 20(4), 765 - 72 RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis; Prior C et al.; The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors . From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast . Measurement of the pyruvate decarboxylase activity in the original rag3-1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K . lactis . The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA . Despite analogies between the RAG3 gene of K . lactis and the PDC2 gene of S . cerevisiae, these genes were unable to reciprocally complement. J Biol Chem, 1996 Apr 12, 271(15), 8851 - 4 A mutant yeast deficient in Golgi transport of uridine diphosphate N-acetylglucosamine; Abeijon C et al.; Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal alpha1-->2-linked N-acetylglucosamine and lack mannose phosphate . In a previous study, Douglas and Ballou (Douglas, R . K., and Ballou, C . E . (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins . The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location . No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc . Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins . Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins . We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K . lactis . Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro . The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent Km of 5.5 microM and a Vmax of 8.2 pmol/mg of protein/3 min . No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells . However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific . In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids. Int J Syst Bacteriol, 1996 Apr, 46(2), 542 - 9 Phylogenetic relationships among members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces deduced by small-subunit rRNA gene sequences; Cai J et al.; A molecular systematic investigation of members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces was performed by using 18S rRNA gene sequence analysis . Our comparative sequence analysis revealed that Brettanomyces anomalus and Brettanomyces bruxellensis were closely related to one another and also to their teleomorphs, Dekkera anomala and Dekkera bruxellensis, respectively . Together with Dekkera custersiana and Dekkera naardenensis, these four species formed a stable and distinct phylogenetic group . The three representative species of the genus Debaryomyces examined (viz., Debaryomyces castellii, Debaryomyces hansenii, and Debaryomyces udenii) were found to be genealogically highly related to each other and exhibited a specific phylogenetic affinity (level of sequence similarity, approximately 99.2%) with Candida guilliermondii (teleomorph, Pichia guilliermondii) . Debaryomyces species and C . guilliermondii formed a distinct phylogenetic group, which displayed a significant association with a phylogenetically coherent cluster encompassing Lodderomyces elongisporus, Candida albicans, and four other Candida species . In contrast to the situation with the genera Brettanomyces and Debaryomyces, the genus Kluyveromyces displayed very marked phylogenetic heterogeneity . Kluyveromyces polysporus, the type species of the genus Kluyveromyces, and six other Kluyveromyces species (viz., Kluyveromyces africanus, Kluyveromyces delphensis, Kluyveromyces lodderae, Kluyveromyces thermotolerans, Kluyveromyces waltii, and Kluyveromyces yarrowii) were phylogenetically intermixed with species of the genera Zygosaccharomyces, Saccharomyces, and Torulaspora . In contrast, Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces wickerhamii, and three Kluyveromyces marxianus varieties, along with their anamorph, Candida kefyr, formed a highly stable monophyletic group worthy of separate generic status . Kluyveromyces blattae and Kluyveromyces phaffii formed two distinct phylogenetic lines that did not exhibit particularly close affinity with each other or other ascomycetous yeast genera . Our phylogenetic findings are discussed in the context of the results of other genotypic and phenotypic studies. Antonie Van Leeuwenhoek, 1996 Apr, 69(3), 267 - 72 Behaviour of the yeast Kluyveromyces marxianus var . marxianus during its autolysis; Amrane A et al.; The lactic yeast Kluyveromyces marxianus var.marxianus (formerly K . fragilis) autolyzates at faster rate than Saccharomyces cerevisiae . During K . marxianus autolysis, quite similar release kinetics were observed for intracellular space markers (potassium ions, nucleotides), cell-wall components (polysaccharides, N-acetyl-D-Glucosamine) and non specific products (amino nitrogen) . By Scanning Electronic Microscopy examination, no cell burst was observed, but a variation in cell shape (from ellipsoidal to cylindrical), as well as a 43% decrease in the internal volume were observed . The mechanism proposed for S . cerevisiae autolysis appeared also likely for K . marxianus. Plant Cell Physiol, 1996 Apr, 37(3), 341 - 6 Resistance to cadmium ions and formation of a cadmium-binding complex in various wild-type yeasts; Inouhe M et al.; The resistance to cadmium ions (Cd-resistance) and possible formation of cadmium-binding complexes were examined in eight different wild-type yeasts . Saccharomyces exiguus, Pichia farinosa, Torulaspora delbrueckii and Schizosaccharomyces octosporus exhibited partial Cd-resistance, as compared to the Cd-resistant strain 301N and the Cu-resistant but Cd-sensitive strain X2180-1B of Saccharomyces cerevisiae . Saccharomyces carlsbergensis, Pichia mogii, Zygosaccharomyces rouxii and Kluyveromyces lactis were all Cd-sensitive . The partially Cd-sensitive species, with the exception of S . exiguus, accumulated Cd2+ ions in the cytoplasmic fraction to varying extents . This fraction from S . octosporus included a Cd-binding complex that contained (gamma EC)nG peptides known as cadystins or phytochelatins, while P . farinosa and T . delbrueckii synthesized Cd-binding proteins that were similar to the Cd-metallothionein produced by S . cerevisiae 301N in terms of molecular weight and amino acid composition . These results suggest that such cytoplasmic molecules play a role in the Cd-tolerance of the above three species of yeast . S . exiguus retained most cadmium in the cell wall fraction and no Cd-binding complex was found in the cytoplasm, an indication of the important role of the cell wall in its Cd-tolerance . Different modes of binding of Cd2+ ions appear to be involved in the Cd-resistance of wild-type yeasts and fungi. Yeast, 1996 Mar 15, 12(3), 241 - 6 The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase; Takeda M et al.; The 36K protein attached at the 5' end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized . The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients . The pGKL2 was present only in the post-microsomal supernatant . Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I . The terminal protein (final ca . 0 center dot 8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence. Appl Microbiol Biotechnol, 1996 Mar, 45(1-2), 102 - 6 Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis; Bui DM et al.; The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector . The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium . The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter . Secreted glucoamylase possesses identical N-terminal amino acid sequences to those