Kluyveromyces

Gene, 1992 Sep 21, 119(1), 75 - 81
Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination; Rossolini GM et al.; Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae . In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector . We have obtained K . lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus . Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40 . Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants . Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event . Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.

Biochim Biophys Acta, 1992 Sep 4, 1159(1), 67 - 73
A protein homologous to glyceraldehyde-3-phosphate dehydrogenase is induced in the cell wall of a flocculent Kluyveromyces marxianus; Fernandes PA et al.; A protein with an apparent molecular weight of 37,000 (p37) is present in very large amounts in the cell wall of Kluyveromyces marxianus, after the induction of flocculation of the yeast . This protein was isolated by preparative gel electrophoresis and its purity checked by SDS-PAGE and reverse-phase HPLC . SDS-PAGE, endoglycosidase-H treatment and peptide sequencing indicated that p37 is a glycoprotein with a high identity to cytosolic glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae . Polyclonal antibodies were used for Western blot analysis and immunocytochemistry, which showed that p37 is present in the cell wall of non-flocculent K . marxianus and, therefore, is a constitutive protein of the cell wall.

Curr Genet, 1992 Sep, 22(3), 191 - 5
Isolation and sequence analysis of the small subunit ribosomal RNA gene from the euryhaline yeast Debaryomyces hansenii; Govind NS et al.; The small subunit ribosomal RNA gene (SSU rDNA) from the euryhaline yeast Debaryomyces hansenii has been isolated and sequenced . After appropriate alignment of this sequence with SSU rDNA sequences from 30 other taxa, phylogenetic reconstruction using distance matrix and maximum parsimony methods indicates that D . hansenii is most closely affiliated with Candida albicans, and occurs in the cluster of the yeasts Saccharomyces cerevisiae, Torulaspora delbruekii, Candida glabrata, and Kluyveromyces lactis . It appears that the capacity to tolerate high salt is independent of phylogenetic affiliations based on SSU rDNA analyses.

Gene, 1992 Sep 1, 118(1), 55 - 63
Sequence of the Kluyveromyces lactis beta-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis; Poch O et al.; The LAC4 gene encoding the beta-galactosidase (beta Gal) of the yeast, Kluyveromyces lactis, was cloned on a 7.2-kb fragment by complementation of a lacZ-deficient Escherichia coli strain . The nucleotide sequence of the structural gene, with 42 bp and 583 bp of the 5'- and 3'-flanking sequences, respectively, was determined . The deduced amino acid (aa) sequence of the K . lactis beta Gal predicts a 1025-aa polypeptide with a calculated M(r) of 117618 and reveals extended sequence homologies with all the published prokaryotic beta Gal sequences . This suggests that the eukaryotic beta Gal is closely related, evolutionarily and structurally, to the prokaryotic beta Gal's . In addition, sequence similarities were observed between the highly conserved N-terminal two-thirds of the beta Gal and the entire length of the beta-glucuronidase (beta Glu) polypeptides, which suggests that beta Glu is clearly related, structurally and evolutionarily, to the N-terminal two-thirds of the beta Gal . The structural analysis of the beta Gal alignment, performed by mean secondary structure prediction, revealed that most of the invariant residues are located in turn or loop structures . The location of the invariant residues is discussed with respect to their accessibility and their possible involvement in the catalytic process.

Int J Food Microbiol, 1992 Sep, 17(1), 9 - 18
Study of surface yeast flora of Roquefort cheese; Besancon X et al.; The change in yeast flora on the surface of two batches of Roquefort cheese was monitored over a period of 6 months . 401 isolates were determined and their technological properties were investigated . The main species isolated were: Debaryomyces hansenii and its non sporulating form Candida famata, Kluyveromyces lactis and its non sporulating form Candida sphaerica and Candida species . The species Debaryomyces hansenii inoculated on the surface of the cheese in one of the batches just before the salting phase was abundant throughout the ripening phases but never exceeded 50% of the yeast count . About 80% of the isolates of each species were resistant to 15% (w/v) of sodium chloride . Most of the species were able to assimilate lactose and lactic acid . 50-90% of the isolates of each species were able to hydrolyze rapeseed oil and glycerol tributyrate . Ten isolates among 401 hydrolyzed gelatin . Most of them were able to assimilate cadaverine, histamine, putrescine and tyramine.

Yeast, 1992 Sep, 8(9), 801 - 4
LEU2 gene homolog in Kluyveromyces lactis; Zhang YP et al.; A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis . The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S . cerevisiae LEU2 gene . The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2 . Further upstream, there is a partial open reading frame homologous to rat ribosomal protein L7.

Mol Microbiol, 1992 Aug, 6(16), 2279 - 86
Ethanol-induced and glucose-insensitive alcohol dehydrogenase activity in the yeast Kluyveromyces lactis; Mazzoni C et al.; The alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis is encoded by four ADH genes . In this paper we report evidence that at least three of these genes are transcribed and translated into protein . KIADH1 and KIADH2, which encode cytoplasmic activities, are preferentially expressed in glucose-grown cells with respect to ethanol-grown cells . KIADH4, which encodes one of the two activities localized within mitochondria, is induced at the transcriptional level in the presence of ethanol as is the ADH2 gene in Saccharomyces cerevisiae . However the regulation of the expression of the K . lactis gene is completely different from that of ADH2 and of other known ADH genes in that KIADH4 is insensitive to glucose repression and is not expressed on non-fermentable carbon sources other than ethanol . This kind of regulation can be clearly observed in non-fermenting strains, where the induction of KIADH4 is dependent on the addition of ethanol to the medium . On the contrary, in fermenting strains KIADH4 is always induced by ethanol or acetaldehyde produced endocellularly and this results in constitutive expression of the gene also in the presence of glucose . The mitochondrial localization of the activity encoded by KIADH4 and the peculiar regulation of this gene could be related to the fact that K . lactis is a petite negative yeast in which some mitochondrial functions seem to be essential for cell viability.

Appl Microbiol Biotechnol, 1992 Aug, 37(5), 604 - 8
Expression of high levels of human tissue plasminogen activator in yeast under the control of an inducible GAL promoter; Martegani E et al.; The human tissue plasminogen activator (h-tPA) cDNA was fused either with the leader sequence of the killer toxin of Kluyveromyces lactis or with the Saccharomyces diastaticus glucoamylase leader peptide and cloned in the yeast expression vector under the control of the inducible USAgal/CYC1 promoter . The recombinant tPA is produced in yeast as a single-chain glycosylated polypeptide of 66-72 kDa, which accumulates intracellularly associated with a membrane fraction . Using two-step fed-batch fermentation, a productivity up to 100 mg/l of active intracellular tPA was obtained.

Biochim Biophys Acta, 1992 Jul 8, 1108(1), 86 - 90
Photodynamic treatment of yeast cells with the dye toluidine blue: all-or-none loss of plasma membrane barrier properties; Paardekooper M et al.; Photodynamic treatment of Kluyveromyces marxianus with the sensitizer Toluidine blue leads to the loss of colony forming capacity . In this paper, the influence of this treatment on the barrier properties of the plasma membrane has been studied . Photodynamic treatment with the dye Toluidine blue resulted in efflux of potassium ions and E260-absorbing material . Moreover, cells became stainable with erythrosine . It is concluded that the permeability change induced by photodynamic treatment proceeds in an all-or-none fashion . Treatment of this yeast strain, with the dye and light, also induced a diminution of the cell volume . This process is most likely not coupled to the cellular potassium content, but rather to the integrity of the vacuole . These data suggest that the vacuole has an important function in the maintenance of cell volume . Finally, it was observed that the loss of cell viability was not induced by the all-or-none loss of barrier properties.

Yeast, 1992 Jul, 8(7), 501 - 17
Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation; Verduyn C et al.; Addition of benzoate to the medium reservoir of glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h-1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g-1 h-1 . Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in qO2 to 13 mmol g-1 h-1 . The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate . Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash-out . As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest to study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes . At D = 0.10 h-1, the residual glucose concentration increased proportionally with increasing benzoate concentration . This suggests that modulation of the glucose flux mainly occurs via a change in the extracellular glucose concentration rather than by synthesis of an additional amount of carriers . Also various intracellular enzyme levels were not positively correlated with the rate of respiration . A notable exception was citrate synthase: its level increased with increasing respiration rate . Growth of S . cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1 h-1 . During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume . Also with the Crabtree-negative yeasts Candida utilis, Kluyveromyces marxianus and Hansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation . In contrast to S . cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h-1 in the presence of benzoate was equal to, or lower than the qO2 attainable at mu(max) without benzoate . Enzyme activities that were repressed by glucose in S . cerevisiae also declined in K . marxianus when the glucose flux was increased by the presence of benzoate.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1992 Jun 15, 267(17), 11714 - 20
Distinct functional roles of two active site thiols in UDPglucose 4-epimerase from Kluyveromyces fragilis; Bhattacharjee H et al.; UDPglucose 4-epimerase from Kluyveromyces fragilis was earlier shown to have two conformationally vicinal thiols at the active site . Upon treatment with diamide, these thiols form a disulfide linkage across the subunits that results in coordinated loss of catalytic activity and coenzyme fluorescence (Ray, M., and Bhaduri, A . (1980) J . Biol . Chem . 255, 10777-10786) . Employing a number of thiol-specific reagents, we now suggest discriminatory and nonidentical roles for these two thiols . Kinetic and statistical analysis of 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide modification reaction of epimerase show that only one thiol is essential for activity . Consecutive modification experiments clearly show that the same active thiol is modified in both cases . However, significant differences are observed when the reactivity of these reagents is monitored in terms of coenzyme fluorescence . Treatment with N-ethylmaleimide leads to a form of inactive enzyme that fully retains its fluorescent properties whereas modification with 5,5'-dithiobis-(2-nitrobenzoic acid), on the other hand, results in the loss of both activity and fluorescence . The closely spaced nonessential second thiol, which is not modified by N-ethylmaleimide is therefore involved in generating and maintaining the coenzyme fluorescence . Modification studies with a series of spin-labeled maleimide shows that only 3-(maleimidomethyl)proxyl causes partial quenching of coenzyme fluorescence . This suggests that the active thiol is situated at a distance of 4.5 A approximately from the coenzyme fluorophore.

J Biol Chem, 1992 Jun 15, 267(17), 11709 - 13
An essential histidine residue for the activity of UDPglucose 4-epimerase from Kluyveromyces fragilis; Mukherji S et al.; UDPglucose 4-epimerase from Kluyveromyces fragilis was completely inactivated by diethylpyrocarbonate following pseudo-first order reaction kinetics . The pH profile of diethylpyrocarbonate inhibition and reversal of inhibition by hydroxylamine suggested specific modification of histidyl residues . Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of 1 essential histidine residue to be responsible for loss in catalytic activity of yeast epimerase . No major structural change in the quarternary structure was observed in the modified enzyme as shown by the identical elution pattern on a calibrated Sephacryl 200 column and association of coenzyme NAD to the apoenzyme . Failure of the substrates to afford any protection against diethylpyrocarbonate inactivation indicated the absence of the essential histidyl residue at the substrate binding region of the active site . Unlike the case of native enzyme, sodium borohydride failed to reduce the pyridine moiety of the coenzyme in the diethylpyrocarbonate-modified enzyme . This indicated the presence of the essential histidyl residue in close proximity to the coenzyme binding region of the active site . The abolition of energy transfer phenomenon between the tryptophan and coenzyme fluorophore on complete inactivation by diethylpyrocarbonate without any loss of protein or coenzyme fluorescence are also added evidences in this direction.

Nucleic Acids Res, 1992 May 11, 20(9), 2211 - 5
Kluyveromyces contains a functional ABF1-homologue; Goncalves PM et al.; ABF1 is a multifunctional protein present in Saccharomyces cerevisiae, involved in transcription-activation and -repression as well as in DNA-replication . Several lines of evidence indicate the occurrence in the related species Kluyveromyces lactis of a protein having similar properties to those of ABF1 in S . cerevisiae . In order to identify conserved functional domains in ABF1, we have cloned and sequenced the gene encoding the ABF1-homologue from K . lactis . KIABF1 is much smaller than ScABF1 (54.6 vs . 81.7 kD) . It exhibits extensive homology with its S . cerevisiae counterpart in the N-terminal region . The C-terminal domain however, is divergent, with the striking exception of a stretch of 20 amino acids, which is virtually identical in the two proteins . KIABF1 can substitute ABF1 in S . cerevisiae, emphasizing the conservation of the multiple functions of this protein.

Mol Gen Genet, 1992 May, 233(1-2), 97 - 105
Glucose transport in the yeast Kluyveromyces lactis . II . Transcriptional regulation of the glucose transporter gene RAG1; Chen XJ et al.; The RAG1 gene encodes a membrane protein involved in the low-affinity glucose/fructose transport system of the yeast Kluyveromyces lactis . Analysis of steady-state mRNA levels analysis and quantitation of expression by beta-galactosidase from RAG1-lacZ fusions assays revealed that the RAG1 gene was poorly expressed in cells grown under gluconeogenesis conditions, but was induced more than ten-fold when they were grown on various sugars . These sugars included glucose, fructose, mannose, sucrose, raffinose, as well as galactose . Nucleotide sequence and deletion analysis of the 5' flanking region of the RAG1 gene showed that an essential cis-acting element required for induced transcription of the RAG1 gene resided between -615 and -750 from the coding sequence . This region contained a 22 bp purine stretch, and a pair of 11 bp direct repeat sequences . The 11 bp repeats harbor a CCAAT motif, a consensus sequence for binding of the yeast and mammalian HAP2/3/4-type protein complex . The transcription of the RAG1 gene was dramatically affected by three unlinked mutations, rag4, rag5 and rag8 . We discuss the possible roles of RAG4, RAG5 and RAG8 gene products in the expression of the RAG1 gene, as well as the importance of the inducible RAG1 gene in the fermentative growth of K . lactis.

Mol Gen Genet, 1992 May, 233(1-2), 89 - 96
Glucose transport in the yeast Kluyveromyces lactis . I . Properties of an inducible low-affinity glucose transporter gene; Wesolowski-Louvel M et al.; In most strains of Kluyveromyces lactis, respiratory function is not required for growth on glucose . However, some natural variant strains are unable to grow when respiration is blocked by specific inhibitors (Rag- phenotype) . This phenotype is due to an allelic variation of the chromosomal gene RAG1 . The sensitive variants have a recessive allele rag1 . The RAG1 gene has been cloned by complementation of a rag1 strain from a genomic bank derived from a Rag+ strain . The nucleotide sequence of the cloned gene indicated that the RAG1 product was a sugar transporter protein . The amino acid sequence deduced from the gene structure contained the 12 hydrophobic segments typical of a transmembrane protein, and showed a high degree of homology with the GAL2 (galactose permease) and HXT2 (a high-affinity glucose transporter) proteins of Saccharomyces cerevisiae . In a rag1 null mutant, as in the natural rag1 variant, uptake of glucose at high external glucose concentrations was impaired . The RAG1 protein appears to correspond to a low-affinity glucose transporter . Transcription of the RAG1 gene, which was undetectable when cells were grown in glycerol, was induced by glucose . It is concluded that respiration-dependent growth on glucose of the Rag- variant strains is due to a defect in this inducible glucose transport system.

Mol Cell Biol, 1992 May, 12(5), 1924 - 31
The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene; Kuzhandaivelu N et al.; Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis . We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more . Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene . This two-base difference is responsible for the strong versus the weak repression phenotype . The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases) . We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS) . According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS . Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon . For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed . Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes . This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

Eur J Epidemiol, 1992 May, 8(3), 471 - 6
Anaerobic yeast killer systems; Polonelli L et al.; The influence of anaerobic conditions on the expression of the killer phenomenon of several yeast isolates belonging to recognized killer systems coded by different genetic determinants (Pichia spp., Kluyveromyces lactis, Saccharomyces cerevisiae) was studied . Anaerobiosis influenced the activity of killer toxins from some individual isolates of the genera Pichia and Saccharomyces on sensitive strains of P . anomala, K . lactis and Candida albicans . However, no influence was detectable on a S . cerevisiae sensitive isolate . Thus, anaerobic conditions seem to interfere more with the metabolic process of sensitive strains than with toxin production by killer yeasts . The selection of a panel of killer yeasts, able to display their activity against reference sensitive yeast isolates under anaerobic conditions in a medium that favored the growth of anaerobes, allowed the use of the killer system to type Bacteroides fragilis isolates for epidemiological purposes.

Microbiologia, 1992 Apr, 8(1), 14 - 20
Effect of aeration rate on the alcoholic fermentation of whey by Kluyveromyces fragilis; Varela H et al.; In this paper, the influence of aeration rate on the alcoholic batch fermentation of whey by Kluyveromyces fragilis NRRL Y-2415 was investigated . Assays in 1.5-L fermentor using concentrated whey permeate containing 100 g/L of lactose were carried out at different oxygen supply rate (KLaC*) from 0 to 82 mmol/Lh . Optimum response was obtained at 14 mmol/Lh: ethanol production rate reached was 3.4 g/Lh yielding 0.46 g of product per gram of initial lactose . An increase of KLaC* from 0 to 14 mmol/Lh improved the ethanol production: maximum specific ethanol production rate (qpm) increased 3.3 times from 0.3 to 1.0 g/gh . For higher aeration levels, ethanol production diminished and biomass formation was stimulated . The declination of qpm and the increase of microns at higher aeration level lead to conclude the importance of a controlled oxygen supply in order to obtain the required balance between yeast biosynthetic needs and ethanol production.

Mol Gen Genet, 1992 Apr, 232(3), 479 - 88
Evolution of the alcohol dehydrogenase (ADH) genes in yeast: characterization of a fourth ADH in Kluyveromyces lactis; Shain DH et al.; Three alcohol dehydrogenase (ADH) genes have recently been characterized in the yeast Kluyveromyces lactis . We report on a fourth ADH in K . lactis (KADH II: KADH2* gene) which is highly similar to other ADHs in K . lactis and Saccharomyces cerevisiae . KADH II appears to be a cytoplasmic enzyme, and after expression of KADH2 in S . cerevisiae enzyme activity comigrated with a K . lactis ADH present in cells grown in glucose or in ethanol . KADH I was also expressed in S . cerevisiae and it comigrated with a major ADH species expressed under glucose growth conditions in K . lactis . The substrate specificities for KADH I and KADH II were shown to be more similar to that of SADH II than to SADH I . SADH I cannot efficiently utilize long chain alcohols, in contrast to other cytoplasmic yeast ADHs, presumably because of the presence of a methionine (residue 271) in its substrate binding cleft . A comparison of the DNA sequences of ADHs among K . lactis, S . cerevisiae and Schizosaccharomyces pombe suggests that the ancestral yeast species contained one cytoplasmic ADH . After divergence from S . pombe, the ADH in the ancestor to K . lactis and S . cerevisiae was duplicated, and one ADH became localized to the mitochondrion, presumably for the oxidative use of ethanol . Following the speciation of S . cerevisiae and K . lactis, the gene encoding the cytoplasmic ADH in S . cerevisiae duplicated, which resulted in the development of the SADH II protein as the primary oxidative enzyme in place of SADH III . In contrast, the K . lactis mitochondrial ADH duplicated to give rise to the highly expressed KADH3 and KADH4 genes, both of which may still play primary roles in oxidative metabolism . These data suggest that K . lactis and S . cerevisiae use different compartments for their metabolism of ethanol . Our results also indicate that the complex regulatory circuits controlling the glucose-repressible SADH2 in S . cerevisiae are a recent acquisition from regulatory networks used for the control of genes other than SADH2.

Curr Genet, 1992 Apr, 21(4-5), 357 - 63
Kluyveromyces lactis killer system: ORF1 of pGKL2 has no function in immunity expression and is dispensable for killer plasmid replication and maintenance; Schaffrath R et al.; To functionally characterize the genes encoded by the larger killer plasmid pGKL2 from Kluyveromyces lactis a previously developed in-vivo recombination system was exploited . An in-vitro modified version of the cytoplasmically expressible LEU2 gene cartridge (LEU2*) flanked by appropriate pGKL2 segments was used to replace the central part of the ORF1 region of pGKL2 . Transformation of a Leu- killer strain resulted in the expected disruption of ORF1 in the resident pGKL2 . The Leu+ transformants obtained can be assigned to three classes . Class I carries both killer plasmids, pGKL1/2, and the recombinant pGKL2 derivative termed pRKL2 . Class II and III additionally harbor palindrome and hairpin-like plasmids, respectively . Upon subculturing of class I transformants under selective pressure, segregation of the native pGKL2 and the recombinant pRKL2 eventually occurs resulting in total loss of pGKL2 . No differences concerning killer and immunity phenotype between a pRKL2-harboring strain and the native pGKL2-carrying recipient could be detected . Thus pGKL2 ORF1 is dispensable for both expression of killer/immunity phenotypes and for the replication and maintenance of the K . lactis killer plasmids.

Antonie Van Leeuwenhoek, 1992 Apr, 61(3), 195 - 205
Physical and genetic characterization of linear DNA plasmids from the heterothallic yeast Saccharomycopsis crataegensis; Bolen PL et al.; Five strains of the heterothallic yeast Saccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al . 1987) . Three DNA plasmids, designated pScrl-1, -2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904 . DNA plasmids were not identified in S . crataegensis strains Y-5910 or YB-192 . Four S . crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al . 1987) . Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3 . Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3 . This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al . (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902 . The pScrl plasmids show no homology to the dsRNA molecules of S . crataegensis, the 2 microM circular DNA of Staccharomyces cerevisiae, the 'killer' plasmids of Kluyveromyces lactis, or the linear DNA plasmids of Pichia inositovora . In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered . Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.

Indian J Biochem Biophys, 1992 Apr, 29(2), 209 - 13
Microenvironment at the substrate binding subsite of the active site of UDPglucose 4-epimerase from Kluyveromyces fragilis using a fluorescent analog of UMP; Ray S et al.; A chromophorics and fluorescent analog of uridine 5'-monophosphate (UMP), a known competitive inhibitor of UDPglucose 4-epimerase was synthesised . This analog, namely 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) uridine 5'-monophosphate, was found to be a powerful reversible inhibitor of UDPglucose 4-epimerase indicating its interaction with the substrate binding site of the enzyme . The extreme sensitivity of the fluorescence emission spectrum of this analog to solvent polarity makes it an excellent probe for the study of the environment at the active site of the enzyme . We report here the effective use of this UMP analog to demonstrate that the hydroxyl groups of the ribose moiety of UMP and presumably the substrates (UDPgalactose and UDPglucose) do not reside in a hydrophobic milieu.

Curr Genet, 1992 Apr, 21(4-5), 365 - 70
Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis; Bergkamp RJ et al.; We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis . This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae . Plasmids containing a portion of K . lactis rDNA for targeted homologous recombination, as well as the S . cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K . lactis, analyzed for both copy number and stability . These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions . Using this vector system, we expressed a fusion construct containing the S . cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for alpha-galactosidase from the plant Cyamopsis tetragonoloba . Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of alpha-galactosidase production (250 mg/l) with a secretion efficiency of about 95% . When compared to extrachromosomal K . lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher alpha-galactosidase production level and a considerably higher stability under non-selective conditions.

J Mol Biol, 1992 Mar 5, 224(1), 1 - 5
Changing the specificity of the sorting receptor for luminal endoplasmic reticulum proteins; Semenza JC et al.; Luminal proteins of the endoplasmic reticulum (ER) share a common carboxy-terminal tetrapeptide which is necessary and sufficient for their retention in the ER . In animal cells this retention signal is usually KDEL, whereas the yeast Kluyveromyces lactis uses the closely related sequences HDEL and DDEL . The yeast ERD2 gene has been shown to determine the capacity and specificity of the retention system, implying that it encodes a sorting receptor . This receptor is thought to retrieve escaped ER proteins from the Golgi, where a human homologue of this protein has been located . This dual function of binding and retrieval requires a receptor with highly specific binding at a specific location in the cell (Golgi but not ER) . Here, a region of the ERD2 protein responsible for the specificity of ligand recognition has been identified using three independent approaches . A single amino acid residue is shown to selectively affect HDEL retention: substitution of residue 51 of the K . lactis receptor is sufficient to abolish recognition of HDEL but not DDEL, generating a novel retention phenotype.

Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1904 - 8
Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate; Yeh P et al.; Due to its remarkably long half-life, together with its wide in vivo distribution and its lack of enzymatic or immunological functions, human serum albumin (HSA) represents an optimal carrier for therapeutic peptides/proteins aimed at interacting with cellular or molecular components of the vascular and interstitial compartments . As an example, we designed a genetically engineered HSA-CD4 hybrid aimed at specifically blocking the entry of the human immunodeficiency virus into CD4+ cells . In contrast with CD4, HSA-CD4 is correctly processed and efficiently secreted by Kluyveromyces yeasts . In addition, its CD4 moiety exhibits binding and antiviral in vitro properties similar to those of soluble CD4 . Finally, the elimination half-life of HSA-CD4 in a rabbit experimental model is comparable to that of control HSA and 140-fold higher than that of soluble CD4 . These results indicate that the genetic fusion of bioactive peptides to HSA is a plausible approach toward the design and recovery of secreted therapeutic HSA derivatives with appropriate pharmacokinetic properties.

Int J Food Microbiol, 1992 Mar-Apr, 15(3-4), 383 - 8
Growth of fermentative and non-fermentative yeasts in natural yoghurt, stored in polystyrene cartons; McKay AM; Permeation of oxygen through polystyrene packaging is a factor in the growth of yeasts in natural yoghurt . Diffusion of oxygen through the packaging material can permit the growth of non-fermentative yeasts in yoghurt stored at refrigeration temperatures . Yarrowia lipolytica, a non-fermentative yeasts which does not utilize lactose was isolated from yoghurt . The growth in natural yoghurt of Yarrowia lipolytica and the lactose-fermenting yeast Kluyveromyces marxianus was investigated . Both yeasts grew in yoghurt with reduced fat content . Storage of yoghurt in an anaerobic atmosphere eliminated growth of Yarrowia lipolytica but permitted fermentative growth of Kluyveromyces marxianus.

J Gen Microbiol, 1992 Feb, 138 ( Pt 2), 337 - 45
Characterization of a circular plasmid from the yeast Kluyveromyces waltii; Chen XJ et al.; A new plasmid was found in the yeast Kluyveromyces waltii . This high-copy-number plasmid, named pKW1, is a double-stranded circular DNA plasmid of 5619 bp . It has several features characteristic of the 2 mu-type plasmids: presence of two inverted repeats and four open reading frames, as well as the interconversion of two isomeric forms . However, the nucleotide sequence shows little homology with known yeast plasmids . An ARS function was localized within a segment of 545 bp near one of the inverted repeats . Chimeric plasmids carrying this segment efficiently transformed K . waltii . A strain of K . waltii cured of the plasmid (cir degree) was also obtained . In the pKW1 sequence, a functionally neutral region was found at which foreign DNA can be inserted with little effect on plasmid stability . Such constructions carrying the full sequence of pKW1 replicated autonomously in a cir degree host and were particularly stable . pKW1-derived full-sequence plasmids also transformed K . thermotolerans, but not K . lactis.

J Bacteriol, 1992 Jan, 174(2), 408 - 14
Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases; Watanabe T et al.; The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12 . Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp . Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1 . The N-terminal 47-amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1 . The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence . A 73-amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit . The evolutionary and functional meanings of these similarities are discussed.

Antonie Van Leeuwenhoek, 1992 Jan, 61(1), 57 - 60
Assignment of Kluyveromyces cellobiovorus nomen nudum to Candida intermedia (Ciferri & Ashford) Langeron et Guerra; Martini A et al.; A yeast culture isolated in Japan from soil and invalidly described as Kluyveromyces cellobiovorus nom . nud . DBVPG 6286 (CBS 7153) was compared for its physiological and morphological properties and by nDNA-nDNA reassociation experiments with the type strains of several species of the genus Kluyveromyces as well as of various Candida species exhibiting similar phenotypic profiles . DBVPG 6286 was found to be conspecific with the type strain of Candida intermedia (Ciferri & Ashford) Langeron et Guerra (1938).

Yi Chuan Xue Bao, 1992, 19(3), 284 - 8
{Expression and secretion of human interferon alpha A in yeast Kluyveromyces lactis}; Chen X et al.; The vector pE1 is derived from the plasmid pKD1 isolated from the yeast Kluyveromyces lactis . It can be maintained stably in the yeast K . lactis at high copy number . We explored the possibility of using the pKD1/K . lactis system for heterologous gene expression . The human interferon alpha A secretion cassette was inserted into pE1 . The obtained plasmid, pE-IFN1, showed slightly instability when compared to pE1 . The data showed that the secretion signal sequence of the alpha factor of Saccharomyces cerevisiae was functionally active in K . lactis . The K . lactis transformants carrying pE-IFN1 secreted efficiently IFN into the growth medium when they were grown in nonselective medium . The total amount of IFN released reached 1-2 mg per liter of culture supernatant.

Nat Toxins, 1992, 1(1), 38 - 47
Trichothecene synergism, additivity, and antagonism: the significance of the maximally quiescent ratio; Koshinsky HA et al.; The interactive effect of the combinations of trichothecene mycotoxins often found in fungus infected plants, contaminated grain, and other biological systems is poorly understood . Growth inhibition of the yeast Kluyveromyces marxianus was used to measure the effects of HT-2 toxin, roridin A, and T-2 toxin as individual toxins or as binary mixtures . A value, the combination index, was derived which indicates the interactive effects of a binary mixture of toxins . The interaction is affected by the ratio of the individual toxins, and the percent inhibition of yeast growth . Generally the interaction of T-2 toxin and roridin A or T-2 toxin and HT-2 toxin changes from antagonistic when they cause a low percent inhibition of yeast growth to synergistic when they cause a high percent inhibition of yeast growth . Additionally, any two trichothecenes have a unique ratio, which we name the maximally quiescent ratio (or MQR), where there is the least change in the type and intensity of their interaction . The maximally quiescent ratio in this case has helped to define the nature of toxin interactions and could be used to provide insights into hormone, immune system, developmental, enzyme, and gene regulation, combined drug therapy, and the action of mixtures of natural or synthetic toxins, carcinogens, pesticides, and environmental pollutants.

Gene, 1991 Nov 15, 107(2), 285 - 95
High-level secretion of correctly processed recombinant human interleukin-1 beta in Kluyveromyces lactis; Fleer R et al.; The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins . Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum {Chen et al., Nucleic Acids Res . 14 (1986) 4471-4481} for the secretion of recombinant human interleukin-1 beta (reIL-1 beta) . High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the 'pre'-region of the K . lactis killer toxin . N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence . Synthesis as prepro-IL-1 beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta . Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K . lactis . As in Saccharomyces cerevisiae {Baldari et al., EMBO J . 6 (1987) 229-234}, but unlike native human IL-1 beta, K . lactis reIL-1 beta is glycosylated . This glycosylation led to a 95% loss of its biological activity . Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity . A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta . The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.

J Biol Chem, 1991 Nov 5, 266(31), 20882 - 7
Kluyveromyces bulgaricus yeast lectins . Isolation of two galactose-specific lectin forms from the yeast cell wall; al-Mahmood S et al.; Incubation of galactose treated Kluyveromyces bulgaricus yeast cells in EDTA/phosphate-buffered saline led to an extract possessing hemagglutinating and yeast flocculating properties . Purification of this extract by affinity chromatography and gel filtration gave two lectin forms, Kb-CWL I and Kb-CWL II, with an apparent molecular mass of 38,000 and 150,000 Da, respectively . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Kb-CWL I and Kb-CWL II were dimeric and octameric of a subunit of 18,900 Da . At high concentration, purified Kb-CWL I associated to give Kb-CWL II . This association seemed to be independent on pH . The two lectin forms were glycoproteins, the peptide counterpart was very rich in Lys, Glu, and Gly, and the carbohydrate part represented 1% of the whole molecule and was composed of Glc, Man, and Ara . The two lectin forms (KB-CWL I and Kb-CWL II) agglutinated human red blood cells and flocculated EDTA-treated K . bulgaricus yeast cells . The activity of both lectin forms required Ca2+ ions, while Sr2+ showed some competitive inhibition . Optimal activity was obtained within a pH range of 4-6.5 for both forms . Temperatures of 80-90 degrees C for 20 min, or proteolytic treatment reduced irreversibly the activity of Kb-CWL I and Kb-CWL II . The role of the cell wall phosphopeptidomannan as a ligand and a potential physiological receptor of these lectin forms was demonstrated.

Mol Cell Biol, 1991 Nov, 11(11), 5454 - 61
Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae; Meyer J et al.; We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose . The data show that the K . lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system . This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209) . Complementation studies in Saccharomyces cervisiae show that K . lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation . We conclude that the regulatory function of GAL1 in K . lactis soon after induction is similar to the function of GAL3 in S . cerevisiae.

Yeast, 1991 Nov, 7(8), 775 - 80
Transport of lactic acid in Kluyveromyces marxianus: evidence for a monocarboxylate uniport; Fonseca A et al.; Lactic acid-grown cells of a strain of Kluyveromyces marxianus transported D- and L-lactic acid by a saturable mechanism that was partially inducible and subject to glucose repression, with the following kinetic parameters at pH 5.4: Vmax = 1.00 (+/- 0.13) mmol h-1 per g dry weight and Ks = 0.42 (+/- 0.08) mM . Lactic acid transport was competitively inhibited by pyruvic, glycolic, acetic and bromoacetic acids . The latter, a non-metabolizable analogue, was transiently accumulated, the extent depending on the extracellular pH . The pH dependence of the Ks values for undissociated lactic acid and for the lactate anion indicated that the latter was the transported species . Lactate uptake was not accompanied by the simultaneous uptake of protons, potassium ions or sodium ions excluding symport mechanisms . Initial lactic acid uptake led to transient membrane hyperpolarization as measured with a fluorescent dye excluding also an electroneutral anion antiport mechanism . It was concluded that lactate anions use a monocarboxylate uniport and that the counter anion, possibly bicarbonate, uses a separate channel, the coupling being electrical and loose.

FEMS Microbiol Lett, 1991 Nov 1, 68(1), 75 - 8
Activity of different 'killer' yeasts on strains of yeast species undesirable in the food industry; Palpacelli V et al.; Killer strains of the genera Saccharomyces, Hansenula and Kluyveromyces were tested for killing activity against yeasts that cause trouble in the food industry (in the genera Zygosaccharomyces, Kloeckera, Saccharomycodes and Schizosaccharomyces) . Saccharomyces strains killed only Zygosaccharomyces rouxii strains, while non-Saccharomyces strains showed a wider anti-yeast spectrum . The Kluyveromyces phaffii killer strain was of particular interest because of its killer action against Kloeckera apiculata, Saccharomycodes ludwigii and Zygosaccharomyces rouxii.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5351 - 8
Coregulation of the Kluyveromyces lactis lactose permease and beta-galactosidase genes is achieved by interaction of multiple LAC9 binding sites in a 2.6 kbp divergent promoter; Godecke A et al.; The coregulated genes LAC4 and LAC12 encoding beta-galactosidase and lactose permease, respectively, are responsible for the ability of the milk yeast Kluyveromyces lactis to utilise lactose . They are divergently transcribed and separated by an unusually large intergenic region of 2.6 kbp . Mapping of the upstream border of the beta-galactosidase gene (LAC4) promoter by introduction of mutations at the chromosomal locus showed that LAC4 and LAC12 share the same upstream activation sites (UAS) . The UASs represent binding sites for the trans-activator LAC9, a K . lactis homologue of GAL4, conforming to the consensus sequence 5'-CGG(N5)A/T(N5)CCG-3' . Two binding sites are located in front of each of the genes at almost symmetrical positions . beta-galactosidase activity measurements as well as quantitation of LAC4 and LAC12 mRNA levels demonstrated that all four sites are required for full induction . LAC4 proximal and a LAC12 proximal sites cooperate in activating transcription of both genes . These sites are more than 1.7 kbp apart and the distal site is located more than 2.3 kbp upstream of the respective start of transcription . Thus, the distance between interacting sites is larger than in any of the well characterised yeast promoters . The contribution to gene activation differs for individual binding sites and correlates with the relative affinity of LAC9 for these sites in vitro suggesting that LAC9 binding is a rate limiting step for LAC promoter function.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5345 - 50
Sequence conservation in the Saccharomyces and Kluveromyces GAL11 transcription activators suggests functional domains; Mylin LM et al.; Efficient transcription of many Saccharomyces cerevisiae genes requires the GAL11 Protein . GAL11 belongs to a class of transcription activator that lacks a DNA-binding domain . Such proteins are thought to activate specific genes by complexing with DNA-bound proteins . To begin to understand the domain structure-function relationships of GAL11 we cloned and sequenced a homologue from the yeast Kluyveromyces lactis, Kl-GAL11 . The two predicted GAL11 proteins show high overall amino acid conservation and an unusual amino acid composition including 18% glutamine, 10% asparagine (S . cerevisiae) or 7% (K . lactis), and 8% proline (K . lactis) or 5% (S . cerevisiae) residues . Both proteins have runs of pure glutamines . Sc-GAL11 has glutamine-alanine runs but in Kl-GAL11 the alanines in such runs are replaced by proline and other residues . The primary sequence similarity is reflected in functional similarity since a gal11 mutation in K . lactis creates phenotypes similar to those seen previously in gal11-defective S . cerevisiae . In addition, Kl-GAL11 complements a gal11-defect in S . cerevisiae by partially restoring induction of GAL1 expression, growth on nonfermentable carbon sources, and phosphorylation of GAL4.

Genes Dev, 1991 Oct, 5(10), 1902 - 11
Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability; Sarge KD et al.; We have cloned two distinct mouse heat shock transcription factor genes, mHSF1 and mHSF2 . The mHSF1 and mHSF2 open reading frames are similar in size, containing 503 and 517 amino acids, respectively . Although mHSF1 and mHSF2 are quite divergent overall (only 38% identity), they display extensive homology in the DNA-binding and oligomerization domains that are conserved in the heat shock factors of Saccharomyces cerevisiae, Kluyveromyces lactis, Drosophila, tomato, and human . The ability of these two mouse heat shock factors to bind to the heat shock element (HSE) is regulated by heat . mHSF1 is expressed in an in vitro translation system in an inactive form that is activated to DNA binding by incubation at temperatures greater than 41 degrees C, the same temperatures that activate heat shock factor DNA binding and the stress response in mouse cells in vivo . mHSF2, on the other hand, is expressed in a form that binds DNA constitutively but loses DNA binding by incubation at greater than 41 degrees C . Both mHSF1 and mHSF2 are encoded by single-copy genes, and neither is transcriptionally regulated by heat shock . However, there is a striking difference in the levels of mHSF1 mRNA in different tissues of the mouse.

Biotechnology (N Y), 1991 Oct, 9(10), 968 - 75
Stable multicopy vectors for high-level secretion of recombinant human serum albumin by Kluyveromyces yeasts; Fleer R et al.; We have designed stable pKD1 derivatives for efficient secretion of recombinant human serum albumin (rHSA) by industrial strains of Kluyveromyces yeasts . A comparison of this multi-copy expression system with isogenic cassettes integrated at chromosomal loci demonstrated that high level secretion of rHSA is a function of gene dosage in K . lactis . Various signal sequences could be used, and the secretion levels were independent of the presence of the native pro peptide . The mitotic stability of the pKD1-based expression vectors was found to be species and strain dependent and was influenced by promoter strength and culture conditions . Vector stability was drastically enhanced when the HSA gene was expressed from an inducible promoter: 90% of the transformed cells still harbored the vector after 100 generations of non-selective growth in uninduced culture conditions . Secretion levels in the range of several grams per liter of correctly folded and processed rHSA were obtained at the pilot scale, thus making the industrial production of pharmaceutical-grade, Kluyveromyces-derived rHSA economically feasible.

Nucleic Acids Res, 1991 Sep 11, 19(17), 4701 - 7
Altered response to growth rate changes in Kluyveromyces lactis versus Saccharomyces cerevisiae as demonstrated by heterologous expression of ribosomal protein 59 (CRY1)
Larson GP, Rossi JJ.
We report the cloning, characterization and preliminary analysis of the regulation of the gene coding for ribosomal protein 59 (RP59) from the budding yeast Kluyveromyces lactis . The RP59 gene is present as a single copy, contains an intron within the amino terminal coding portion of the gene, and harbors conserved S . cerevisiae splicing signals . Sequence elements upstream of the transcriptional start site are homologous to UASRPG, known to regulate the transcription of numerous genes in S . cerevisiae via their interaction with the trans-activating factor RAP1 . These elements are necessary for transcription of RP59 in both K.lactis and S.cerevisiae hosts . UASRPG in S.cerevisiae rp genes also modulate the transcription of rp RNA synthesis in response to a growth rate upshift . In K.lactis, the RP59 gene does not respond to growth rate upshift . Reciprocal expression of RP59 and CRY1 in heterologous hosts demonstrates that glucose upshift occurs in S.cerevisiae but not K.lactis . These results demonstrate that a factor or factors required for growth upshift are lacking in K.lactis, and provide further evidence that the UASRPG are sufficient signals for modulating this response.

FEBS Lett, 1991 Sep 2, 289(1), 64 - 8
Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var . marxianus ATCC 12424; Laloux O et al.; Cell wall inulinase (EC 3.2.1.7) was purified from Kluyveromyces marxianus var . marxianus (formerly K . fragilis) and its N-terminal 33-amino acid sequence was established . PCR amplification of cDNA with 2 sets of degenerate primers yielded a genomic probe which was then used to screen a genomic library established in the YEp351 yeast shuttle vector . One of the selected recombinant plasmids allowed an invertase-negative Saccharomyces cerevisiae mutant to grow on inulin . It was shown to contain an inulinase gene (INU 1) encoding a 555-amino acid precursor protein with a typical N-terminal signal peptide . The sequence of inulinase displays a high similarity (67%) to S . cerevisiae invertase, suggesting a common evolutionary origin for yeast beta-fructosidases with different substrate preferences.

Mol Gen Genet, 1991 Sep, 228(3), 401 - 9
A phosphoglucose isomerase gene is involved in the Rag phenotype of the yeast Kluyveromyces lactis; Goffrini P et al.; The rag2 mutant of Kluyveromyces lactis cannot grow on glucose when mitochondrial functions are blocked by various mitochondrial inhibitors, suggesting the presence of a defect in the fermentation pathway . The RAG2 gene has been cloned from a K . lactis genomic library by complementation of the rag2 mutation . The amino acid sequence of the RAG2 protein deduced from the nucleotide sequence of the cloned RAG2 gene shows homology to the sequences of known phosphoglucose isomerases (PGI and PHI) . In vivo complementation of the pgi1 mutation in Saccharomyces cerevisiae by the cloned RAG2 gene, together with measurements of specific PGI activities and the detection of PGI proteins, confirm that the RAG2 gene of K . lactis codes for the phosphoglucose isomerase enzyme . Complete loss of PGI activity observed when the coding sequence of RAG2 was disrupted leads us to conclude that RAG2 is the only gene that codes for phosphoglucose isomerase in K . lactis . The RAG2 gene of K . lactis is expressed constitutively, independently of the growth substrates (glycolytic or gluconeogenic) . Unlike the pgi1 mutants of S . cerevisiae, the K . lactis rag2 mutants can still grow on glucose, however they do not produce ethanol.

Biochimie, 1991 Sep, 73(9), 1195 - 203
Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast; Chen XJ et al.; The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp) . The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase . A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity . Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903 . These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host . Lac+ and G418-resistant transformants were obtained with either orientation of Pk1 . The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids . It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid . The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.

Yeast, 1991 Aug-Sep, 7(6), 617 - 25
Intracellular expression of Kluyveromyces lactis toxin gamma subunit mimics treatment with exogenous toxin and distinguishes two classes of toxin-resistant mutant; Butler AR et al.; The Kluyveromyces lactis toxin is a heterotrimeric protein which irreversibly arrests proliferation of sensitive Saccharomyces cerevisiae cells in the G1 phase of the cell cycle . By expressing the gamma subunit of the toxin in sensitive yeast cells from a conditional promoter, it was previously demonstrated that it alone is required for inhibition (Tokunaga et al . (1989) . Nucleic Acids Res . 17, 3435-3446) . Here we show that, like native exogenous toxin, intracellular gamma subunit expression promotes a striking arrest of sensitive cells in G1 . However, unlike the G1 arrest caused by native toxin, that induced by the gamma subunit alone does not result in reduced cellular viability and is fully and rapidly reversible, suggesting that the G1 arrest and the irreversibility of action may reflect different aspects of the toxin's interaction with sensitive cells . We have selected a large number of S . cerevisiae mutants which are highly resistant to the toxin in order to study its mode of action in more detail . Complementation analysis demonstrated that all but one of the mutants were recessive and these defined four separate genes . Members of two complementation groups concurrently acquired resistance to intracellular gamma subunit expression, suggesting that they contain a modified toxin target site . The other two genes appear to be required for entry of the gamma subunit into the sensitive cells since these mutants, while refractory to exogenous toxin, were fully sensitive to intracellular gamma subunit expression.

Eur J Biochem, 1991 Jul 15, 199(2), 483 - 8
Kluyveromyces lactis toxin has an essential chitinase activity; Butler AR et al.; The Kluyveromyces lactis toxin is a protein containing three subunits (alpha, beta and gamma) which causes sensitive yeast cells to arrest proliferation in the G1 phase of the cell cycle . Despite the toxin's complex structure, the gamma subunit appears to be the only component required for it to arrest proliferation since intracellular expression of the gamma polypeptide alone in a sensitive yeast strain mimics the effect of the exogenous native toxin . The toxin alpha subunit shows sequence similarity to a variety of chitinases and here we report that the toxin is a potent exochitinase . The exochitinase activity is absolutely required for its biological activity against sensitive Saccharomyces cerevisiae cells and allosamidin, a specific inhibitor of chitinases, abolishes the biological activity of the toxin . However, since the alpha subunit is not required for the G1 arrest induced by the toxin, the chitinase activity of the toxin cannot be directly responsible for the ultimate effect of the toxin and most likely plays a role in the initial interaction of the toxin with sensitive cells.

J Biol Chem, 1991 Jul 5, 266(19), 12146 - 51
Proton-motive force-driven D-galactose transport in plasma membrane vesicles from the yeast Kluyveromyces marxianus; Van Leeuwen CC et al.; Galactose transport was studied in membrane vesicles, prepared by fusion of plasma membranes from the yeast Kluyveromyces marxianus with proteoliposomes containing beef heart cytochrome c oxidase as a proton-motive force-generating system . Sugar transport studies performed under nonenergized conditions revealed that, even at high protein to phospholipid ratios, not all vesicles contained a D-galactose-specific transporter . The amount of vesicles containing an active carrier proved to be proportional to the amount of plasma membrane protein present in the fusion mixture . By addition of a suitable electron donor system a proton-motive force of -160 mV could be generated, inside alkaline and negative . Moreover, D-galactose accumulation was observed . It was found that D-galactose accumulation was highly dependent on the phospholipid composition of the vesicles, whereas generation of a proton-motive force was not . Best results were obtained with vesicles prepared with Escherichia coli phospholipid, giving a galactose accumulation of 14 times . Uphill transport could be established under conditions where only the pH gradient or the electrical gradient was present . Moreover, kinetic analysis of the galactose transport activity in energized vesicles revealed influx with a Km value of 540 microM, which is in good agreement with the apparent affinity constant obtained with whole cells . These results establish that galactose transport of K . marxianus is a proton-motive force-driven process . Moreover it demonstrates that plasma membrane vesicles co-reconstituted with cytochrome c oxidase are a valuable resource for the analysis of proton-motive force-driven sugar transport systems of yeast.

Genes Dev, 1991 Jul, 5(7), 1252 - 63
Unexpected point mutations activate cryptic 3' splice sites by perturbing a natural secondary structure within a yeast intron; Deshler JO et al.; The 3' splice site of the budding yeast Kluyveromyces lactis actin gene (ACT) intron is distally spaced (122 nucleotides) from its branchpoint and is also preceded by a silent PyAG located 43 nucleotides upstream . We devised a genetic screen that resulted in the isolation of several randomly induced cis-acting mutations that activate the silent PyAG as a 3' splice site . These mutations fall within a region surrounding this PyAG, which can hypothetically fold into a higher-order structure . Site-directed mutational analyses demonstrate that a hairpin structure in this region is required for correct 3' splice-site selection . Analysis of the point mutations suggests that local breathing of the hairpin near the first PyAG can lead to its activation . These data demonstrate that 3' splice-site selection is not a consequence of a linear, directional scanning mechanism, but support the notion of a critical positioning requirement for 3' splice-site selection . We speculate on the possible origin of this intron-encoded structural motif, which has homology to a bacterial transposon and suggests one possible origin for alternative splicing mechanisms in higher eukaryotes.

J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1749 - 57
Analysis of the response of Saccharomyces cerevisiae cells to Kluyveromyces lactis toxin; Butler AR et al.; The response of Saccharomyces cerevisiae cells to the toxin produced by certain strains of Kluyveromyces lactis was studied . The toxin caused an arrest of sensitive cells in the unbudded (G1) phase of the cell cycle, consistent with the accumulation of cells with an unreplicated (G1) content of DNA in treated populations . However, toxin-treated cells were not proficient for mating . The effects of the toxin were dependent on its continuous presence for over an hour and removal of cells into fresh medium at earlier times prevented inhibition . Following toxin treatment, cells increased in volume and continued to synthesize protein and RNA, suggesting that they were able to continue growth in the absence of division . However, several lines of evidence suggested that the toxin does not simply block proliferation in G1, but that another continuous or post-G1 event is also affected . Possible models to explain these observations are discussed.

Curr Genet, 1991 Jul, 20(1-2), 99 - 114
Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron; Hardy CM et al.; The cytochrome oxidase subunit 1 gene (COX1) in K . lactis K8 mtDNA spans 8,826 bp and contains five exons (termed E1-E5) totalling 1,602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S . cerevisiae . The four introns (termed K1 cox1.1-1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes . The first intron belongs to group II whereas the remaining three are group I type B . Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5 a, and 1.5 b respectively from S . cerevisiae . Horizontal transfer of an intron between recent progenitors of K . lactis and S . cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching . Sequence comparisons between K1 cox1.3/Sc cox1.5 a and K1 cox1.4/Sc cox1.5 b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts . Intron K1 cox1.2 is not found in S . cerevisiae and appears at an unique location in K . lactis . A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions . Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S . cerevisiae mtDNA.

Curr Genet, 1991 Jul, 20(1-2), 115 - 20
A mobile group II intron of a naturally occurring rearranged mitochondrial genome in Kluyveromyces lactis; Skelly PJ et al.; Mitochondrial intron content is variable in the yeast Kluyveromyces lactis . Strains can be divided into three classes depending on the structure of the cytochrome oxidase subunit 1 (COX1) gene: (1) those containing intron K1 cox1.1, (2) those containing K1 cox1.2, 3 and 4 and, (3) those that contain all four introns . In addition, strains belonging to the first class (designated Type B strains), have an altered mitochondrial gene order relative to strains from classes (2) and (3) (Type A, Hardy et al . 1989) . Crossing experiments reveal that K1 cox1.1 (a group II intron) transfers at high frequency (89%) to mitochondrial genomes lacking this intron . By contrast, the mobility of the remaining introns (all group I) is of the order of 7%.

Cell, 1991 May 31, 65(5), 797 - 804
Structural basis for the regulation of splicing of a yeast messenger RNA; Eng FJ et al.; In S . cerevisiae, ribosomal protein L32 regulates the splicing of the transcript of its own gene, RPL32 . We have identified an RNA structure within the transcript that is responsible for this regulation . Initial deletions limited essential sequences to the 5' exon and the first few nucleotides of the intron . To take advantage of phylogenetic comparison of RNA structures, RPL32 was cloned from the closely related species, Kluyveromyces lactis . The splicing of its transcript is similarly regulated . Sequences conserved between the S . cerevisiae and K . lactis transcripts suggested a structure involving base pairing of a region encompassing the 5' splice site with another near the 5' end of the transcript . Analysis of numerous site-directed mutations supports this structure . We infer that stabilization of this structure by L32 inhibits splicing by precluding the interaction of U1 RNA with the 5' splice site.

Curr Genet, 1991 May, 19(5), 389 - 93
Linear DNA plasmids of Pichia inositovora are associated with a novel killer toxin activity; Hayman GT et al.; Pichia inositovora, strain NRRL Y-18709, which contains three linear double-stranded DNA plasmids, pPinl-1, pPinl-2 and pPinl-3, was cured of these plasmids both by growing the strain in the presence of 50 micrograms/ml bisbenzimide, and by exposure to ultraviolet light . Both cured and uncured strains were tested for growth on a variety of carbon sources . No differences in growth response were detected, indicating no discernible involvement of the linear plasmids in the catabolism of these compounds . Culture supernatants of Pichia inositovora were shown to contain a substance larger than 100 kDa that is toxic to Saccharomyces cerevisiae, strain GS 1688 . Toxin activity was optimal in YEPD assay plates containing 50 mM citrate buffer with a pH between 3.4 and 4.2 . Culture supernatants from P . inositovora were also weakly active against Cephaloascus albidus, strain NRRL Y-18710, and Citeromyces matritensis, strain NRRL Y-18711 . Concentrated supernatants from cured P . inositovora strains did not exhibit these activities, consistent with the hypothesis that this toxic activity is linear plasmid-encoded . Unlike the well-known Kluyveromyces lactis system, or the newly identified P . acaciae system, P . inositovora strains cured of their linear plasmids do not become detectably sensitive to toxin produced by the wild-type strain suggesting a nonplasmid-encoded immunity function.

Yeast, 1991 May-Jun, 7(4), 391 - 400
Two genes encoding putative mitochondrial alcohol dehydrogenases are present in the yeast Kluyveromyces lactis; Saliola M et al.; Four structural genes encoding isozymes of the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis have been identified by hybridization to ADH2 DNA probes from Saccharomyces cerevisiae . In this paper we report on the isolation of KlADH4 and the complete sequencing of KlADH3 and KlADH4, two genes which show high homology to KlADH1, the ADH gene previously isolated in K . lactis, and to the ADH genes of S . cerevisiae . When compared with KlADH1, both KlADH3 and KlADH4 encode amino-terminal extensions which show the characteristics of the mitochondrial targeting sequences . These extensions are poorly conserved both at the nucleotide and the amino acid level . Surprisingly, the KlADH4 extension shows a higher identity at the amino acid level to the one encoded by ADH3 of S . cerevisiae than to the KlADH3 presequence . KlADH3 and KlADH4, in contrast to the ADH3 gene of S . cerevisiae, show a strong bias in the choice of codons.

J Gen Microbiol, 1991 May, 137 ( Pt 5), 1223 - 30
Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences; Hendriks L et al.; The classification of species belonging to the genus Candida Berkhout is problematic . Therefore, we have determined the small ribosomal subunit RNA (srRNA) sequences of the type strains of three human pathogenic Candida species; Candida krusei, C . lusitaniae and C . tropicalis . The srRNA sequences were aligned with published eukaryotic srRNA sequences and evolutionary trees were inferred using a matrix optimization method . An evolutionary tree comprising all available eukaryotic srRNA sequences, including two other pathogenic Candida species, C . albicans and C . glabrata, showed that the yeasts diverge rather late in the course of eukaryote evolution, namely at the same depth as green plants, ciliates and some smaller taxa . The cluster of the higher fungi consists of 10 ascomycetes and ascomycete-like species with the first branches leading to Neurospora crassa, Pneumocystis carinii, Candida lusitaniae and C . krusei, in that order . Next there is a dichotomous divergence leading to a group consisting of Torulaspora delbrueckii, Saccharomyces cerevisiae, C . glabrata and Kluyveromyces lactis and a smaller group comprising C . tropicalis and C . albicans . The divergence pattern obtained on the basis of srRNA sequence data is also compared to various other chemotaxonomic data.

Biotechnol Appl Biochem, 1991 Apr, 13(2), 212 - 6
Effects of growth temperature on toxicity of T-2 toxin and roridin A to yeast; Gu TS et al.; In yeasts, growth temperature is known to affect the membrane phospholipid content . The effect of temperature on the growth inhibition of Kluyveromyces marxianus and Saccharomyces cerevisiae by the trichothecene mycotoxins, T-2 toxin and roridin A, was investigated . Examination of EC50 values for T-2 toxin and roridin A showed that these toxins were least inhibitory to both yeasts at 30 and 25 degrees C, respectively . Increasing or decreasing growth temperature from these temperatures gradually increased the inhibitory effect of the trichothecene mycotoxins . Temperature may affect the toxicity of the trichothecenes to the yeasts by regulating the composition of yeast cell membranes.

Mol Gen Genet, 1991 Apr, 226(1-2), 97 - 106
Genome organization of the linear plasmid, pSKL, isolated from Saccharomyces kluyveri; Hishinuma F et al.; We have determined the complete nucleotide sequence of the linear DNA plasmid, pSKL, isolated from Saccharomyces kluyveri . Sequence analysis showed that pSKL has a high (A + T) content of 71.7%, and that there are 10 open reading frames (ORFs) larger than 250 nucleotides . All 10 ORFs were shown to be transcribed in S . kluyveri cells by S1 nuclease mapping analysis . The localization of ORFs, direction of transcription, and the predicted amino acid sequences of each ORF were quite similar to that of pGKL2, one of the killer plasmids found in Kluyveromyces lactis . The amino acid sequences of the largest two ORFs (ORF2 and ORF6) have homology with several DNA polymerases and RNA polymerases, respectively.

J Bacteriol, 1991 Apr, 173(7), 2250 - 5
Evolutionary relationships among pathogenic Candida species and relatives; Barns SM et al.; Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis {Candida} glabrata and Yarrowia {Candida} lipolytica) and for Hansenula polymorpha . Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var . lactis, and Aspergillus fumigatus indicate that Candida albicans, C . tropicalis, C . parapsilosis, and C . viswanathii form a subgroup within the genus . The remaining significant pathogen, T . glabrata, falls into a second, distinct subgroup and is specifically related to S . cerevisiae and more distantly related to C . kefyr (psuedotropicalis) and K . marxianus var . lactis . The 18S rRNA sequence of Y . lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them . As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.

Mol Cell Biol, 1991 Apr, 11(4), 1777 - 84
Identification of base and backbone contacts used for DNA sequence recognition and high-affinity binding by LAC9, a transcription activator containing a C6 zinc finger; Halvorsen YD et al.; The LAC9 protein of Kluyveromyces lactis is a transcriptional regulator of genes in the lactose-galactose regulon . To regulate transcription, LAC9 must bind to 17-bp upstream activator sequences (UASs) located in front of each target gene . LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae, and the two proteins must bind DNA in a very similar manner . In this paper we show that high-affinity, sequence-specific binding by LAC9 dimers is mediated primarily by 3 bp at each end of the UAS: {Formula: see text} . In addition, at least one half of the UAS must have a GC or CG base pair at position 1 for high-affinity binding; LAC9 binds preferentially to the half containing the GC base pair . Bases at positions 2, 3, and 4 in each half of the UAS make little if any contribution to binding . The center base pair is not essential for high-affinity LAC9 binding when DNA-binding activity measured in vitro . However, the center base pair must play an essential role in vivo, since all natural UASs have 17, not 16, bp . Hydroxyl radical footprinting shows that a LAC9 dimer binds an unusually broad region on one face of the DNA helix . Because of the data, we suggest that LAC9 contacts positions 6, 7, and 8, both plus and minus, of the UAS, which are separated by more than one turn of the DNA helix, and twists part way around the DNA, thus protecting the broad region of the minor groove between the major-groove contacts.

Yeast, 1991 Apr, 7(3), 245 - 52
Killer system of Kluyveromyces lactis: the open reading frame 10 of the pGK12 plasmid encodes a putative DNA binding protein; Tommasino M; ORF 10 of the K2 plasmid from Kluyveromyces lactis encodes a small basic protein (22.3% lysine) . The function of its product has been investigated . Western blot analysis, using an antibody against MS2 RNA polymerase/ORF 10 fusion protein, reveals a protein band with an apparent molecular weight of 14 kDa . The protein can bind a DNA-Sepharose column, and is eluted by 350 mM-salt . Immunoprecipitation experiments show that the ORF 10 protein coprecipitates with the linear genomic DNAs of the two killer plasmids (K1 and K2) . From Western/Southern blot data, it is possible to conclude that the interaction between protein and DNA occurs directly, rather than via other protein(s) . ORF 10 is easily detected by Western blot and its transcript is one of the most abundant of the K2 plasmid, suggesting that this protein may have a structural rather than a regulatory function . This possibility is also suggested by the observed sequence homology between the ORF 10 protein and the family of histone-like proteins.

Int J Syst Bacteriol, 1991 Apr, 41(2), 249 - 54
Characterization of yeast strains of the genera candida, Hansenula, Kluyveromyces, Pichia, Rhodotorula, and Saccharomyces by mixed-dye fluorometry; Geyer W et al.; An analytical method in which we used the selective adsorption of several fluorophores by yeast cells is described . The suitability of using binary mixtures of 1-pyrene butyric acid, 3,6-dimethylamino acridine, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, and rhodamine B isothiocyanate for the characterization and identification of microorganisms was tested with 98 yeast strains belonging to the genera Candida, Hansenula, Kluyveromyces, Pichia, Rhodotorula, and Saccharomyces . The application of multivariate statistical methods and pattern recognition methods to the allocation of the yeast strains into genus-species-strain structures and to a comparison of fluorescence data sets for differentiation and identification purposes showed the usefulness of the method.

Antonie Van Leeuwenhoek, 1991 Apr, 59(3), 199 - 206
Occurrence and taxonomic aspects of proton movements coupled to sugar transport in the yeast genus Kluyveromyces; Kilian SG et al.; The occurrence of proton symport mechanisms for the transport of glucose, galactose, fructose, raffinose and sucrose in 21 yeast strains representing the species of the genus Kluyveromyces was surveyed . Proton symport of one or more sugars occurred in 57% of the strains . Similarly, all the sugars investigated were transported by symports by several strains . Symport systems for non-utilisable sugars were rare . Starvation of cells frequently resulted in the appearance of a symport absent in non-starved glucose-grown cells, indicating that repression of proton symports by glucose and subsequent derepression by starvation is a general phenomenon in members of Kluyveromyces . The addition of a sugar to cell suspensions resulted in acidification in 80% of cases, indicating the activity of a membrane-bound ATPase . Acidification was also observed with a number of sugars that cannot be utilised by the particular species . Interesting correlations between the number of proton symports and the abundance of other phenotypic characteristics in members of the genus emerged . Most members of the infertile group of species showing an increase in the number of small chromosomes, inability to produce well-developed pseudomycelium, linoleic and linolenic acid, a decrease in the number of carbon compounds utilised and inability to utilise ethylamine also had no proton symports, whereas most members of the interfertile species produced one or more proton symports . It was concluded that the distribution of the number of proton symports amongst Kluyveromyces species coincided with that of other positive characteristics and may therefore be of taxonomic value.

Appl Environ Microbiol, 1991 Mar, 57(3), 655 - 9
Fermentation and aerobic metabolism of cellodextrins by yeasts; Freer SN; The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored . When grown aerobically, Candida wickerhamii, C . guilliermondii, and C . molischiana metabolized cellodextrins of degree of polymerization 3 to 6 . C . wickerhamii and C . molischiana also fermented these substrates, while C . guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3 . Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically . These yeasts also fermented these substrates, except for K . lactis, which fermented only glucose and cellobiose . The remaining species/strains tested, K . lactis, Brettano-myces claussenii, B . anomalus, K . dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose . Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose . Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose . However, with two exceptions, R . minuta and P . guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.

J Dairy Sci, 1991 Mar, 74(3), 758 - 63
Antimicrobial activity of Microgard against food spoilage and pathogenic microorganisms; al-Zoreky N et al.; Microgard, a commercially available fermented milk product containing antimicrobial metabolites, was a potent inhibitor for Gram-negative bacteria such as Pseudomonas, Salmonella, and Yersinia when 1% concentration was incorporated into agar media . Gram-positive Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes were insensitive to Microgard . Kluyveromyces marxianus, an unidentified black yeast, and Penicillium expansum were partially suppressed, whereas Aspergillus niger and a yogurt spoilage yeast were tolerant to 5% Microgard . Optimum activity of Microgard was at pH 5.3 and below; the concentration that gave complete inhibition depended upon the number of bacteria present as well as the genus tested . Blood agar base reversed the antagonistic activity of Microgard against Pseudomonas putida compared with plate count agar.

Curr Genet, 1991 Mar, 19(3), 155 - 61
Plasmid functions involved in the stable propagation of the pKD1 circular plasmid in Kluyveromyces lactis; Bianchi MM et al.; Plasmid factors involved in the stable propagation of pKD1-derived vectors in Kluyveromyces lactis transformants have been identified . Three genes (A, B and C) have been found to be present in pKD1: the interruption of the B and C genes led to high plasmid instability . Stability could be restored in trans when host cells contained pKD1 as the resident plasmid (pKD1+ strains) . The A gene, which codes for a site-specific recombinase, did not affect plasmid partitioning . Vectors bearing only the pKD1 replication origin (or a chromosomal ARS), and no other pKD1 sequence, were very unstable both in the presence and absence of the resident plasmid in host cells . These vectors could be stabilized in pKD1+ strains, but not in pKD1 degree strains, by the insertion of a 200 bp-long pKD1 sequence . This sequence, called the cis-acting stability locus (CSL), together with the products of the B and C genes, ensured plasmid partitioning at cell division . Possible hairpin structures and direct repeats were regularly spaced within the CSL . This region, and the corresponding cis-acting stabilizing elements of other yeast plasmids, did not have sequence homology but shared some structural regularities.

Arch Latinoam Nutr, 1991 Mar, 41(1), 72 - 8
{Biological value of the unicellular protein of Kluyveromyces marxianus var . lactis}; Carrasco de Mendoza MS et al.; The ever increasing problem of environmental pollution and protein scarcity led us to begin producing protein biomass from cheese whey, having selected for this aim, Kluyveromyces marxianus va . lactis strain, which represents the right relationship between its protein content (53.3% d.w.) and that of the RNA (4.63% d.w.) . On the other hand, the distribution of its essential amino acids is balanced, although it shows a deficiency in methionine, its value being 1.5 g/16 gN . The purpose of this work was to assess the biological quality of this protein concentrate, so as to use it in animal feeding . Protein quality was determined by the NPU method (net protein utilization), according to Miller and Bender's technique . Three balanced diets were prepared (10% protein), the test sample with casein, and the rest with protein biomass, one of then supplemented with methionine (0.5 g/100 g food) . Based on the results it can be concluded that the biomass analyzed has an adequate biological value (55.37%) for use in animal feeding, especially when methionine as added (60.23%).

Curr Genet, 1991 Mar, 19(3), 163 - 7
Distribution of mitochondrial r1-type introns and the associated open reading frame in the yeast genus Kluyveromyces; Wilson C et al.; We have sequenced the intron in the large subunit ribosomal RNA gene from the mitochondrion of Kluyveromyces lactis . It is a typical group I intron but, unlike the corresponding intron (r1) in Saccharomyces cerevisiae, it does not contain an open reading frame . This intron is widespread in the genus Kluyveromyces although intron-less strains were also found in some species of this genus . Sequences homologous to the open reading frame of the S . cerevisiae ribosomal intron were detected in some strains of K . waltii, K . thermotolerans and K . africanus.

Curr Genet, 1991 Feb, 19(2), 109 - 18
Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis; Kamper J et al.; Linear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae . Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5' ends . The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination . Nuclear promoters are not recognised in the cytoplasm.

Appl Environ Microbiol, 1991 Feb, 57(2), 557 - 62
Production and localization of beta-fructosidase in asynchronous and synchronous chemostat cultures of yeasts; Rouwenhorst RJ et al.; In synchronized continuous cultures of Saccharomyces cerevisiae CBS 8066, the production of the extracellular invertase (EC 3.2.1.26) showed a cyclic behavior that coincided with the budding cycle . The invertase activity increased during bud development and ceased at bud maturation and cell scission . The cyclic changes in invertase production resulted in cyclic changes in amounts of invertase localized in the cell wall . However, the amount of enzyme invertase present in the culture liquid remained constant throughout the budding cycle . Also, in asynchronous continuous cultures of S . cerevisiae, the production and localization of invertase showed significant fluctuation . The overall invertase production in an asynchronous culture was two to three times higher than in synchronous cultures . This could be due to more-severe invertase-repressive conditions in a synchronous chemostat culture . Both the intracellular glucose-6-phosphate concentration and residual glucose concentration were significantly higher in synchronous chemostat cultures than in asynchronous chemostat cultures . In the asynchronous and synchronous continuous cultures of S . cerevisiae, about 40% of the invertase was released into the culture liquid; it has generally been believed that S . cerevisiae releases only about 5% of its invertase . In contrast to invertase production and localization in the chemostat cultures of S . cerevisiae, no significant changes in inulinase (EC 3.2.1.7) production and localization were observed in chemostat cultures of Kluyveromyces maxianus CBS 6556 . In cultures of K . marxianus about 50% of the inulinase was present in the culture liquid.

EMBO J, 1991 Feb, 10(2), 369 - 75
A conserved heptapeptide restrains the activity of the yeast heat shock transcription factor; Jakobsen BK et al.; In yeast, expression of heat shock genes is regulated by a factor (HSF) which binds constitutively to DNA, but activates transcription efficiently only after heat shock . We have compared the HSFs from Saccharomyces cerevisiae and Kluyveromyces lactis . Both factors contain an activation domain whose activity is masked at low temperature, but the amino acid sequences of these activators are unrelated . Masking requires the evolutionarily conserved DNA binding and oligomerization domains, as well as a short conserved element close to the activator . Although this element contains potential phosphorylation sites, they are not required for induction . We suggest that the conserved element binds either to the structural core of the protein or to another polypeptide, holding the activator in an inactive configuration, and that high temperatures disrupt this interaction . Our results emphasize the importance of global protein structure in the regulation of transcription factor activity.

Yeast, 1991 Feb, 7(2), 127 - 35
Constitutive expression of the Saccharomyces cerevisiae CUP1 gene in Kluyveromyces lactis; Macreadie IG et al.; Shuttle plasmids, pE1.CUP1B and pE1.CUP1E of 10.6 kb, have been constructed between the metallothionein-encoding CUP1 gene of Saccharomyces cerevisiae and a vector capable of replication in Kluyveromyces lactis . Introduction of these plasmids into K . lactis confers resistance to copper as well as to cadmium and silver . Resistance to these latter metal ions, in the absence of induction by copper, suggested that the CUP1 gene is constitutively expressed in the foreign background . Introduction of the lacZ reporter gene from Escherichia coli into a cloning site downstream from the CUP1 promoter showed that expression of this gene is constitutive in K . lactis but in S . cerevisiae induction by copper is necessary . Sequences upstream from the CUP1 promoter are involved in the constitutive expression since deletion of 91 nucleotides from this region abolishes metal resistance . It is suggested that a K . lactis protein, normally involved in activating transcription of the resident CUP1 gene in the presence of copper, can promote transcription in the absence of metal ion by binding to the upstream activation sequence of the introduced CUP1 gene.

Appl Biochem Biotechnol, 1991 Spring, 28-29, 307 - 15
Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol; Ballesteros I et al.; A total of 27 yeast strains belonging to the groups Candida, Saccharomyces, and Kluyveromyces were screened for their ability to grow and ferment glucose at temperatures ranging 32-45 degrees C . K . marxianus and K . fragilis were found to be the best ethanol producing organisms at the higher temperature tested and, so, were selected for subsequent simultaneous saccharification and fermentation (SSF) studies . SSF experiments were performed at 42 and 45 degrees C, utilizing Solkafloc (10%) as cellulose substrate and a cellulase loading of 15 FPU/g substrate . Best results were achieved at 42 degrees C with K . marxianus L . G . and K . fragilis L . G., both of which produced close to 38 g/L ethanol and 0.5 ethanol yield, in 78 h.

J Hyg Epidemiol Microbiol Immunol, 1991, 35(1), 41 - 9
T-2 toxin degradation by micromycetes; Jesenska Z et al.; The biodegradation of T-2 toxin was studied by strains of micromycetes which were isolated from the environment . The 26 tested strains were divided into three groups . Group contains strains which degraded T-2 toxin very fast . This toxin could not be chromatographically determined in the medium even after 48 hours of incubation and the antifungal activity of residua against Kluyveromyces fragilis CCY-51-1-2 was low or zero . There were strains of Alternaria sp., Ulocladium sp., Aspergillus candidus, Cladosporium cladosporioides, Rhodotorula sp., Aspergillus flavus and Cladosporium macrocarpum . Group II contains with a low activity and in group III the results were variable and non stable.

J Biol Chem, 1990 Dec 15, 265(35), 21427 - 9
The transcription factor LAC9 from Kluyveromyces lactis-like GAL4 from Saccharomyces cerevisiae forms a Zn(II)2Cys6 binuclear cluster; Pan T et al.; The DNA binding domain of the transcription factor LAC9 contains 6 cysteine residues with spacing in the primary peptide sequence identical to that found in the DNA binding domain of the GAL4 transcription factor . In GAL4, the CysX2CysX6CysX6CysX2CysX6Cys motif has been shown to form a Zn(II)2Cys6 binuclear cluster (Pan, T . and Coleman, J . E . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 2077-2081), representing a new structure for a Zn(II)-containing transcription factor which differs from the "zinc finger" motif first described for TFIIIA . LAC9 has been shown to bind two Zn(II) ions (Halvorsen, Y . C., Nandabalan, K., and Dickson, R . D . (1990) J . Biol . Chem . 265, 13283-13289) . The similarity of the amino acid sequence and the Cys spacing within the DNA binding domain suggest that LAC9 should also be capable of forming the Zn(II)2Cys6 cluster found in GAL4 . A fragment of LAC9 consisting of 144 amino acid residues spanning the DNA binding domain has been prepared with 113Cd(II) substituted for the two native Zn(II) ions . 113Cd NMR of this fragment (denoted LAC9(85-228*} has been carried out in an attempt to test the hypothesis that LAC9, like GAL4, forms a binuclear cluster . The chemical shifts of the two bound 113Cd(II) ions, 705 and 692 ppm respectively, are consistent with ligation of each 113Cd(II) ion to 4 sulfur atoms . The best model for such ligation is that two of the cysteine S- form bridges between the two Cd(II) ions . Formation of a Zn(II)-Cd(II) hybrid form of LAC9(85-228*) has also been observed . We conclude that LAC9 contains a Zn(II)2Cys6 binuclear cluster as previously reported for GAL4.

Int J Food Microbiol, 1990 Dec, 11(3-4), 289 - 303
The yeasts of cheese brines; Seiler H et al.; A total of 365 yeasts were isolated from the brines of soft, semihard and hard cheeses from different manufacturers . Identification was based on 131 characteristics, primarily employing a method with microtitration plates . Most brines exhibited a characteristic yeast flora . The predominant strains proved to be mainly Debaryomyces hansenii and Candida versatilis . In a few brines Trichosporon beigelii, C . rugosa, C . intermedia, Kluyveromyces marxianus, Saccharomyces sp . and C . tenuis/polymorpha were predominant . Also of importance were C . tropicalis, C . parapsilosis, C . zeylanoides, Issatchenkia orientalis and Geotrichum klebahnii . Not all strains could be clearly identified . Lists of characters are provided for subdividing D . hansenii and T . beigelii . The specificity of the yeast flora of brines is assumed to contribute to the sensory variety of cheeses.

Arch Latinoam Nutr, 1990 Dec, 40(4), 594 - 602
{Chemical composition of the cellular biomass of yeasts}; Scarinci HE et al.; This work is aimed at analyzing yeast strains, possibly used in animal feeding, obtained by batch cultivation from cheese whey as main carbohydrated substrate . For that purpose 10 yeast strains selected for its biomass production capacity were chemically analyzed . From the results, it can be observed that the chemical composition of the strains is quiet variable, showing in all cases high protein content, good solubility and enzymatic digestibility . In all of them, the RNA content is low, being this important if the biomass is utilized in human feeding . On the other hand, they have an adequate content of essential amino acids, although the sulphur amino acids content is deficient . Among the tested yeasts, Kluyveromyces marxianus var . lactis (No . 10) stands out for the good relationship it has between protein and RNA content, as well as for the detection of methionine among its essential amino acids.

Curr Genet, 1990 Dec, 18(6), 517 - 22
Centromeric DNA of Kluyveromyces lactis; Heus JJ et al.; A direct selection method was used to isolate centromeres from a genomic library of the yeast Kluyveromyces lactis . The method is based on the lethality at high copy number of the ochre-suppressing tRNA gene SUP11 . Five different chromosomal fragments were found that confer mitotic stability to plasmids containing a replication origin of K . lactis (KARS) . In addition, KARS plasmids containing these fragments have a copy number of approximately one, and each of the five fragments hybridizes to a different chromosome of K . lactis . From these results we conclude that five of the six centromeres of K . lactis have been isolated . These centromeres do not function in S . cerevisiae.

Appl Environ Microbiol, 1990 Nov, 56(11), 3329 - 36
Localization of inulinase and invertase in Kluyveromyces species; Rouwenhorst RJ et al.; In vivo hydrolysis of inulin and sucrose was examined in selected yeasts of the genus Kluyveromyces . Cells, grown in sucrose-limited chemostat cultures, were subjected to treatments for the removal of inulinase, the enzyme responsible for the hydrolysis of both inulin and sucrose . The effects of these treatments were studied by measurement of inulin-dependent and sucrose-dependent oxygen consumption by cell suspensions . In Kluyveromyces marxianus var . marxianus, inulinase was partially secreted into the culture fluid . Removal of culture fluid inulinase by washing had no effect on sucrose-dependent oxygen consumption by this yeast . However, this treatment drastically reduced inulin-dependent oxygen consumption . Treatment of washed cells with sulfhydryls removed part of the cell wall-retained inulinase and reduced inulin-dependent oxygen consumption by another 80% . Sucrose-dependent oxygen consumption was less affected, decreasing by 40% . Cell suspensions of K . marxianus var . drosophilarum, K . marxianus var . vanudenii, and Saccharomyces kluyveri rapidly utilized sucrose but not inulin . This is in accordance with the classification of these yeasts as inulin negative . Supernatants of cultures grown at pH 5.5 did not catalyze the hydrolysis of inulin and sucrose . This suggested that these yeasts contained a strictly cell-bound invertase, an enzyme not capable of inulin hydrolysis . However, upon washing, cells became able to utilize inulin . The inulin-dependent oxygen consumption further increased after treatment of the cells with sulfhydryls . These treatments did not affect the sucrose-dependent oxygen consumption of the cells . Apparently, these treatments removed a permeability barrier for inulin that does not exist for sucrose.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1990 Nov, 56(11), 3337 - 45
Structure and properties of the extracellular inulinase of Kluyveromyces marxianus CBS 6556; Rouwenhorst RJ et al.; In the yeast Kluyveromyces marxianus two forms of inulinase were present, namely, an inulinase secreted into the culture fluid and an inulinase retained in the cell wall . Both forms were purified and analyzed by denaturing and nondenaturing polyacrylamide gel electrophoresis . With the use of endo-beta-N-acetyl-glucosaminidase H, it was established that the enzyme retained in the cell wall and the enzyme secreted into the culture fluid have similar subunits consisting of a 64-kDa polypeptide with varying amounts of carbohydrate (26 to 37% of the molecular mass) . The two forms of inulinase differed in size because of their differences in subunit aggregation . The enzyme present in the culture fluid was a dimer, and the enzyme retained in the cell wall was a tetramer . The differences in oligomerization did not affect the apparent Km values towards the substrates sucrose and raffinose . These findings support the hypothesis that the retention of glycoproteins in the yeast cell wall may be caused by a permeability barrier towards larger glycoproteins . The amino-terminal end of inulinase was determined and compared with the amino terminus of the closely related invertase . The kinetic and structural evidence indicates that in yeasts two distinct beta-fructosidases exist, namely, invertase and inulinase.

Yeast, 1990 Nov-Dec, 6(6), 483 - 90
An assay of relative cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe; De Nobel JG et al.; We have developed a new assay to determine relative cell wall porosity in yeasts, which is based on polycation-induced leakage of UV-absorbing compounds . Polycations with a small hydrodynamic radius as measured by gel filtration (poly-L-lysine) caused cell leakage independent of cell wall porosity whereas polycations with a large hydrodynamic radius (DEAE-dextrans) caused only limited cell leakage due to limited passage through the cell wall . This allowed the ratio between DEAE-dextran- and poly-L-lysine-induced cell leakage to be used as a measure of cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe . Using this assay, we found that the composition of the growth medium affected cell wall porosity in S . cerevisiae . In addition, we could show that cell wall porosity is limited by the number of disulphide bridges in the wall and is dependent on cell turgor . It is argued that earlier methods to estimate cell wall porosity in S . cerevisiae resulted in large underestimations.

Antonie Van Leeuwenhoek, 1990 Nov, 58(4), 227 - 34
Restriction site variation, length polymorphism and changes in gene order in the mitochondrial DNA of the yeast Kluyveromyces lactics; Coria R et al.; The purpose of this work was to compare mitochondrial DNA restriction endonuclease patterns in strains of the yeast Kluyveromyces lactis, from different sources, to see how conserved is the organization of this organellar genome . The mitochondrial DNA of five independently-isolated strains and one of unknown origin were compared . Strains NRRL Y-1205, NRRL Y-8279 and NRRL Y-1140 gave identical patterns . Strain NRRL Y-1564 showed an insertion, with respect to the other three, of approximately 1250 bp . Strain W600B had also an insertion with extra restriction sites for EcoRI, HpaI, HaeIII, HincII and XbaI . On the other hand, strain Y-123 showed a restriction pattern quite different from the others . Sequences putatively encoding apocytochrome b, ATPase subunit 9 and ribosomal RNA large subunit, were localized on the physical maps of three strains . Results demonstrated that the order of these three genes shows a common feature in strains W600B and WM37 (auxotroph of Y-1140) but a different distribution in WM27 (auxotroph derived from Y-123) . All these facts explain the extensive intraspecific polymorphism observed in the mtDNA of this yeast.

Rev Argent Microbiol, 1990 Oct-Dec, 22(4), 175 - 81
{Beta-galactosidase activity of strains of Kluyveromyces spp . and production of ethanol from lactose}; de Figueroa LC et al.; We investigated the behavior of yeast of the genus Kluyveromyces (K . fragilis 507, K . lactis 29 and K . lactis 10), which grow on lactose as sole carbon source, since they possess an enzyme system for the utilization of this sugar . We determined the beta-galactosidase activity of these strains, grown in the logarithmic phase in media containing glucose and lactose . On addition of 0 to 12% v/v ethanol to cells treated with toluene, we did not observe inhibition of the enzyme in strain 10 of Kluyveromyces lactis, which showed the greatest activity (704.4 Units) . Since there exist the possibility of industrial utilization of concentrated whey (4 times), we performed fermentation tests of the three strains, at 30 C, in media containing initial lactose concentrations of 16.5 and 24.5% . After 48 h the residual lactose concentration was practically zero, and the ethanol concentrations had reached 7.60 and 10.10% v/v . It might be expected that the rate of fermentation of a disaccharide such as lactose would be related to the rate of hydrolysis of the sugar, so that strains having a higher rate of enzymatic hydrolysis should show a higher fermentation rate . However, we did not observe such behavior, as strains of Kluyveromyces having enzymatic activities as different as K . lactis 10 (704.4 U) and K . lactis 29 (189.7 U) did not show any great difference in the production of ethanol from lactose.

Mol Cell Biol, 1990 Oct, 10(10), 5128 - 37
The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1; Witte MM et al.; LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis . The DNA-binding domain is composed of a zinc finger and nearby amino acids (M . M . Witte and R . C . Dickson, Mol . Cell . Biol . 8:3726-3733, 1988) . The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R . M . Evans and S . M . Hollenberg, Cell 52:1-3, 1988) . The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear . To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein . A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9 . However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding . Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced . Thus, in a qualitative sense C6 fingers perform a similar function(s) . However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence . This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Nucleic Acids Res, 1990 Sep 11, 18(17), 5213 - 7
Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9; Dickson RC et al.; The GAL4 protein of Saccharomyces cerevisiae and the LAC9 protein of Kluyveromyces lactis are transcription activator proteins with similar structure and function . Greatest similarity occurs in the C region near the carboxy terminus, where 16 of 18 amino acids are identical . The function of the C region is unclear . Here we show that the structural similarity is reflected in functional similarity . Single amino acid changes in the C region of GAL4 and LAC9 create a similar phenotype: constitutive gene expression . In S . cerevisiae the constitutive phenotype caused by GAL4 mutants can be abolished by overproduction of GAL80 . These results support a model in which the C region of GAL4 and LAC9 constitute similar negative regulatory domains that interact with GAL80 in S . cerevisiae and an unidentified GAL80 homolog in K . lactis . This protein-protein interaction prevents expression of the galactose operon in the uninduced state.

Mol Gen Genet, 1990 Sep, 223(2), 342 - 4
Magnification of the rDNA cluster in Kluyveromyces lactis; Maleszka R et al.; By employing pulsed field gel electrophoresis we find that slow growing strains of Kluyveromyces lactis have only 43%-55% of the wild-type level of ribosomal DNA (rDNA) repeats . When subjected to prolonged vegetative growth these strains can increase both the number of rDNA repeats and their growth rate.

Yeast, 1990 Sep-Oct, 6(5), 417 - 27
Mating type locus-dependent stability of the Kluyveromyces linear pGKL plasmids in Saccharomyces cerevisiae; Gunge N et al.; The linear killer plasmids, pGKL1 and pGKL2, from Kluyveromyces lactis stably replicated in mitochondrial DNA-deficient (rho 0) MATa or MAT alpha haploids of Saccharomyces cerevisiae, but were unstable and frequently lost in rho 0 MATa/MAT alpha diploids, suggesting that the replication of pGKL plasmids was under the control of the MAT locus . In MATa/MAT alpha cells of S . cerevisiae, the MAT alpha gene product (alpha 2) is combined with the MATa gene product (a1) and the resultant protein, a1-alpha 2, acts to repress the expression of haploid specific genes . Experiments showed that the K . lactis linear plasmids were stably maintained in rho 0 mata1/MAT alpha diploids, indicating that the a1-alpha 2 repressor interfered with the stability of pGKL2 . It was revealed by computer analysis that the consensus sequence homologous to the a1-alpha 2 repressor binding site occurred within the coding regions of pGKL2 genes which were presumed to be essential for the plasmid replication . Since the plasmids were stably maintained in diploids of K . lactis, the mating type control must not be working there.

Nucleic Acids Res, 1990 Aug 25, 18(16), 4877 - 82
A palindromic mutation of the linear killer plasmid k2 of yeast; Wesolowski-Louvel M et al.; Production of the killer toxin in Kluyveromyces lactis is dependent on the presence of two linear DNA plasmids, k1 and k2 . We isolated a non-killer mutant, VM5, with a modified plasmid composition . It had lost k1, but conserved k2, and acquired, in addition, three new DNA species . The new species were found to be rearranged derivatives of the k2 plasmid . One of them, pVM5-1, was made of the left terminal 4720 bp sequence of k2, including the inverted terminal repeat, and was organized as a large palindromic dimer molecule . The second, pVM5-2, was made of one strand of the pVM5-1 palindrome, folded into a hairpin structure . Like normal k2, pVM5-1 and 2 were present in a high copy number . The third species, pVM5-x, of variable size, was also a deletion product of k2, but not palindromic, and did not contain the terminal repeat . Genetic analysis showed that the presence of the palindromic derivatives appeared to destabilize the normal k2 genome, leading to gradual accumulation of plasmid-less cells.

J Biol Chem, 1990 Aug 5, 265(22), 13283 - 9
LAC9 DNA-binding domain coordinates two zinc atoms per monomer and contacts DNA as a dimer; Halvorsen YC et al.; The LAC9 protein of Kluyveromyces lactis activates transcription by binding to upstream activating sequences lying in front of genes of the lactose-galactose regulon . LAC9 belongs to a family of fungal proteins having a conserved domain containing 6 cysteines . This domain, termed a C6 zinc finger, is thought to bind one zinc atom and to play a vital role in DNA binding . To further characterize the DNA-binding domain of LAC9, we have developed a procedure to produce and to purify milligram amounts of LAC9 peptides . The two larger peptides, one containing amino acids 1-228 and the other containing amino acids 85-228, formed dimers in solution and bound DNA specifically as a dimer . The smallest LAC9 peptide, amino acids 85-160, failed to dimerize and did not bind DNA . Atomic absorption spectroscopy revealed that each LAC9 monomer coordinated two zinc atoms, not one, as had been predicted . This result suggests, as does previously published data, that the C6 zinc finger domain has a unique conformation that may represent a new type of DNA-binding motif.

J Bacteriol, 1990 Aug, 172(8), 4510 - 21
Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae; Salama SR et al.; The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for secretory protein movement from the Golgi complex . That some conservation of SEC14p function may exist was initially suggested by experiments that revealed immunoreactive polypeptides in cell extracts of the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe . We have cloned and characterized the K . lactis SEC14 gene (SEC14KL) . Immunoprecipitation experiments indicated that SEC14KL encoded the K . lactis structural homolog of SEC14p . In agreement with those results, nucleotide sequence analysis of SEC14KL revealed a gene product of 301 residues (Mr, 34,615) and 77% identity to SEC14p . Moreover, a single ectopic copy of SEC14KL was sufficient to render S . cerevisiae sec14-1(Ts) mutants, or otherwise inviable sec14-129::HIS3 mutant strains, completely proficient for secretory pathway function by the criteria of growth, invertase secretion, and kinetics of vacuolar protein localization . This efficient complementation of sec14-129::HIS3 was observed to occur when the rates of SEC14pKL and SEC14p synthesis were reduced by a factor of 7 to 10 with respect to the wild-type rate of SEC14p synthesis . Taken together, these data provide evidence that the high level of structural conservation between SEC14p and SEC14pKL reflects a functional identity between these polypeptides as well . On the basis of the SEC14p and SEC14pKL primary sequence homology to the human retinaldehyde-binding protein, we suggest that the general function of these SEC14p species may be to regulate the delivery of a hydrophobic ligand to Golgi membranes so that biosynthetic secretory traffic can be supported.

Gene, 1990 Jul 2, 91(1), 43 - 50
Expression of a foreign KmR gene in linear killer DNA plasmids in yeast; Tanguy-Rougeau C et al.; The killer plasmids of the yeast Kluyveromyces lactis, pGKL1 and 2 (k1 and k2 for short), are linear double-stranded DNAs . The expression of genes of these plasmids is thought to depend on their own transcription system . Cloning the plasmid genes in conventional circular vectors is therefore not suitable for transcriptional studies, because such vectors use the host nuclear transcription system . In vitro modification of the linear plasmid genomes in order to introduce transcription reporter genes has been difficult because the structure of the plasmids, with covalently bound terminal proteins, does not allow their manipulation in vitro and amplification in Escherichia coli . We introduced the kanamycin/G418 resistance gene, KmR, into the k1 plasmid in vivo, by transforming the yeast with the linearized KmR gene bordered with short k1 sequences (part of the region encoding the toxin) to allow homologous recombination with the resident k1 . In the linear recombinants obtained, however, the KmR was not expressed, while it was expressed if carried on circularized plasmids . By replacing the native promoter of KmR by the ORF1 promoter from k1, the KmR gene could be expressed in linear recombinants and conferred on the host a high level of resistance to the drug . All the linear recombinant plasmids were extremely stable under nonselective conditions . As a rare event, the integration of KmR produced a palindromic rearrangement of the k1 plasmid.

Curr Genet, 1990 Jul, 18(1), 77 - 80
Killer toxin production in Pichia acaciae is associated with linear DNA plasmids; Worsham PL et al.; We have identified a strain of the yeast Pichia acaciae which produces a "killer" toxin active against the yeast Debaryomyces tamarii . The killer phenotype was associated with the presence of two DNA plasmids, pPacl-1 (13.6 kilobase pairs) and pPacl-2 (7.3 kilobase pairs) . P . acaciae strains, cured of these plasmids by irradiation with ultraviolet light, lacked killer activity and were sensitive to toxin produced by the parental strain . A partially cured strain, GS-1215, missing only the smaller plasmid, pPacl-2, also exhibited loss of both toxin activity and immunity . Exonuclease studies revealed that both plasmids were linear double-stranded DNA molecules with 5' protected ends . The P . acaciae system differs from that of the well-studied Kluyveromyces lactis "killer" system both in the range of susceptible strains and in the sizes of the plasmids involved . Our studies contradict previous reports that Pichia killer systems are invariably chromosomal.

J Bacteriol, 1990 Jun, 172(6), 2871 - 6
Continuous-culture study of the regulation of glucose and fructose transport in Kluyveromyces marxianus CBS 6556; Postma E et al.; Regulation of transport of D-glucose and D-fructose was studied in Kluyveromyces marxianus grown in continuous culture . Both substrates could be transported by at least two different transport systems, low-affinity transport and high-affinity proton-sugar symport . The low-affinity transporter, specific for both glucose and fructose, was constitutively present and was apparently not regulated by carbon catabolite repression . Regulation of the activity of the glucose- and fructose-specific proton symport systems appeared to proceed mainly through catabolite repression . Activation of symport did not need the presence of specific inductor molecules in the medium . Nevertheless, the capacities of the proton-sugar symporters varied in cells grown on a wide variety of carbon sources . The possibility that the control of proton symport activity is related to the presence of specific intracellular metabolites is discussed.

Appl Environ Microbiol, 1990 May, 56(5), 1386 - 91
Trehalose levels and survival ratio of freeze-tolerant versus freeze-sensitive yeasts; Hino A et al.; Five freeze-tolerant yeast strains suitable for frozen dough were compared with ordinary commercial bakers' yeast . Kluyveromyces thermotolerans FRI 501 cells showed high survival ability after freezing when their resting cells were fermented for 0 to 180 min in modified liquid medium, and they grew to log and stationary phases . Among the freeze-tolerant strains of Saccharomyces cerevisiae, FRI 413 and FRI 869 showed higher surviving and trehalose-accumulating abilities than other S . cerevisiae strains, but were affected by a prolonged prefermentation period and by growth phases . The freeze tolerance of the yeasts was, to some extent, associated with the basal amount of intracellular trehalose after rapid degradation at the onset of the prefermentation period . In the freeze-sensitive yeasts, the degree of hydrolysis of trehalose may thus be affected by the kind of saccharide, unlike in freeze-tolerant yeasts.

Yeast, 1990 May-Jun, 6(3), 193 - 204
The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis; Saliola M et al.; We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis . Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K . lactis . Two of these genes have been isolated from a genomic bank by hybridization to ADH2 . The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S . cerevisiae and Schizosaccharomyces pombe respectively . One K . lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S . cerevisiae, was named K1ADH1 . The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence . We named this gene K1ADH3 . The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K . lactis genome . ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K . lactis cells depending on the carbon source.

FEMS Microbiol Lett, 1990 May, 57(1-2), 7 - 11
Fluid-phase endocytosis in yeasts other than Saccharomyces cerevisiae; Fernandez N et al.; A FITC-dextran internalization assay with Saccharomyces cerevisiae as positive control was used to determine whether fluid-phase endocytosis is a general characteristic of yeasts . Schizosaccharomyces pombe, Pichia polymorpha, Kluyveromyces phaseolosporus, Yarrowia lipolytica and Candida albicans were clearly positive, whereas results obtained with Debaryomyces marama were inconclusive . In all cases internalized FITC-dextran was found to be localized in the vacuoles and the process was always time- and temperature-dependent . Lower eucaryotes, particularly yeasts, appear to have the ability to incorporate substances from the extracellular medium through fluid-phase endocytosis.

Indian J Biochem Biophys, 1990 Apr, 27(2), 112 - 7
Interaction of hexadecyltrimethylammonium bromide with yeast cells; Divakar S; The interaction of hexadecyltrimethylammonium bromide (CTAB) with two yeast cells, Kluyveromyces fragilis and Saccharomyces cerevisiae, has been studied . Strong binding of CTAB to the cell was inferred from 1H and 13C NMR studies, the probable site of binding being the cell-surface . 13C and 31P NMR studies have indicated facilitation of free passage of molecules from outside to inside the cell and vice versa on treatment with CTAB . 31P NMR studies showed that intracellular pH (pHi) was affected in presence of CTAB and the rate of exchange of H+ and PO4(-3) between outside and inside of the cell was 508 s-1 . CTAB treatment of yeast cells also affected pH and conductance measurements of the cell-suspension . There was a marked difference in the pH changes around the critical micellar concentration (CMC) of CTAB . The observed pH changes were dependent on (i) CTAB concentration, (ii) pH of the cell-suspension and (iii) pK values of groups from molecules released from the cell . Also, it was shown that ionisation of phosphate diester from polar head groups of membranes constituting cell surface enhanced CTAB binding . Conductance measurements have shown that observed changes were independent of the concentration of yeast cells, but probably dependent on CMC of CTAB.

Ital J Biochem, 1990 Mar-Apr, 39(2), 71 - 82
Extraction, purification and characterization of ADH1 from the budding yeast Kluyveromyces marxianus; Pessione E et al.; The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae . K . marxianus ADH1 has an optimal temperature higher than the S . cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions . In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15 . Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.

Nucleic Acids Res, 1990 Feb 25, 18(4), 745 - 51
A mutation in the Zn-finger of the GAL4 homolog LAC9 results in glucose repression of its target genes; Kuger P et al.; The transcriptional activator LAC9, a GAL4 homolog of Kluyveromyces lactis which mediates lactose and galactose-dependent activation of genes involved in the utilization of these sugars can also confer glucose repression to those genes . Here we report on the isolation and characterization of LAC9-2, an allele which encodes a glucose-sensitive activator in contrast to the one previously cloned . A single amino acid exchange of leu-104 to tryptophan is responsible for the glucose-insensitive phenotype . The mutation is located within the Zn-finger-like DNA binding domain which is highly conserved between LAC9 and GAL4 . Glucose repression is also eliminated by duplication of the LAC9-2 allele . The data indicate that LAC9 is a limiting factor for beta-galactosidase gene expression under all growth conditions and that glucose reduces the activity of the activator.

Antonie Van Leeuwenhoek, 1990 Feb, 57(2), 77 - 81
Modes of lactose uptake in the yeast species Kluyveromyces marxianus; Carvalho-Silva M et al.; Twelve lactose-assimilating strains of the yeast species Kluyveromyces marxianus and its varieties marxianus, lactis and bulgaricus were studied with respect to transport mechanisms for lactose, glucose and galactose, fermentation of these sugars and the occurrence of extracellular lactose hydrolysis . The strains fell into three groups . Group I (two strains): Fermentation of lactose, glucose and galactose, extracellular lactose hydrolysis, apparent facilitated diffusion of glucose and galactose; Group II (two strains): Lactose not fermented, glucose and galactose fermented and transported by an apparent proton symport, extracellular hydrolysis of lactose present (one strain) or questionable; Group III (eight strains): Lactose, glucose and galactose fermented, lactose transported by an apparent proton symport mechanism, extracellular hydrolysis of lactose and transport modes for glucose and galactose variable.

Am J Clin Nutr, 1990 Feb, 51(2), 197 - 201
Effect of temperature on the lactose hydrolytic capacity of a lactase derived from Kluyveromyces lactis; Schneider RE et al.; In vitro studies of lactose hydrolysis in milk with 20-125 neutral lactase units (NLUs) carried out at 38.0 degrees C for 15 min with a beta-galactosidase derived from Kluyveromyces lactis (Lactaid, Lactaid Inc, Pleasantville, NJ) resulted in 85-95% of the hydrolysis observed with standard incubation conditions (24 h at 4-5 degrees C with 1000 NLU/L) . Thirty-three lactose-maldigesting Guatemalan subjects, 16 children and 17 adults, were challenged with oral doses of lactose in milk (children aged less than 12 mo, 2 g/kg body wt; children aged 12-24 mo, 15 g/kg body wt; older children and adults, 18 g/kg body wt) preincubated for 20 min at 38 +/- 0.5 degrees C with 50-125 NLU Lactaid . Under these conditions the subjects consumed milk without presenting any signs of intolerance . Furthermore, their breath-hydrogen excretion showed a 91-93% reduction when compared with a similar load of milk containing nonhydrolyzed lactose.

Biotechnology (N Y), 1990 Feb, 8(2), 135 - 9
Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin; van den Berg JA et al.; We have developed the yeast Kluyveromyces lactis as a host organism for the production of the milk-clotting enzyme chymosin . In contrast to Saccharomyces cerevisiae, we found that this yeast is capable of the synthesis and secretion of fully active prochymosin . Various signal sequences could be used to efficiently direct the secretion of prochymosin in Kluyveromyces, but not in S . cerevisiae . We conclude that the efficient synthetic and secretory capacity of this heterologous protein is a property of the yeast Kluyveromyces . These results have led to the development of a large scale production process for chymosin.

J Basic Microbiol, 1990, 30(1), 31 - 5
Occurrence of the general control of amino acid biosynthesis in yeasts; Bode R et al.; The response of three amino acid biosynthetic enzymes, threonine dehydratase, tyrosine aminotransferase and saccharopine dehydrogenase, to conditions of histidine, tryptophan or lysine limitation was investigated in 15 yeast species . The activities of all these enzymes increased about two- to fourfold as a result of action of the general control of amino acid biosynthesis in Brettanomyces anomalus, Candida maltosa, Hansenula polymorpha, Rhodosporidium toruloides, Saccharomyces cerevisiae and Yarrowia lipolytica . No evidence for the existence of the general control was found in Candida brumptii, Candida utilis, Hansenula anomala, Hansenula henricii, Kluyveromyces marxianus, Pichia guilliermondii, Saccharomycopsis capsularis, Trichosporon adeninovorans and Trigonopsis variabilis.

Adv Biochem Eng Biotechnol, 1990, 43, 75 - 102
Transfer and expression of heterologous genes in yeasts other than Saccharomyces cerevisiae; Reiser J et al.; In the past few years, yeasts other than those belonging to the genus Saccharomyces have become increasingly important for industrial applications . Species such as Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe, Yarrowia lipolytica and Kluyveromyces lactis have been modified genetically and used for the production of heterologous proteins . For a number of additional yeasts such as Schwanniomyces occidentalis, Zygosaccharomyces rouxii, Trichosporon cutaneum, Pachysolen tannophilus, Pichia guilliermondii and members of the genus Candida genetic transformation systems have been worked out . Transformation was achieved using either dominant selection markers based on antibiotic resistance genes or auxotrophic markers in conjunction with cloned biosynthetic genes involved in amino acid or nucleotide metabolism.

Yeast, 1990 Jan-Feb, 6(1), 69 - 76
Isolation and sequence analysis of a K . lactis chromosomal DNA element able to autonomously replicate in S . cerevisiae and K . lactis; Fabiani L et al.; We have undertaken a search for autonomously replicating (ARSs) from Kluyveromyces lactis chromosomal DNA able to sustain plasmid replication in K . lactis and in Saccharomyces cerevisiae . The discovery of such sequences might be interesting for the comparison of ARSs from different sources and possibly useful for the construction of multivalent vectors . HindIII fragments from K . lactis chromosomal DNA were inserted in the YIp5 plasmid (lacking an origin of replication) and the resulting chimaeric plasmids were selected for the ability to transform S . cerevisiae . Four plasmids were identified and further analysed . Two contained the same 1.8 kb K . lactis fragment and transformed both K . lactis and S . cerevisiae with the same efficiency and stability, whereas the third transformed only S . cerevisiae and the fourth transformed K . lactis with a higher efficiency than S . cerevisiae . A detailed study was performed on the 1.8 kb fragment which exhibited ARS function in both yeasts . The fragment was subcloned using different restriction enzymes and Bal31 exonuclease . Subclones were tested for ARS function . ARS activities in the two yeasts were localized in the same 100 bp region . Sequencing demonstrated the presence in this region of the dodecanucleotide 5'ATTTATTGTTTT3' differing from the ARS core consensus of S . cerevisiae only by a T insertion . A similar nucleotide sequence is present in the putative replication origin of the 2 mu-like plasmid pKD1 which stably replicates in K . lactis . Homologies with ARSs from S . cerevisiae were also found in the regions flanking the above-mentioned dodecanucleotide.

Int J Food Microbiol, 1990 Jan, 10(1), 73 - 89
Effect of polymyxin B nonapeptide and polymyxin B sulphate on trichothecene mycotoxin sensitivity of yeasts using a conductimetric instrument; Connolly P et al.; The addition of polymyxin B sulphate (PBS), or an inactive by-product, polymyxin B nonapeptide (PBN) to a yeast bioassay system, increased its sensitivity to various toxic agents . The nil effect level (NEL) of T-2 toxin was reduced from 0.1 to 0.01 microgram/ml for Kluyveromyces fragilis GK 1005 in the presence of these agents when using a Malthus AT 192 conductimetric instrument . Other synergistic agents (DMSO, ethanol, cetyl trimethyl ammonium bromide and Triton X-100) gave poor results in the conductimetric system . PBN also increased sensitivity of K . fragilis GK 1005 towards cycloheximide in the Malthus system, and PBS reduced the NEL of T-2 toxin for K . fragilis GK 1005 in a disc diffusion assay from 0.2 to 0.04 microgram per disc . No yeasts were found sensitive to the trichothecene deoxynivalenol (DON) even at a DON concentration of 10 micrograms/ml, except in the presence of PBN and PBS . The minimal inhibitory concentration (mic) of DON in the presence of PBS was 2 micrograms/ml for K . fragilis GK 1005.

Biotechnology (N Y), 1990 Jan, 8(1), 42 - 6
The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences; Sleep D et al.; We demonstrate the secretion of human serum albumin into the culture supernatant from the yeast Saccharomyces cerevisiae . Studies with five KEX2 processed leader sequences, namely the S . cerevisiae alpha factor, the natural human serum albumin, the Kluyveromyces lactis killer, a natural human serum albumin/alpha factor fusion, and a Kluyveromyces lactis killer/alpha factor fusion leader, are described . We show that the leader sequence used to direct secretion influences the quantity and quality of the secreted product . In designing secretion systems for heterologous proteins, one aims to maximise both the yield and fidelity of the product . Our results indicate that the choice of leader sequence and its relationship to the structural protein under study are crucial to the success of this process.

Curr Genet, 1989 Dec, 16(5-6), 419 - 27
Deletions and rearrangements in Kluyveromyces lactis mitochondrial DNA; Hardy CM et al.; Three classes of respiratory deficient mutants have been isolated from a fusant between Kluyveromyces lactis and Saccharomyces cerevisiae that contains only K . lactis mtDNA . One class (15 isolates), resemble rho 0 mutants of S . cerevisiae as they lack detectable mtDNA . A second class (16 isolates), resemble point mutations (mit-) or nuclear lesions (pet-) of S . cerevisiae as no detectable change is found in their mtDNA . The third class (five isolates), with deletions and rearrangements in their mtDNA are comparable to S . cerevisiae petite (rho-) mutants . Surprisingly, three of the five deletion mutants have lost the same 8.0 kb sector of the mtDNA that encompasses the entire cytochrome oxidase subunit 2 gene and the majority of the adjacent cytochrome oxidase subunit 1 gene . In the other strains, deletions are accompanied by complex rearrangements together with substoiciometric bands and in one instance an amplified sector of 800 bp . By contrast to G + C rich short direct repeats forming deletion sites in S . cerevisiae mtDNA, excision of the 8.0 kb sector in K . lactis mtDNA occurs at an 11 bp A + T rich direct repeat CTAATATATAT . The recovery of three strains manifesting this deletion suggests there are limited sites for intramolecular recombination leading to excision in K . lactis mtDNA.

Curr Genet, 1989 Dec, 16(5-6), 429 - 32
A petite positive strain of Kluyveromyces lactis has a 300 kb deletion in the rDNA cluster; Maleszka R et al.; By employing Pulsed Field Gel Electrophoresis we have shown that the chromosomal DNA pattern of a petite-positive strain of Kluyveromyces lactis, designated KF4, differs from the karyotype of the reference strain . Digestion with SfiI and hybridization with an rDNA probe, demonstrate that a novel chromosomal band in KF4 results from a deletion of 35-40 rDNA cistrons from the rDNA cluster . The significance of this finding to possible alterations in cell physiology and the ability to generate "petite" mutants is discussed.

Mycopathologia, 1989 Dec, 108(3), 211 - 5
Serological study of yeast killer toxins by monoclonal antibodies; Polonelli L et al.; Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F . Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used . In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.

Genetics, 1989 Nov, 123(3), 477 - 84
The maltose permease encoded by the MAL61 gene of Saccharomyces cerevisiae exhibits both sequence and structural homology to other sugar transporters; Cheng Q et al.; The MAL61 gene of Saccharomyces cerevisiae encodes maltose permease, a protein required for the transport of maltose across the plasma membrane . Here we report the nucleotide sequence of the cloned MAL61 gene . A single 1842 bp open reading frame is present within this region encoding the 614 residue putative MAL61 protein . Hydropathy analysis suggests that the secondary structure consists of two blocks of six transmembrane domains separated by an approximately 71 residue intracellular region . The N-terminal and C-terminal domains of 100 and 67 residues in length, respectively, also appear to be intracellular . Significant sequence and structural homology is seen between the MAL61 protein and the Saccharomyces high-affinity glucose transporter encoded by the SNF3 gene, the Kluyveromyces lactis lactose permease encoded by the LAC12 gene, the human HepG2 glucose transporter and the Escherichia coli xylose and arabinose transporters encoded by the xylE and araE genes, indicating that all are members of a family of sugar transporters and are related either functionally or evolutionarily . A mechanism for glucose-induced inactivation of maltose transport activity is discussed.

Mol Microbiol, 1989 Nov, 3(11), 1599 - 604
Mitochondrial cytochrome b genes with a six-nucleotide deletion or single-nucleotide substitutions confer resistance to antimycin A in the yeast Kluyveromyces lactis; Coria R et al.; Extrachromosomal mutants resistant to antimycin, from the yeast Kluyveromyces lactis, have been isolated, genetically characterized, and assigned to two specific genetic loci (Brunner et al., 1987) . In the present work the cytochrome b nucleotide sequence from six of these mutants was determined . Five mutants had single point mutations, corresponding to transversions . In one mutant, a six-base-pair deletion, beginning at nucleotide 689, was observed . The amino acid sequence derived from the coding strand showed that, in three independent antimycin-resistant mutants, a change of asparagine 31 into lysine took place (two of these mutants are also resistant to diuron) . Two other mutants showed a change from lysine 228 into isoleucine (or methionine) . Leucine 230, isoleucine 231, and threonine 232, were lost in the deletion mutant and were replaced by serine.

Eur J Biochem, 1989 Oct 1, 184(3), 699 - 706
Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera; Strasser AW et al.; We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis . A cosmid gene library of S . occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion . The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500 . The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases . The AMY l gene was found to be expressed from its original promoter in S . cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.

Nucleic Acids Res, 1989 Sep 12, 17(17), 6927 - 37
Genetic instability of an oligomycin resistance mutation in yeast is associated with an amplification of a mitochondrial DNA segment; Ragnini A et al.; In the yeast Kluyveromyces lactis, mutations affecting mitochondrial functions are often highly unstable . In order to understand the basis of this genetic instability, we examined the case of an oligomycin resistant mutant . When the mutant was grown in the absence of the drug, the resistance was rapidly lost . This character showed a typical cytoplasmic inheritance . The unstable resistance was found to be associated with the presence of a repetitive DNA in which the repeating unit was a specific segment of the mitochondrial DNA . The amplified molecules were co-replicating with the wild type genome in the mutant cells . The spontaneous loss of the drug resistance was accompanied by the disappearance of the amplified DNA . The repetitive sequence came from a 405 base-pair segment immediately downstream of a cluster of two transfer RNA genes (threonyl 2 and glutamyl) . Modified processing of these tRNAs was detected in the mutant . A possible mechanism by which these events could lead to drug resistance is discussed.

Mol Cell Biol, 1989 Sep, 9(9), 3931 - 7
In vivo construction of linear vectors based on killer plasmids from Kluyveromyces lactis: selection of a nuclear gene results in attachment of telomeres; Kamper J et al.; Linear vectors based on plasmids pGKL1 and pGKL2 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae and selected for integration of the nuclear LEU2 gene . The linear hybrid molecules obtained had no proteins attached to their 5' ends, as is found for native pGKL plasmids . However, telomere-specific sequences were added to the ends of pGKL1 . In contrast to the cytoplasmically localized pGKL plasmids, the newly obtained linear hybrid vectors probably replicate within the nucleus and provide evidence that the nuclear LEU2 gene cannot be expressed in the cytoplasm.

Curr Genet, 1989 Sep, 16(3), 153 - 8
Sequence and transcript analysis of the C . albicans URA3 gene encoding orotidine-5'-phosphate decarboxylase; Losberger C et al.; The human pathogen Candida albicans grows either as a yeast or in filamentous form . We have determined the sequence of a 1.365 kb genomic C . albicans fragment that complements Saccharomyces cerevisiae ura3 and Escherichia coli pyrF mutations . An open reading frame within this fragment corresponds to a protein of 270 amino acids that shows homology to orotidine-5'-phosphate decarboxylases (ODCases) of other fungal species . The C . albicans ODCase is most closely related to the ODCases of the budding yeasts Kluyveromyces lactis and S . cerevisiae (74% and 71% homology, respectively) . Most 5' ends of URA3 transcripts in the authentic host and in the heterologous host S . cerevisiae were found to be identical . These results demonstrate a close taxonomic relationship between non-pathogenic budding yeasts and C . albicans.

Curr Genet, 1989 Aug, 16(2), 95 - 8
The host range of the pKD1-derived plasmids in yeast; Chen XJ et al.; pKD1 is a 2 mu-like circular plasmid found in the yeast Kluyveromyces drosophilarum that can also stably replicate in Kluyveromyces lactis . We have found a short intergenic region in this genome that appears to be functionally neutral; that is, the introduction of foreign sequences into the single EcoRI restriction site located near one of the inverted repeats did not affect the high stability of the natural plasmid . By introducing a G418 resistance gene at this site, we constructed an autonomous recombinant plasmid . Since this vector did not require cir+ hosts for its stable maintenance, it could be used to examine the transformation host range of pKD1 among all the species belonging to the genus Kluyveromyces . Both species closely related to K . drosophilarum as well as a few other species that are very different in chromosomal GC% could be transformed to yield highly stable transformant clones.

Mol Cell Biol, 1989 Jul, 9(7), 2950 - 6
Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80; Salmeron JM Jr et al.; In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4 . The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S . cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein . Here we show that GAL80 does repress LAC9-activated transcription in S . cerevisiae if overproduced . We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80 . Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S . cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo . The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini . We discuss the implications of these results for the mechanism of galactose metabolism regulation in S . cerevisiae and K . lactis.

Nucleic Acids Res, 1989 Jun 26, 17(12), 4485 - 91
Analysis of the regions coding for transfer RNAs in Kluyveromyces lactis mitochondrial DNA; Wilson C et al.; The major regions coding for the transfer RNA genes in the mitochondrial DNA of K . lactis were studied . Twenty one, out of a supposed twenty four tRNA genes were identified and localized with respect to other mitochondrial genes . Most of the tRNA genes were found in a cluster downstream of the large ribosomal RNA gene . The order of a few groups of genes is conserved with respect to S . cerevisiae and T . glabrata . The highly diverged intergenic sequences contained a large number of guanine-cytosine clusters which frequently formed long palindromic sequences.

Yeast, 1989 May-Jun, 5(3), 209 - 18
Cloning and sequencing of the gene for apocytochrome b of the yeast Kluyveromyces lactis strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140); Brunner A et al.; The apocytochrome b genes from two strains of the yeast Kluyveromyces lactis, have been isolated and sequenced . The coding sequences in strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140) were identical but the upstream noncoding regions were slightly different . The sequences demonstrated the presence of a continuous open reading frame with no introns . The amino acid sequence, derived from the coding strand, showed 82% homology to the apocytochrome b of Saccharomyces cerevisiae strain D273-10B and only 58% homology to the protein from Schizosaccharomyces pombe strain 50 . CUN and CGN codon families were absent from the K . lactis gene . Codon usage was very similar to that of other mitochondrial genomes with mostly U or A in the third position . There were two unusual features . All threonines were coded by ACA(U) and all arginines by AGA.

Mol Cell Biol, 1989 May, 9(5), 2208 - 13
Kluyveromyces lactis maintains Saccharomyces cerevisiae intron-encoded splicing signals; Deshler JO et al.; The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined . The gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces cerevisiae introns . We demonstrated splicing of heterologous ACT transcripts in both K . lactis and S . cerevisiae.

Plasmid, 1989 May, 21(3), 185 - 94
Physical and biological characterization of linear DNA plasmids of the yeast Pichia inositovora; Ligon JM et al.; Three cryptic DNA plasmids have been identified in a strain of the yeast Pichia inositovora that are 18, 13, and 10 kbp in size . All are sensitive to digestion by DNase I, restriction endonucleases, and exonuclease III, but are resistant to the activities of RNase A and lambda exonuclease . These results indicate that each plasmid is a linear DNA molecule whose 5' ends are protected . A restriction map has been developed for each of the plasmids, demonstrating that each is unique and confirming their linear nature . The plasmids are a major constituent of DNA prepared from whole cells, but are absent from DNA preparations of purified mitochondria and nuclei, indicating that the plasmids are located in the cytoplasm . These plasmids share many of the physical characteristics described for the linear plasmids of the yeasts Kluyveromyces lactis and Saccharomycopsis crataegensis . Unlike the linear plasmids of K . lactis, however, they appear not to be capable of killer toxin production.

Yeast, 1989 Apr, 5 Spec No, S379 - 83
Restriction mapping of rDNA and the taxonomy of Kluyveromyces van der Walt emend . van der Walt; Lachance MA; Ribosomal DNA from the type strains of 13 nomenspecies of Kluyveromyces and from other strains were mapped with 11 restriction endonucleases . The length of the repeating unit ranged from ca . 8.4 kb (in K . aestuarii) to ca . 10.9 kb (in K . phaffii) . The length variation resided as expected in the nontranscribed spacer . The patterns confirmed some of the inferences articulated by various students of the genus . The closely related species K . marxianus and K . lactis constituted a core to which could be linked first K . wickerhamii and K . dobzhanskii and then K . aestuarii . The presumed relatedness between K . waltii and K . thermotolerans was endorsed by rDNA mapping as well, but evidence linking these two species to the rest of the genus is wanting . The restriction patterns suggest that the multispored species together with K . delphensis form a loose assemblage acting as a bridge between the "core" species and the species K . phaffii and K . lodderi.

Curr Genet, 1989 Apr, 15(4), 261 - 9
Functional relationship among TATA sequences, gene induction and transcription initiation in the beta-galactosidase, LAC4, gene from Kluyveromyces lactis; Ficca AG et al.; In the 5' non-coding region of the beta-galactosidase, LAC4, gene of Kluyveromyces lactis, three TATA-like sequences are present at -230, -170 and -142 from the ATG translation start site . By means of deletion mutations in the TATA region, at least two of these TATA sequences, those at -230 and -142, were shown to be required for normal gene expression . Evidence is presented for a functional hierarchy and cooperation between these TATA sequences . The deletion or a change in the position of the TATA sequences affects both beta-galactosidase induction and the location of RNA initiation sites . The TATA sequence at -230 alone is sufficient for correct gene induction when it is moved to a position 41 bp from the major RNA initiation sites located around -110; the -142 TATA alone contributes only partly to gene induction . We suggest a functional distinction between these two related regulatory sequences . This functional distinction might be established by sequence differences and/or targets of unlike specific DNA binding protein(s) . A conformational analysis of the LAC4 promoter showed that under torsional stress the functional elements UAS, TATA boxes RNA initiation sites and ATG can be detected as P1-sensitive sites . Possible functions of DNA structural alterations on gene expression are discussed.

Mol Gen Genet, 1989 Apr, 216(2-3), 422 - 7
Glucose repression of LAC gene expression in yeast is mediated by the transcriptional activator LAC9; Breunig KD; In the yeast Kluyveromyces lactis the beta-galactosidase gene is induced by lactose or galactose . As shown here it can also be repressed by glucose but only in some strains . When the LAC9 gene of a repressible strain is substituted by an allele of a non-repressible strain, the beta-galactosidase gene is no longer glucose repressed . LAC9 codes for a regulatory protein homologous to GAL4 which activates transcription in the presence of the inducer . Since the LAC9 product is also present in the repressed strain and binds to DNA in vitro, as shown by DNA footprinting, glucose repression cannot be caused by repression of LAC9 gene expression . Instead, our results demonstrate that glucose repression is mediated by the LAC9 gene product, and is separable from the ability of LAC9 to activate transcription.

J Cell Biol, 1989 Apr, 108(4), 1271 - 81
The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex; Bankaitis VA et al.; We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex . Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth . Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass . In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments . These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi . Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe . Furthermore, the K . lactis SEC14p was able to functionally complement S . cerevisiae sec14ts defects . These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.

Yeast, 1989 Mar-Apr, 5(2), 99 - 106
RAG1 and RAG2: nuclear genes involved in the dependence/independence on mitochondrial respiratory function for growth on sugars; Goffrini P et al.; The analysis of five independent isolates of Kluyveromyces lactis shows that CBS 2359, CBS 683 and CBS 4574 could grow in the presence of mitochondrial inhibitors (antimycin A, oligomycin or erythromycin) and that CBS 2360 and CBS 141 were unable to grow in the presence of drugs . The resistant growth was observed only on glucose and not on other fermentable carbon sources (galactose, lactose) . The phenotype 'growth on glucose in the presence of mitochondrial inhibitors' was called Rag+ . This phenotype was found to be controlled by two unlinked nuclear genes: RAG1 and RAG2 . Either of their recessive alleles, rag1 and rag2, led to the Rag- phenotype (i.e . the failure of growth on glucose in the presence of antimitochondrial drugs) . Rag- strains represent the case in which fermentative growth becomes absolutely dependent on the functioning of the normal respiratory chain.

Mikrobiologiia, 1989 Mar-Apr, 58(2), 291 - 7
{Killer-yeast strains with a broad spectrum of action: a search among collection strains and preliminary classification}; Kazantseva DI et al.; Killer strains were looked for among certain yeast species belonging to five spore-forming genera (Hansenula, Pichia, Kluyveromyces, Williopsis and Zygowilliopsis) and one imperfect genus (Candida) . 171 killers were found among 272 strains, and many of them had a high activity and a broad action spectrum . These as well as S . cerevisiae K1 and K2 killers (173 killers all in ail) were classified according to differences in their activity and the spectra of action on susceptible strains . Thirty-four groups belonging to 13 classes were isolated.

Biochim Biophys Acta, 1989 Feb 9, 1010(2), 191 - 8
Polyphosphate synthesis in yeast; Schuddemat J et al.; Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus . Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length . With increasing incubation times, polyphosphates with longer chain lengths were formed . Polyphosphates were synthesized faster during incubation with glucose than with ethanol . Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast . Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S . cerevisiae . However, in K . marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source . This was confirmed in experiments, conducted in the absence of an exogenous energy source.

Curr Genet, 1989 Feb, 15(2), 143 - 8
Multicopy plasmids containing the gene for the transcriptional activator LAC9 are not tolerated by K . lactis cells; Breunig KD; The high copy number 2-microns DNA-like Kluyveromyces plasmid pKD1 was extremely unstable in Kluyveromyces lactis when carrying the gene for the regulatory protein LAC9, a transcriptional activator involved in the induction of the LAC and GAL genes . Transformants of a lac9 mutant strain normally contained rearranged plasmids and all were Lac-, indicating that the LAC9 gene was inactive . Lac+ "revertants" could be obtained from Lac- transformants by selection on lactose plates . In some of these, the pKD1-based plasmid was stably maintained by being integrated into the chromosome of the cell; in others, the disrupted chromosomal gene was restored by a gene conversion event . None of the Lac+ revertants had more than one intact LAC9 gene, an indication that LAC9 overexpression affects cell viability.

J Basic Microbiol, 1989, 29(10), 707 - 16
Characterization of yeasts with high L{+}-lactic acid production: lactic acid specific soft-agar overlay (LASSO) and TAFE-patterns; Witte V et al.; Only few yeast strains are known for the high level production of L{+}-lactate . We report indications for the conspecifity of Kluyveromyces thermotolerans (formerly Saccharomyces veronae) strain CBS 4728 with Stamm 42 (formerly Saccharomyces pretoriensis, RADLER 1984) . We suggest that Stamm 42 has little, if any relationship to Saccharomyces cerevisiae . Furthermore, we have optimized the method of Subden et al . (1982) for the detection of lactate producing microorganisms . Using this method in a screening with 100 yeast strains of our institute collection, we could not find additional strains with high L{+}-lactate production . This method may provide a useful tool for the molecular cloning of the unique yeast L{+}-LDH1) gene (s).

Yeast, 1989 Jan-Feb, 5(1), 1 - 10
Analysis of chromosomal DNA patterns of the genus Kluyveromyces; Sor F et al.; Using an improved procedure of pulsed field gel electrophoresis, yeast chromosomes were separated over a wide range of molecular size (250-4000 kbp) on single gels . The chromosomal DNA patterns of all the species belonging to the genus Kluyveromyces were examined . Within the species K . marxianus, the varieties lactis, drosophilarum and vanudenii showed closely related patterns: very different from them, the varieties bulgaricus and marxianus were related to each other, forming a distinct group; the strains commonly called 'K . lactis' and 'K . fragilis' were unambiguously different from each other in chromosome patterns . These differences were correlated with the presence of characteristic repetitive sequence elements in the mitochondrial DNA of the former group and not in the latter . Analysis of Candida macedoniensis, which had been considered to be an anamorph of K . marxianus var . marxianus, showed that these two yeast species were indeed similar in chromosome patterns and in mitochondrial DNA restriction patterns.

Arch Microbiol, 1989, 152(5), 441 - 6
Molecular probes for the detection of Kluyveromyces marxianus chromosomal DNA in electrophoretic karyotypes of intergeneric protoplast fusion products; Witte V et al.; Random genomic DNA fragments from Kluyveromyces marxianus were cloned in order to identify chromosomal bands in pulsed field electrophoresis patterns of intergneric hybrid strains which were obtained by protoplast fusion . Molecular hybridization data indicated that the K . marxianus parental strain might be triploid, and it showed strong chromosome length polymorphism . We analyzed the karyotype of two Saccharomyces cerevisiae/K . marxianus hybrid strains (St . 1.St.46) with our DNA probes and with a Ty1 specific probe . We found indications for recombinational events which lead to the formation of hybrid chromosomal DNA molecules.

Yeast, 1989 Jan-Feb, 5(1), 35 - 50
Cloning and analysis of the Kluyveromyces lactis TRP1 gene: a chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histone H3; Stark MJ et al.; The TRP1 gene of the yeast Kluyveromyces lactis has been cloned from a genomic library by complementation of the Saccharomyces cerevisiae trp1-289 mutation . The gene was located within the clone by transposon mutagenesis and the coding region identified by DNA sequencing . This has indicated that K . lactis TRP1 encodes a 210-amino acid polypeptide which shows 53% identity to the homologous S . cerevisiae protein . The K . lactis TRP1 gene has been disrupted by substituting the S . cerevisiae URA3 gene for a large part of the TRP1 coding sequence . Replacement of the chromosomal TRP1 locus with this construction has enabled the production of non-reverting trp1- strains of K . lactis, while a genetic analysis of the disrupted allele confirmed that the TRP1 gene had been cloned . DNA sequencing has also shown that the K . lactis TRP1 sequence is flanked by genes encoding inorganic pyrophosphatase and histone H3, which we have designated IPP and HHT1 respectively . Hybridization studies have shown that in common with S . cerevisiae, K . lactis has two copies of the histone H3 gene . Each H3 gene is closely linked to a gene encoding histone H4 and in both yeast species the IPP gene is tightly linked to one of the histone gene pairs.

Mol Gen Genet, 1988 Dec, 215(1), 46 - 52
Palindrome-hairpin linear plasmids possessing only a part of the ORF1 gene of the yeast killer plasmid pGKL1; Kitada K et al.; The yeast Kluyveromyces lactis harboring linear DNA plasmids pGKL1 and pGKL2 exhibits killer and killer-resistant phenotypes . Two new linear plasmids pK192L and pK192S were found in the weak killer mutant KUV192 induced by UV irradiation . pK192S was always accompanied by pK192L in subclones of KUV192 . Both plasmids were derived from pGKL1 by deletion of the large right part of it . pK192L was 4.9 kb in size and had a palindromic structure consisting of 2.35 kb inverted terminal repetitions and a 215 base unique sequence . Analysis of denatured and renatured DNA strands suggested that pK192S was a hairpin-like form of pK192L . The pK192 plasmids were maintained only in cells haboring either pGKL1 or pGKL1Sin addition to pGKL2 and completed with pFKL1 or pGKL1S for their maintenance . Since no complete ORF1 was conserved in pK192 plasmids, these results lead to the conclusion that the ORF1 gene is necessary for the replication and/or maintenance of pGKL1.

Anal Biochem, 1988 Dec, 175(2), 531 - 6
In situ assay of intracellular enzymes of yeast (Kluyveromyces fragilis) by digitonin permeabilization of cell membrane; Gowda LR et al.; The yeast, Kluyveromyces fragilis was permeabilized to a number of low-molecular-weight substrates using digitonin . The activities of intracellular yeast enzymes, viz., alcohol dehydrogenase (ADH), beta-galactosidase, glucose-6-phosphate dehydrogenase, aspartase, and hexokinase were found to be much higher in the permeabilized cells than the untreated cells . The optimum conditions for permeabilization with reference to ADH were 0.1% digitonin at 37 degrees C for 15 min . The ADH activity in permeabilized cells was several-fold higher than that in cell free extracts prepared by either physical or chemical methods.

Eur J Epidemiol, 1988 Dec, 4(4), 409 - 14
Replication and maintenance of the Kluyveromyces linear pGKL plasmids; Gunge N et al.; Two new linear plasmids, pK192L (4.9 kb) and pK192S (2.4 kb), were isolated from a Kluyveromyces lactis killer strain carrying pGKL1 and pGKL2 . pK192L was a deletion plasmid of pGKL1, derived from a part of the ORF1, and had a palindrome structure of a 215 bp unique sequence flanked by 2.35 bp inverted repeats . pK192S was a hairpin plasmid produced by self-annealing of a single-stranded pK192L DNA . In genetic analysis, pK192L and pK192S always coexisted and replicated in cells harboring pGKL2 and pGKL1, in contrast to other pGKL1-derived deletion plasmids, such as F1, F2 and pGKLIS, which could replicate in cells carrying pGKL2 only . Based on these and other lines of evidence, it was concluded that the reason for the pGKL1 dependent replication of the pK192L/S plasmids was the absence of the intact pGKL1-ORF1 gene and that the ORF1 function was necessary for the replication of the pGKL1 genome . This finding is in good agreement with a recent view reporting that ORF1 may encode a DNA polymerase of pGKL1 . In a separate experiment, four new linear plasmids were isolated from a Saccharomyces cerevisiae strain carrying pGKL1 and pGKL2 . Structural analysis showed that they consisted of two pairs of hairpin-palindrome type plasmids, each derived from different parts of pGKL2, respectively . pGKL1 stability replicated in cells carrying both these pGKL2 derived deletion plasmids.

J Biol Chem, 1988 Nov 15, 263(32), 16696 - 703
Primary structure of the lactose permease gene from the yeast Kluyveromyces lactis . Presence of an unusual transcript structure; Chang YD et al.; The LAC12 gene of Kluyveromyces lactis codes for an inducible lactose permease . We have determined the nucleotide sequence of a DNA fragment which includes the complete LAC12 gene . The 4.7-kilobase (kb) mRNA carrying LAC12 contained two open reading frames, ORFI (1761 bases) and ORFII (1266 bases), separated by a 573-base pair noncoding region . Mung bean and exonuclease VII mapping showed that there was no splicing of the 4.7-kb transcript and thus no intron between the two open reading frames . Chromosomal disruption of ORFI with the URA3 gene destroyed lactose transport activity, suggesting that ORFI codes for a component of the permease . Disruption of ORFII and the noncoding region between the two open reading frames did not affect the lactose permease function, indicating that they do not comprise a part of the permease . We do not know if ORFII is translated, but in either case, the structure of the 4.7-kb mRNA is unusual . We discuss possible origins for it . The peptide predicted from ORFI is hydrophobic as would be expected for a membrane-bound protein . Compared with other membrane proteins, LAC12 (ORFI) protein showed sequence similarity to the human glucose and the Escherichia coli xylose-H+ and arabinose-H+ transporters . No obvious amino acid sequence similarity was found with the lactose permease of E . coli.

Proc Natl Acad Sci U S A, 1988 Nov, 85(22), 8386 - 90
Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants; Perentesis JP et al.; Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins . The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid . Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A . In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced . Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin . Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.

Am J Gastroenterol, 1988 Oct, 83(10), 1145 - 9
Efficacy of addition of exogenous lactase to milk in adult lactase deficiency; Lami F et al.; The efficacy of lactase by Kluyveromyces lactis in hydrolyzing milk lactose and reducing milk intolerance symptoms was tested in 52 proved lactose malabsorbers . The enzyme was added to milk administered to the patients, and H2 breath excretion (as an index of carbohydrate malabsorption), was determined by gas chromatograph technique, and milk intolerance symptoms were recorded . H2 mean excretion was 78.3 +/- 5.49 ppm after administration of intact whole milk 500 ml (test A), 43.5 +/- 4.99 ppm when lactase 2000 U was added to milk 500 ml immediately before administration (test B); 36.7 +/- 5.01 ppm when milk 500 ml was incubated for 12 h with lactase 1000 U (test C), and 29.7 +/- 4.35 ppm when the incubation was prolonged for 24 h (test D) . Symptoms score was: test A = 5.85 +/- 0.56, test B = 3.71 +/- 0.45, test C = 2.77 +/- 0.63, test D = 1.7 +/- 0.68 . A correlation index of r = 0.44 (p less than 0.01) was obtained between reduction in H2 mean excretion and reduction in symptoms score of a single individual . The addition of this lactase to milk seems to be effective in correcting lactose malabsorption, thus representing a convenient approach in milk intolerance.

Gene, 1988 Sep 30, 69(2), 181 - 92
A gene fusion system using the aminoglycoside 3'-phosphotransferase gene of the kanamycin-resistance transposon Tn903: use in the yeast Kluyveromyces lactis and Saccharomyces cerevisiae; Chen XJ et al.; The aminoglycoside 3'-phosphotransferase type I (APHI)-coding gene of the bacterial transposon Tn903 confers resistance to kanamycin on bacteria and resistance to geneticin (G418) on many eukaryotes . We developed an APHI fusion system that can be used in the study of gene expression in these organisms, particularly in yeasts . The first 19 codons of the KmR (APHI) gene can be deleted, and replaced by other genes in a continuous reading frame, without loss of APH activity . Examples of vector constructions are given which are adapted to the yeast Kluyveromyces lactis transformation system . Their derivatives containing the 2 mu origin of replication can also be used in Saccharomyces cerevisiae.

Nucleic Acids Res, 1988 Sep 12, 16(17), 8433 - 42
Mitochondrial DNA of the yeast Kluyveromyces: guanine-cytosine rich sequence clusters; Ragnini A et al.; Mitochondrial DNA from the yeast Kluyveromyces marxianus var . lactis (K.lactis) is a circular molecule of 39 kilobase-pairs . A genetic and physical map was constructed . We found that this genome contained a large number of guanine-cytosine (GC)-rich sequence clusters, many of which are characterized by the presence of SacII restriction sites (CCGCGG) . The primary sequence of the GC clusters often showed a palindromic structure . These GC clusters were present in several varieties of K.marxianus, but not in others . The presence of these clusters is a major feature that distinguishes K.lactis strains from those of K.marxianus var . marxianus (including K.fragilis).

Yeast, 1988 Sep, 4(3), 191 - 8
Karyotyping of yeast strains of several genera by field inversion gel electrophoresis; Johnston JR et al.; Field inversion gel electrophoresis has been used to improve the resolution of the large chromosomes (greater than 1000 kb) present in Saccharomyces kluyveri and in several genera of yeasts other than Saccharomyces cerevisiae, and thus establish more accurately the electrophoretic karyotype of these yeasts . Field inversion gel electrophoresis has also been used to demonstrate the presence of chromosome length polymorphisms in several of the yeasts studied . By Southern blotting techniques the greater degree of relatedness of S . kluyveri and Kluyveromyces lactis to S . cerevisiae, as compared to that of the other genera of yeasts studied, has been established.

Mol Cell Biol, 1988 Sep, 8(9), 3726 - 33
Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis; Witte MM et al.; LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis . LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae . Both proteins have a single "zinc finger" which plays a role in DNA binding . We previously hypothesized (L . V . Wray, M . M . Witte, R . C . Dickson, and M . I . Riley, Mol . Cell . Biol . 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger . In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein . Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding . Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity . These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix . Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

Nucleic Acids Res, 1988 Aug 25, 16(16), 8097 - 112
Extranuclear gene expression in yeast: evidence for a plasmid-encoded RNA polymerase of unique structure; Wilson DW et al.; Strains of the yeast Kluyveromyces lactis that produce killer-toxin have been found to contain two linear dsDNA plasmids, k1 (8.9 Kb) and k2 (13.4 Kb) . The four transcribed open reading frames of plasmid k1 contain no recognisable yeast nuclear expression signals . Moreover, a toxin subunit gene fused with the lacZ gene of Escherichia coli is not detectably expressed when introduced to K.lactis or Saccharomyces cerevisiae on a nuclear vector, even when native k1 and k2 are present in the cell . This and other evidence is consistent with the hypothesis that k1 and k2 reside in an extranuclear location, and do not utilise the nuclear RNA polymerases I, II or III for transcription of their genes . Sequencing of plasmid k2, which is thought to encode factors necessary for the maintenance or expression of k1, reveals an open reading frame predicted to encode a 974 amino acid polypeptide with homology to several DNA-directed RNA polymerases . We suggest that this is a component of a novel plasmid-specific extranuclear gene expression system.

Nucleic Acids Res, 1988 Aug 25, 16(16), 8011 - 28
The organization and transcription of the galactose gene cluster of Kluyveromyces lactis; Webster TD et al.; The yeast Kluyveromyces lactis grows on galactose by inducing the Leloir pathway enzymes-kinase, epimerase, and transferase . To investigate the molecular mechanism for regulating expression of this metabolic pathway we isolated GAL1, GAL7, GAL10, which code for kinase, transferase, and epimerase, respectively, and characterized their size, organization, and transcriptional regulation . Our results indicate that induction of the Leloir pathway in K . lactis occurs at the level of transcription and that the organization and regulation of the GAL gene cluster in K . lactis is closely related to the homologous gene cluster in Saccharomyces cerevisiae . Likewise, the Upstream Activator Sequences that regulate induction of the GAL genes are similar in base sequence, number and relative location in the two yeasts.

Nucleic Acids Res, 1988 Aug 11, 16(15), 7333 - 50
A transcriptional barrier to expression of cloned toxin genes of the linear plasmid k1 of Kluyveromyces lactis: evidence that native k1 has novel promoters; Romanos MA et al.; The killer toxin of Kluyveromyces lactis consists of three polypeptides encoded by the linear plasmid k1 . We re-introduced the entire k1 sequence, cloned on a circular replicating plasmid, into K . lactis strains lacking k1, and found that the resulting transformants did not produce toxin . The barrier to expression was found to be transcriptional: the four transcripts of native k1 were absent, and instead shorter, aberrant k1 transcripts were made . We determined the precise initiation sites of the four transcripts of native k1: these had very short untranslated leaders and mapped about 14bp downstream of an "upstream conserved sequence" (UCS) . It appears that k1 has novel promoters which are inactive on circular plasmids which replicate in the nucleus . This is consistent with the suggestion that native k1 resides in the cytoplasm.

Genome, 1988 Aug, 30(4), 501 - 5
Comparison of the orotidine 5'-monophosphate decarboxylase sequences of eight species; Radford A et al.; Predicted amino acid sequences of the enzyme orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) from eight different organisms are compared . The comparisons are made on the basis of primary structural differences, primary amino acid sequence, hydropathy profiles, and secondary structure predictions . The organisms compared are Mus musculus, Aspergillus nidulans, Neurospora crassa, Kluyveromyces lactis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli, and Salmonella typhimurium.

FEBS Lett, 1988 Jul 18, 234(2), 464 - 70
The Kluyveromyces lactis KEX1 gene encodes a subtilisin-type serine proteinase; Tanguy-Rougeau C et al.; KEX1 is a chromosomal gene required for the production of the killer toxin encoded by the linear DNA plasmid pGKL-1 of Kluyveromyces lactis . The nucleotide sequence of the cloned KEX1 gene has been determined . The deduced structure of the KEX1 protein, 700 amino acids long, indicated that it contained an internal domain with a striking homology to the sequences of the subtilisin-type proteinases, and a probable transmembrane domain near the carboxyl terminus . The results confirm the hypothesis that the product of the gene KEX1 of K . lactis is a proteinase involved in the processing of the toxin precursor.

Nucleic Acids Res, 1988 Jul 11, 16(13), 5863 - 78
Genome organization of the killer plasmid pGK12 from Kluyveromyces lactis; Tommasino M et al.; We have determined the entire sequence of the plasmid K2 from Kluyveromyces lactis which is involved in the maintenance of both killer plasmids in the cell . K2 shares many of the characteristics of the smaller killer plasmid K1: high A+T content (74.7%) and very compact genomic organization . K2 contains ten open reading frames . Some of them overlap on different strands and some on the same strand . Northern blotting of K2 transcripts shows that at least eight ORFs are transcribed . Analysis of the predicted aminoacid sequence of ORF2 from K2 reveals homology with the aminoacid sequence of ORF 1 from K1 and with several viral DNA polymerases . The sequence of K2 from Saccaromyces cerevisiae F102-2 was also determined . Only one nucleotide difference was found between the K2 sequence from the two yeasts . This mutation does not change the genome organization of the plasmid and has only minimal effect on the structure of the encoded proteins.

Mol Gen Genet, 1988 Jul, 213(1), 150 - 4
The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae; Iborra F et al.; The relationship between the promoter length of the Kluyveromyces fragilis beta-glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless beta-galactosidase gene of Escherichia coli . The removal of a region from position -425 to -232 led to a tenfold increase in the expression of the gene . The same results were obtained for the reconstructed beta-glucosidase gene with the same promoter length . It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae . This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the beta-glucosidase gene . It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.

Can J Microbiol, 1988 Jun, 34(6), 787 - 92
{Variation of the sterols from Kluyveromyces lactis and from a resistant mutant by culture in the presence of sublethal doses of amphotericin B}; Hakkou A et al.; A mutant strain of Kluyveromyces lactis resistant to amphotericin B and weakly to nystatin has been isolated from subcultures of the wild strain grown in the presence of sublethal doses of amphotericin B . The mutant and the wild strain were equally sensitive to pimaricin, filipin, and candicidin . The efficacy of fungizone was very low . In comparison with the wild strain the level of sterols was two times lower in the resistant strain but the composition of these sterols was about the same in the two strains . The action of sublethal doses of amphotericin B on the composition of the sterols was the same in these two yeasts and brought a 40% decrease of the total sterol level and a modification in their distribution . This variation cannot fully explain the resistance of the yeast but it may be associated to other changes of the membranes.

Biochim Biophys Acta, 1988 Apr 22, 939(3), 569 - 76
Regulation of sugar transport systems of Kluyveromyces marxianus: the role of carbohydrates and their catabolism; De Bruijne AW et al.; In Kluyveromyces marxianus grown on a glucose-containing synthetic medium four different sugar transporters have been identified . In cells, harvested during the exponential phase, only the constitutive glucose/fructose carrier, probed with 6-deoxy-D-glucose or sorbose, appeared to be active . In cells from the stationary phase three proton symporters can be active, recognizing 6-deoxyglucose (a glucose/galactose carrier), sorbose (a fructose carrier) and galactosides (lactose carrier), respectively . These symporters appeared to be sensitive to catabolite inactivation . This process is induced by incubating cells in the presence of glucose, fructose or mannose . Catabolite inactivation was not influenced by the inhibitor of protein synthesis, anisomycin . Derepression of the proton/sorbose and the proton/galactoside symporters proceeded readily when cells were incubated in a medium without glucose . Activation of the proton/galactose symporter needed, in addition, the presence of specific molecules (inducers) in the medium . The activation of each of these active transport systems was inhibited by anisomycin, showing the involvement of protein synthesis.

J Biol Chem, 1988 Mar 15, 263(8), 3930 - 4
Kluyveromyces bulgaricus yeast lectins . Isolation of N-acetylglucosamine and galactose-specific lectins: their relation with flocculation; Al-Mahmood S et al.; Kluyveromyces bulgaricus is a yeast which, upon culture in a calcium-enriched glucose-peptone medium, flocculates . Its flocculation can be reversed by the addition of galactose . In this paper, it is shown that two lectins can be isolated either from the concentrated culture broth or from the supernatant of deflocculated cells suspended in galactose solution . The N-acetylglucosamine-specific lectin, at pH 7.4, agglutinates untreated sheep red blood cells, but agglutinates neither untreated rabbit red blood cells nor glutaraldehyde-fixed sheep or rabbit red blood cells . Conversely, at pH 4.5, this lectin agglutinates glutaraldehyde-fixed sheep red blood cells . The galactose-specific lectin, at pH 7.4, agglutinates both untreated and glutaraldehyde-fixed rabbit red blood cells but does not agglutinate untreated or glutaraldehyde-fixed sheep red blood cells . At pH 4.5, this lectin agglutinates both glutaraldehyde-fixed sheep and rabbit red blood cells and induces flocculation of deflocculated K . bulgaricus cells . In all cases, the agglutination and the flocculation induced by one of these two lectins were inhibited by free or conjugated N-acetyl-D-glucosamine or by free or conjugated D-galactose, respectively . No glycosylhydrolase activity could be detected in the purified lectins.

Eur J Epidemiol, 1988 Mar, 4(1), 99 - 103
Biotyping of aerobic actinomycetes by modified killer system; Morace G et al.; Forty-four yeasts belonging to the genera Pichia, Candida, Saccharomyces and Kluyveromyces were tested for their potential killer effect on 13 aerobic actinomycetes (6 Nocardia asteroides, 1 N . brasiliensis, 1 N . caviae and 5 Actinomadura madurae) . Only a few yeast strains did not display any killer activity against the aerobic actinomycetes studied, thus confirming that the killer phenomenon is widespread among microorganisms . For epidemiological purposes, a killer system was developed . According to their susceptibility to the 9 killer yeasts chosen, it was possible to differentiate the Nocardia and Actinomadura isolates into biotypes . Fitting conditions of the killer system to potential sensitive microorganisms with different characteristics of growth are also discussed.

Yeast, 1988 Mar, 4(1), 61 - 9
Changes in ribosomal properties during adenylate deprivation in the cells of Kluyveromyces lactis; Ishiguro J et al.; In an adenine-requiring mutant strain of the yeast, Kluyveromyces lactis, the intracellular content of ATP is one-third to one-fifth that in a prototrophic wild strain under growing conditions . The quantitative differences becomes rather small in resting stationary-phase cells . Temporary changes in the two-dimensional protein patterns of mutant ribosomes occur when the ATP content is lowest during the transition phase of growth . The transfer of exponentially growing cells to a synthetic complete medium void of adenine induces the same changes in mutant ribosomes within several hours . Identification of ribosomal proteins by two-dimensional gel electrophoresis indicated all changeable proteins (at least five proteins) to belong to 40S ribosomal subunits . The mutant ribosomes prepared from the transition-phase cells have much lower activity (below 60%) for poly(U)-directed polyphenylalanine synthesis than those in exponentially growing or resting stationary-phase cells . Thus, changes in ribosomal components associated with the differences in ribosome activity in a cell-free system were noted in the adenylate-deprived cells of K . lactis.

Yeast, 1988 Mar, 4(1), 71 - 81
A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis; Wesolowski-Louvel M et al.; The killer system of Kluyveromyces lactis is associated with two linear DNA plasmids, pGKL1 and pGKL2 . The killer toxin and the immunity determinant are coded for by pGKL1 . Mutations which block the expression of the killer character have been isolated . These mutations reside in a single chromosomal gene which we have named KEX1 . The KEX1 gene of K . lactis has been cloned by complementation of kex1 mutations by using a recombinant plasmid pool containing the entire Kluyveromyces lactis genome, on a multicopy plasmid KEp6, which contains the Saccharomyces cerevisiae URA3 gene as a marker . Genetic analyses of strains carrying a disrupted kex1 allele demonstrated that the cloned DNA corresponded to the KEX1 gene . The cloned KEX1 gene of K . lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae . In vivo complementation of the kex1 mutation of K . lactis by the KEX2 gene of S . cerevisiae, and complementation of the kex2 mutation of S . cerevisiae by the KEX1 gene of K . lactis, demonstrated that KEX1 of K . lactis is functionally related to the KEX2 gene of S . cerevisiae . K . lactis diploids homozygous for kex1 are deficient for sporulation.

Antonie Van Leeuwenhoek, 1988, 54(2), 127 - 38
Impairment of insulin assimilation and beta-fructosidase activity due to a petite mutation in Kluyveromyces marxianus; Guiraud JP et al.; A respiratory deficient mutant of Kluyveromyces fragilis was isolated using ethidium bromide mutagenesis . It was characterized by a loss of cytochromes a + a3 and deficiency in cytochrome b . This petite mutant has brought about modifications in the excretion pattern of beta-fructosidase active on saccharose and inulin . The mutant practically no longer excretes the enzyme, and is incapable of growth and fermentation in the presence of inulin . The study of the activities of different enzyme extracts (culture medium, whole and disrupted cells) on inulin and saccharose suggests the existence of an unique enzyme system capable of taking several forms, and also shows the influence of the growth substrate on the I/S activity ratio.

J Basic Microbiol, 1988, 28(4), 211 - 20
A gene-cloning system for Kluyveromyces lactis and isolation of a chromosomal gene required for killer toxin production; Chen XJ et al.; A transformation system derived from the circular plasmid pKD1 has been developed for Kluyveromyces lactis . The principle is essentially equivalent to that of the 2 microns/Saccharomyces cerevisiae transformation system . The main features of the system are presented . Using a pKD1-based DNA bank of K . lactis, the KEX1 gene involved in the killer system was isolated by complementation.

Acta Biol Hung, 1988, 39(4), 335 - 44
Lysosomal enzyme release from macrophages: a model of food yeast toxicity evaluation; Hernandez Rosales F et al.; The role of lysosomal enzyme released by macrophages was examined in relation to the toxic effect caused by food yeast . Mouse peritoneal macrophages exposed to yeast in culture showed marked release of N-acetyl glucosaminidase, beta-galactosaminidase and beta-glucuronidase below the median lethal dose (LD50) . LD50 was measured from the dose response curves of the cytoplasmic lactate dehydrogenase enzyme . Saccharomyces cerevisiae showed the highest LD50 followed by Kluyveromyces fragilis and Candida utilis yeast . LD50 values obtained as well as the in vitro lysosomal release by mouse peritoneal macrophages may be relevant to assess the toxic capacity of food yeast intended for human consumption.

Folia Biol (Praha), 1988, 34(4), 277 - 81
Method for estimating activity of killer toxin from Kluyveromyces lactis; Palkova Z et al.; At the exponential growth phase, nystatin-treated Saccharomyces cerevisiae cells stain with Rhodamine B if they have not previously been exposed to killer toxin produced by Kluyveromyces lactis . This finding constitutes the principle of a method for estimating the activity of this toxin.

J Basic Microbiol, 1988, 28(4), 241 - 7
OFAGE banding patterns of different yeast genera and of intergeneric hybrids; Zimmermann M et al.; Electrophoretic karyotypes of yeasts belonging to the species Saccharomyces cerevisiae, Kluyveromyces marxianus and Candida macedoniensis were established by means of OFAGE . Hybrids between S . cerevisiae and K . marxianus as well as between K . marxianus and C . macedoniensis were analyzed by comparing their OFAGE-banding-pattern with the parental banding-patterns . Thus, evidence for exchanges of intact chromosomes and for chromosomal rearrangements could be gained on a molecular level.

Mol Cell Biol, 1987 Dec, 7(12), 4369 - 76
Identification of upstream activator sequences that regulate induction of the beta-galactosidase gene in Kluyveromyces lactis; Leonardo JM et al.; Transcription of the Kluyveromyces lactis beta-galactosidase gene, LAC4, is inducible by galactose and lactose . We examined the effects of deletion mutations within the LAC4 promoter on the expression of beta-galactosidase activity . The results of these experiments indicate that at least two upstream activator sequences (UAS) mediate maximum induction by galactose . These UAS sequence elements are homologous to UAS that regulate induction of the melibiose-galactose regulon of Saccharomyces cerevisiae . We also show that a synthetic copy of one of the K . lactis UAS restores the inducibility of a deleted, noninducible LAC4 promoter . Since the uninduced or basal level of LAC4 expression was increased in several promoter deletion strains and in deletion strains carrying one or two synthetic UAS, we examined the contribution of the LAC9 positive regulatory protein to this effect . The LAC9 protein is thought to bind to UAS and activate transcription of LAC4 (L.V . Wray, M.M . Witte, R.C . Dickson, and M.I . Riley, Mol . Cell . Biol . 7:1111-1121, 1987) . Our results demonstrate that LAC9 protein plays a role in setting the uninduced level of gene expression, but other factors also participate . For example, in a lac9 background a LAC4 promoter deletion mutant with two copies of a synthetic 17-base-pair UAS yields a sevenfold higher level of uninduced LAC4 expression than the same strain with one UAS . These and other data indicate that the basal level of gene expression is strongly influenced by the base sequence of the promoter.

Mol Cell Biol, 1987 Dec, 7(12), 4400 - 6
Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site; Breunig KD et al.; As shown previously, the beta-galactosidase gene of Kluyveromyces lactis is transcriptionally regulated via an upstream activation site (UASL) which contains a sequence homologous to the GAL4 protein-binding site in Saccharomyces cerevisiae (M . Ruzzi, K.D . Breunig, A.G . Ficca, and C.P . Hollenberg, Mol . Cell . Biol . 7:991-997, 1987) . Here we demonstrate that the region of homology specifically binds a K . lactis regulatory protein . The binding activity was detectable in protein extracts from wild-type cells enriched for DNA-binding proteins by heparin affinity chromatography . These extracts could be used directly for DNase I and exonuclease III protection experiments . A lac9 deletion strain, which fails to induce the beta-galactosidase gene, did not contain the binding factor . The homology of LAC9 protein with GAL4 (J.M . Salmeron and S . A . Johnston, Nucleic Acids Res . 14:7767-7781, 1986) strongly suggests that LAC9 protein binds directly to UASL and plays a role similar to that of GAL4 in regulating transcription.

Biochim Biophys Acta, 1987 Oct 16, 903(3), 425 - 33
Characterization of low- and high-affinity glucose transports in the yeast Kluyveromyces marxianus; Gasnier B; Glucose transport in the yeast Kluyveromyces marxianus proceeds by two functionally and presumably structurally distinct transporters depending on the carbon source of the culture medium . In lactose-grown cells, glucose was taken up through a high-affinity H+-sugar symporter (Km = 0.09 mM), whereas a low-affinity transporter (Km = 3.5 mM) was utilized in glucose-grown cells . The two transporters exhibited different substrate specificities . Galactose was demonstrated to be a selective substrate of the H+-glucose symporter (Km = 0.14 mM) and did not significantly enter glucose-grown cells . Fructose was a preferential substrate of the low-affinity carrier (Km = 3.5 mM), but it entered lactose-grown cells through a high-affinity H+-fructose symporter distinct from the H+-glucose one . Other putative substrates of the two glucose transporters were identified by competition experiments . 2-Deoxyglucose recognized both carriers with a similar affinity, while the non-phosphorylatable analogues 6-deoxyglucose, 3-O-methylglucose and D-fucose exhibited a 10-30 fold preference for the high-affinity transporter.

Biotechnol Appl Biochem, 1987 Oct, 9(5), 410 - 22
Comparison of the properties of the purified beta-glucosidase from the transformed strain of Saccharomyces cerevisiae TYKF2 with that of the donor strain Kluyveromyces fragilis Y610; Leclerc M et al.; Saccharomyces cerevisiae TYKF2 was obtained by cloning in S . cerevisiae the gene coding for beta-glucosidase in Kluyveromyces fragilis Y610 (ATCC 12424) . The beta-glucosidases of both organisms were purified and their biochemical characteristics were determined . The two beta-glucosidases had the same enzymatic properties as those previously described in the literature . The strain S . cerevisiae TYKF2 is able to produce enhanced amounts of enzyme.

Mol Gen Genet, 1987 Jun, 208(1-2), 145 - 51
Isolation and characterization of mutants of Kluyveromyces lactis defective in lactose transport; Riley MI et al.; Mutants of Kluyveromyces lactis defective in lactose transport were identified among lactose-resistant revertants of lactose-sensitive strains . The mutations are closely linked to the beta-galactosidase gene, LAC4, and they are located in a previously identified gene, LAC12, which has been shown to code for a lactose permease . Our data establish that LAC12 is the only lactose permease gene in K . lactis . The lactose permease also transports galactose . LAC12 is transcribed in a direction opposite to that of LAC4, there being about 2.5 kb between their transcription start sites . Transcription of LAC12 is inducible as is that of all other structural genes in the lactose-galactose regulon of K . lactis.

Pediatrics, 1987 May, 79(5), 766 - 72
Effective reduction of lactose maldigestion in preschool children by direct addition of beta-galactosidases to milk at mealtime; Barillas C et al.; We examined the efficiency of two beta-galactosidase preparations--one derived from the yeast, Kluyveromyces lactis (Lactaid), the other derived from the fungus, Aspergillus oryzae (Takamine)--to assist the in vivo digestion of lactose consumed by healthy Guatemalan preschool children . Milk prehydrolyzed by in vitro incubation with enzymes was used as the standard of reference, and the degree of incomplete digestion of lactose from 240 mL of milk was determined using the hydrogen breath test . In in vivo dose-response studies, both 3,250 neutral lactose units of Lactaid and 6,635 food and chemical codex lactose units of Takamine completely eliminated excess H2 excretion in a small sample of lactose-maldigesting subjects . When evaluated in a controlled, clinical trial setting, the same dose of Lactaid added directly to the milk at consumption produced an 82% relative reduction in H2 excretion, whereas Takamine was equally as effective as the prehydrolyzed milk . Thus, intraluminal conditions and gastrointestinal transit in the preschool child support the effective assisted digestion of milk lactose in an efficient manner and with the same enzyme to milk ratios as observed previously in adults.

Biochem J, 1987 Mar 15, 242(3), 729 - 34
The role of ATP in the control of H+-galactoside symport in the yeast Kluyveromyces marxianus; Van den Broek PJ et al.; Transport of methyl beta-D-thiogalactoside and p-nitrophenyl beta-D-galactoside is shown to proceed through the H+-lactose symporter of Kluyveromyces marxianus . Uptake of these compounds is strongly reduced under anaerobic conditions or aerobically in the presence of antimycin . It is shown that antimycin treatment affects p-nitrophenyl beta-D-galactoside uptake in a similar way as it affects the cellular amount of ATP, suggesting regulation of p-nitrophenyl beta-D-galactoside transport by ATP . Also, manipulation of cellular ATP by antimycin treatment followed by glucose incubation, or by aerobic incubation of cells with 2-deoxy-D-glucose, showed a similar dependence of galactoside uptake on the ATP level . Transport of the lipophilic cation tetraphenylphosphonium is affected by ATP variations in a similar way as galactoside influx . It is concluded that ATP regulates H+-galactoside symport by its influence on charge translocation . It is discussed that a membrane ATPase probably plays a central role in the control of the activity of H+-sugar symport.

Mol Cell Biol, 1987 Mar, 7(3), 1111 - 21
Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae; Wray LV Jr et al.; Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis . We show here that the regulon is not induced in strains defective in LAC9 . We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription . The LAC9 gene was isolated by complementation of a lac9 defective strain . DNA sequence analysis of the gene gave a deduced protein of 865 amino acids . Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology . One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences . We speculate that a portion of this region, adjacent to the "metal-binding finger," specifies DNA binding . We discuss possible functions of the two other regions of homology . The functional implications of these structural similarities were examined . When LAC9 was introduced into a gal4 defective strain of S . cerevisiae it complemented the mutation and activated the galactose-melibiose regulon . However, LAC9 did not simply mimic GAL4 . Unlike normal S . cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL1 and MEL1, two genes in the regulon . The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4 . We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.

Mol Cell Biol, 1987 Mar, 7(3), 991 - 7
Positive regulation of the beta-galactosidase gene from Kluyveromyces lactis is mediated by an upstream activation site that shows homology to the GAL upstream activation site of Saccharomyces cerevisiae; Ruzzi M et al.; In contrast to the Escherichia coli lac operon, the yeast beta-galactosidase gene is positively regulated . In the 5'-noncoding region of the Kluyveromyces lactis LAC4 gene, we mapped an upstream activation site (UAS) that is required for induction . This sequence, located between positions -435 and -326 from the start of translation, functions irrespective of its orientation and can confer lactose regulation to the heterologous CYC1 promoter . It is composed of at least two subsequences that must act in concert . One of these subsequences showed a strong homology to the UAS consensus sequence of the Saccharomyces cerevisiae GAL genes (E . Giniger, S . M . Varnum, and M . Ptashne, Cell 40:767-774, 1985) . We propose that this region of homology located at about position -426 is a binding site for the product of the regulatory gene LAC9 which probably induces transcription of the LAC4 gene in a manner analogous to that of the GAL4 protein.

Nucleic Acids Res, 1987 Feb 11, 15(3), 1031 - 46
Expression and identification of immunity determinants on linear DNA killer plasmids pGKL1 and pGKL2 in Kluyveromyces lactis; Tokunaga M et al.; The linear dsDNA plasmids, pGKL1 (8.9 kb) and pGKL2 (13.4 kb) discovered in Kluyveromyces lactis, confer killer and immunity characteristics upon various yeast strains . We have devised an immunity assay and have been able to show the expression of an immunity phenotype in the K . lactis transformants harbouring conventional circular plasmids which contain DNA fragments of pGKL1 . Using this expression system, the immunity determinant on pGKL1 was identified as ORF5 . In addition, the presence of pGKL2 was proved to be essential for the expression of the immunity phenotype . This is the first demonstration of this new pGKL2 function, as distinct from its known functions for the replication and maintenance of pGKL1 in yeast cells.

Mol Cell Biol, 1987 Feb, 7(2), 780 - 6
GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon; Riley MI et al.; A Kluyveromyces lactis mutant defective in lac9 cannot induce beta-galactosidase or galactokinase activity and is unable to grow on lactose or galactose . When this strain was transformed with the GAL4 positive regulatory gene of Saccharomyces cerevisiae it was able to grow on lactose or galactose as the sole carbon source . Transformants bearing GAL4 exhibited a 4.5-h generation time on galactose or lactose, versus 24 h for the nontransformed lac9 strain . A K . lactis lac9 strain bearing two integrated copies of GAL4 showed 3.5-fold induction of beta-galactosidase activity and 1.8-fold induction of galactokinase activity compared with 15.6-fold and 4.4-fold induction, respectively, for the LAC9 wild-type strain . In transformants bearing 10 integrated copies of GAL4, the induced level of beta-galactosidase was nearly as high as in the LAC9 wild-type strain . In addition to restoring lactose and galactose gene expression, GAL4 in K . lactis lac9 mutant cells conferred a new phenotype, severe glucose repression of lactose and galactose-inducible enzymes . Glucose repressed beta-galactosidase activity 35- to 74-fold and galactokinase activity 14- to 31-fold in GAL4 transformants, compared with the 2-fold glucose repression exhibited in the LAC9 wild-type strain . The S . cerevisiae MEL1 gene was repressed fourfold by glucose in LAC9 cells . In contrast, the MEL1 gene in a GAL4 lac9 strain was repressed 20-fold by glucose . These results indicate that the GAL4 and LAC9 proteins activate transcription in a similar manner . However, either the LAC9 or GAL4 gene or a product of these genes responds differently to glucose in K . lactis.

Rev Argent Microbiol, 1987 Jan-Mar, 19(1), 1 - 7
{Optimizing conditions for the discontinuous production of unicellular protein using whey}; Bainotti AE et al.; The cheese whey is one of the most important effluents which is being disposed off in our area . That is why the study for optimizing conditions in the process of recovering whey to produce protein biomass in a batch fermenter was undertaken . A yeast strain (Kluyveromyces marxianus var . lactis) was propagated on a medium based on whey from cheese making plants, the following conditions for cell development being optimized: temperature, pH and initial concentration of lactose . A methodology based on performing several tests ordered according to a "Latin Squares" structure was proposed; this enables the simultaneous study of three variables with a small number of experiences . Such experiences were performed in a cylindrical (air lift type) glass fermenter, obtaining a maximum yield (4.78 g/l by dry weight) when working with an initial lactose concentration equal to 4.8% . For all temperatures (27, 30 and 35 degrees C) and pH (3, 4 and 5) employed, an increase in the cell number occurred with the initial lactose concentration increasing from 2% to 4.8% (Table 2) . It is preferable to work at 27 degrees C and pH 4 (since these conditions minimize the bacterial contamination) and with a lactose concentration equal to 4.8%, i.e . the concentration in the residual cheese whey . Thus, an optimum yield in protein biomass is obtained, enabling a good utilization of this effluent, and also diminishing its initial BOD from 60,000 to 15,000 ppm.

Antonie Van Leeuwenhoek, 1987, 53(2), 119 - 24
Kluyveromyces fragilis SS-437: an associatively-profiled thermotolerant yeast; Fatichenti F et al.; The lactose-utilizing Kluyveromyces fragilis SS-437 was found to have an associative temperature profile, but a thermotolerant growth yield behaviour . Cardinal growth temperatures were: 3 degrees C minimum for growth; 41.5 degrees C optimum; 44.5 degrees C final maximum (growth and death rates equalize); 46.1 degrees C initial maximum (maximum limit for growth).

J Gen Microbiol, 1987 Jan, 133 ( Pt 1), 15 - 23
Uptake of galactose and lactose by Kluyveromyces lactis: biochemical characteristics and attempted genetical analysis; Boze H et al.; Study of the lactose and galactose transport systems in Kluyveromyces lactis has shown that lactose uptake is by active transport . The transport system is under monogenic control and is inducible . Galactose uptake is also by active transport but the system is controlled by two genes which, in the four strains we studied, are present only in K . lactis CBS 2359 . Galactose uptake in the other K . lactis strains is by a simple diffusion process.

Zentralbl Mikrobiol, 1987, 142(3), 263 - 8
Kinetics of spheroplasts formation from selected yeast strains; van Huynh N et al.; The formation of spheroplasts from several Kluyveromyces and Saccharomyces strains was studied with the Coulter counter technique . When yeasts were incubated with zymolyase, the number of intact cells decreased according to first-order kinetics after an initial lag . In the concentration ranges studied, the rates were proportional to the cell-lytic enzyme concentrations . The rate (-0.099 min-1) of the most susceptible strain, K . fragilis, was 80-fold higher than that of S . cerevisiae CBS 5495 . Compared to the cell sizing technique, the spectrophotometric determination leads to the underestimation of the reaction course.

Curr Genet, 1987, 11(6-7), 475 - 82
Extrachromosomal genetics in the yeast Kluyveromyces lactis . Isolation and characterization of antimycin-resistant mutants; Brunner A et al.; Antimycin-resistant (AR) mutants of the yeast Kluyveromyces lactis, obtained either spontaneously or after manganese treatment, were isolated and genetically characterized . Most of the mutants obtained after manganese mutagenesis and two spontaneous mutants, tolerated high antimycin concentrations (more than 10 micrograms/ml) and were extrachromosomal . One mutant which grew only in low antimycin (1 microgram/ml) showed a Mendelian type of inheritance . The extrachromosomal mutants could be assigned to at least two genetic loci (ARI and ARII) . Mutants representative of these two groups showed increased resistance to the antibiotic when the respiration of whole cells or mitochondria was studied . Extrachromosomal mutants of Saccharomyces cerevisiae resistant to antimycin were also induced with manganese, isolated and characterized . Comparative studies of the antimycin-resistant mutants of K . lactis and S . cerevisiae permitted the following observations: a) K . lactis is more resistant to antimycin, funiculosin, mucidin and diuron than S . cerevisiae, as are the AR mutants; b) K . lactis shows correlated sensitivity to funiculosin differing in this aspect from S . cerevisiae; c) the antimycin-resistant mutants of K . lactis belonging to group II (ARII) were also resistant to diuron, tolerating concentrations of more than 200 micrograms/ml; d) all extrachromosomal antimycin-resistant-mutants of S . cerevisiae and some of the AR mutants of K . lactis were more sensitive to mucidin than the wild type.

Curr Genet, 1987, 12(2), 99 - 104
Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomous replication of hybrid plasmids in Saccharomyces cerevisiae; Fujimura H et al.; By use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker . The replication properties of hybrid plasmids in yeasts were investigated . We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids . Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.

Acta Microbiol Hung, 1987, 34(1), 73 - 83
The role of genes LAC1 and LAC2 in the biosynthesis of lactose metabolism enzymes by Kluyveromyces lactis; Boze H et al.; By crossing different Kluyveromyces lactis strains, the role of genes LAC1, LAC2 and of gene C were analyzed . These genes are involved in the biosynthesis of enzymes for the metabolism of lactose and galactose . They control the biosynthesis of the lactose and galactose transport, of beta-galactosidase and of the three enzymes of the Leloir pathway . The presence of at least one of the LAC gene is required for the biosynthesis to occur . The gene C seems to code for a negative factor which blocks the expression of the LAC1 and LAC2 genes in the absence of an inducer.

EMBO J, 1987 Jan, 6(1), 229 - 34
A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1 beta in Saccharomyces cerevisiae; Baldari C et al.; Killer strains of Kluyveromyces lactis secrete a toxin which presumably is processed during secretion from a larger precursor . Analysis of the sequence of the K . lactis killer toxin gene predicts that the first 16 amino acids at the amino terminus of the protein should represent its leader peptide . We have tested the capability of this leader peptide to direct secretion of a protein fused to it by inserting a synthetic oligonucleotide identical to the sequence of the putative leader peptide into a yeast expression vector . Subsequently, the cDNA coding for the secreted active portion of the human interleukin 1 beta (IL-1 beta) was fused to the leader peptide sequence of the killer toxin . This construction in Saccharomyces cerevisiae is capable of directing synthesis and secretion of correctly processed IL-1 beta into the culture medium.

Curr Genet, 1987, 12(3), 175 - 84
Sequence and transcription of the beta-glucosidase gene of Kluyveromyces fragilis cloned in Saccharomyces cerevisiae; Raynal A et al.; The complete nucleotide sequence of the beta-glucosidase gene of Kluyveromyces fragilis has been determined . This sequence contains an open reading frame of 2535 base pairs encoding a protein of 845 amino acids . Analysis of the transcription products revealed only one transcript of about 3 kb identical in both Kluyveromyces fragilis and in the expression host Saccharomyces cerevisiae . The protein molecular weight of 93,811 Kd deduced from the sequence is consistent with the 90,000 Kd determined by SDS polyacrylamide gel electrophoresis with the purified protein . Mapping of the starts of transcription shows that two starting points are used in the natural host Kluyveromyces fragilis . A comparison of the amino acid sequence with that of other beta-glucosidases revealed three regions of homology . One of these regions contains an amino acid sequence very similar to a peptide isolated from the active site of beta-glucosidase A3 from Aspergillus wentii and could be implicated in the catalytic mechanism of these glucolytic enzymes.

Antibiot Med Biotekhnol, 1986 Nov, 31(11), 828 - 30
{Nisin biosynthesis during the joint cultivation of Streptococcus lactis strain MGU and yeasts}; Egorov NS et al.; In simultaneous inoculation of the medium for nisin biosynthesis with Streptococcus lactis (strain MSU) and yeasts such as Rhodotorula colostri, Zigowilliopsis californicus, Hansenuia anomala, Saccharomyces ludvigii, Kluyveromyces lactis and Endomyces magnusi the quantitative ratio of the inoculates in the mixed cultures is not in principle important for the antibiotic synthesis by the Streptococcus . When 6-, 12-, 18- and 24-hour inoculates of the yeasts were added simultaneously to 3-, 6-, 9-, 12- and 18-hour cultures of S . lactis biosynthesis of nisin was at the control level . The fractional composition of the fermentation broth of the above yeast species had no significant effect on biosynthesis of nisin.

Arch Microbiol, 1986 Nov, 146(2), 115 - 7
Biosynthesis regulation of the beta-glucosidase produced by a yeast strain transformed by genetic engineering; Leclerc M et al.; The biosynthesis of the beta-glucosidase enzyme was studied in a transformed yeast obtained by cloning in Saccharomyces cerevisiae the structural gene coding for beta-glucosidase in Kluyveromyces fragilis . The enzyme biosynthesis was found to be non-adaptative, and repressed by glucose . These features are similar to those observed in K . fragilis . beta-Glucosidase activity in the transformed yeast was much higher than in K . fragilis . We attempted to ferment cellobiose with the transformed yeast: practically no cellobiose was consumed, growth and ethanol production were negligible . Warburg experiments showed that cellobiose fermentation did not occur when the respiratory chain was not functioning.

Nucleic Acids Res, 1986 Oct 10, 14(19), 7767 - 81
Analysis of the Kluyveromyces lactis positive regulatory gene LAC9 reveals functional homology to, but sequence divergence from, the Saccharomyces cerevisiae GAL4 gene; Salmeron JM Jr et al.; The galactose metabolism positive regulatory gene from Kluyveromyces lactis, LAC9, has been isolated through its ability to activate expression of galactose metabolism enzyme genes in Saccharomyces cerevisiae . The LAC9 gene also activates expression of the S . cerevisiae alpha-galactosidase (MEL1) and K . lactis beta-galactosidase (LAC4) genes in S . cerevisiae . Although LAC9-activated gene expression in K . lactis is not glucose repressed, activation of MEL1 gene expression by LAC9 in S . cerevisiae is . The LAC9 gene is expressed at an extremely low level as a approximately 2.9-kb mRNA, and encodes a protein of 865 amino acids . Although the LAC9 gene is functionally analogous to the S . cerevisiae GAL4 gene, the bulk of its protein sequence shows little homology to that of GAL4 . Two of the three regions of homology that do exist, however, are restricted to areas of GAL4 protein already implicated in nuclear localization, DNA binding, and transcriptional activation.

Nucleic Acids Res, 1986 Sep 11, 14(17), 6871 - 84
Efficient isolation of the linear DNA killer plasmid of Kluyveromyces lactis: evidence for location and expression in the cytoplasm and characterization of their terminally bound proteins; Stam JC et al.; Differential centrifugation of an osmotic lysate of K . lactis protoplasts showed that the linear DNA killer plasmids of K . lactis, pGKL1 and pGKL2, are almost exclusively present in the cytoplasmic fraction . This fractionation procedure allows the rapid isolation of large amounts of plasmid DNA without contamination by chromosomal and mitochondrial DNA . With these DNA preparations the size of the terminally bound proteins was estimated to be 28 and 36 kDal for pGKL1 and pGKL2, respectively . The entire pGKL1 sequence (except for 21 base pairs at the right terminus) was cloned in a shuttle vector that permits autonomous replication in the nucleus of K . lactis . However, killer gene expression could not be established in transformants . In connection with the observed cytoplasmic localization, this result suggests that gene expression of the killer DNA plasmids is entirely cytoplasmic.

Can J Microbiol, 1986 Sep, 32(9), 738 - 42
{Effect of amphotericin B on the sterol composition of Kluyveromyces bulgaricus and Kluyveromyces lactis}; Coulon J et al.; The degree of sensitivity of the yeasts Kluyveromyces bulgaricus and K . lactis to amphotericin B is linked to a difference in the sterol composition of their membranes . No direct proportionality was found between sensitivity and the quantity of sterols present . At sublethal doses, amphotericin B perturbed sterol synthesis, resulting in ergosterol precursor accumulation . An ergosterol pathway is proposed for Kluyveromyces.

Yeast, 1986 Sep, 2(3), 179 - 91
Physical separation and functional interaction of Kluyveromyces lactis and Saccharomyces cerevisiae ARS elements derived from killer plasmid DNA; Thompson A et al.; Two DNA fragments which have autonomously replicating sequence (ARS) activity in both Saccharomyces cerevisiae and Kluyveromyces lactis have been isolated from the K . lactis kl killer plasmid . One fragment (Kla1) is 700 base pairs (bp) in length and plasmids carrying it are mitotically unstable in both hosts . In K . lactis, this instability leads to colonies having a 'nibbled' phenotype when grown on selective media and appears to be the result of inefficient plasmid segregation . The other fragment (Kla2) is an artificial junction fragment of 1100 bp which was produced during the cloning procedure . Kla2 has been divided into two sub-fragments Kla2A and Kla2B which have, respectively, ARS activity in K . lactis and S . cerevisiae but not the other species . This indicates that these two closely related yeasts have different sequence requirements for ARS activity . Kla2B contains a perfect match to the S . cerevisiae ARS consensus but Kla2A does not . Both Kla2A and Kla1 share a 10 bp sequence as the sole region of homology between them . This sequence, 5'TCATAATATA3', is tentatively offered as defining the ARS consensus sequence for K . lactis.

EMBO J, 1986 Aug, 5(8), 1995 - 2002
The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them; Stark MJ et al.; The killer character of the yeast Kluyveromyces lactis is associated with the presence of the linear DNA plasmids k1 and k2 and results from the secretion of a protein toxin into the growth medium . We find that toxin activity co-purifies with three polypeptides which we have termed the alpha- (mol . wt 99,000), beta- (mol . wt 30,000) and gamma- (mol . wt 27,500) subunits . The alpha-subunit appears to contain a single asparagine-linked oligosaccharide chain but neither of the smaller subunits is glycosylated . The N-terminal amino acid sequence of each subunit has been determined . Comparison of these data with the DNA sequence of plasmid k1 indicates that it encodes all three subunits . The alpha- and beta-subunits must be processed from the primary translation product of a single gene by an enzyme related to the KEX2 endopeptidase of Saccharomyces cerevisiae.

Nucleic Acids Res, 1986 Jun 11, 14(11), 4471 - 81
Sequence organization of the circular plasmid pKD1 from the yeast Kluyveromyces drosophilarum; Chen XJ et al.; pKD1 is the only circular plasmid known in the genus Kluyveromyces . Nucleotide sequence analysis has revealed that this 4757 base-pairs long plasmid contained three major open reading frames, A, B, and C, and a pair of inverted repeats of 346 base-pairs . The molecule exists in two isomeric forms generated by internal recombination at these repeats . The functional organization of pKD1 genome appears to be quite analogous to that of the 2u plasmid of Saccharomyces cerevisiae . There is however little homology of sequences between these plasmids, except that the gene A has a dispersed but significant homology with the FLP recombinase gene of the 2u plasmid . S.cerevisiae cells can be transformed by derivatives of pKD1 carrying URA3 gene as a selection marker.

Plasmid, 1986 May, 15(3), 248 - 52
Analysis of a 1.6-micron circular plasmid from the yeast Kluyveromyces drosophilarum: structure and molecular dimorphism; Falcone C et al.; A new plasmid has been found in the yeast Kluyveromyces drosophilarum . It is a double-stranded circular DNA, 1.6 micron in length (4.8 kilobase pairs) . As in the case of Saccharomyces 2 mu circles, this plasmid occurs in two isomeric forms corresponding to the inversion of a segment between two 346-bp-long inverted repeats within the molecule . Each form has been separately cloned into bacterial plasmids . The new yeast plasmid, called pKD1, contains sequences that allow its replication in Saccharomyces cerevisiae.

Basic Life Sci, 1986, 40, 5 - 27
Mitochondrial introns as mobile genetic elements: the role of intron-encoded proteins; Dujon B et al.; Introns of organelle genes share distinctive RNA secondary structures that allow their classification into two known families . These structures are believed to play an essential role in splicing, and members of both structural classes have recently been shown to perform self-splicing reactions in vitro . In lower eukaryotes, many structured introns also contain long internal open reading frames (ORFs), which are able to code for hydrophilic proteins . Several properties of self-splicing structured introns suggest that they resemble mobile genetic elements, even though no actual transposition event involving these introns has yet been found . We report here on the characterization of two intron-encoded proteins that strongly support this attractive idea . First, we show that the class I intron of the 21S ribosomal RNA (rRNA) gene of Saccharomyces cerevisiae omega+ strains (rl intron) encodes a specific transposase . This protein has been partially purified from Escherichia coli cells that overexpress it from an artificial universal code equivalent to the rl intronic ORF . The omega transposase shows a double-strand endonuclease activity in vitro . This activity creates a 4-bp staggered cut with 3' OH overhangs within a specific sequence of the 21S rRNA gene of omega- strains . It is precisely within this sequence that the rl intron inserts by a duplicative transposition . Second, we report on the synthesis, in E . coli, of a putative reverse transcriptase encoded by the class II intron of the cytochrome b gene of Schizosaccharomyces pombe . This synthesis was obtained from E . coli expression vectors, using the class II intronic ORF linked to an artificial initiator sequence . As further support of the idea that structured introns are mobile, we show, from a systematic screening of introns in various yeast species, that the rl intron has transposed into the ATPase subunit 9 gene of Kluyveromyces fragilis . Structural features observed at the new intron homing site may be relevant to the transposition event.

Proc Natl Acad Sci U S A, 1985 Dec, 82(23), 7909 - 13
Construction of strains of Saccharomyces cerevisiae that grow on lactose; Sreekrishna K et al.; We have constructed strains of Saccharomyces cerevisiae that grow on lactose (Lac+) . S . cerevisiae strain YNN27, which, like all S . cerevisiae, is unable to grow on lactose, was transformed with pKR1B-LAC4-1 . This plasmid has a selectable marker gene conferring resistance to the antibiotic G418 and carries a 13-kilobase region of the Kluyveromyces lactis genome including LAC4, a beta-galactosidase gene . Transformants were selected first for G418 resistance and then for growth on lactose . Southern hybridization experiments showed that Lac+ transformants had integrated 15-25 tandem copies of the vector into a host chromosome . Several lines of evidence indicate that the Lac+ phenotype in pKR1B-LAC4-1-transformed S . cerevisiae is due to expression of a K . lactis lactose permease gene that lies between 2 and 8.6 kilobase upstream of LAC4 and also to expression of LAC4 . The permease gene has been designated LAC12.

Appl Environ Microbiol, 1985 Nov, 50(5), 1330 - 2
Identification and characterization of antimicrobial activity in two yeast genera; Bilinski CA et al.; A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera . Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue . Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited . No antibacterial activity was evident against four gram-negative bacteria used in this study . Optimal activities were found to be expressed after yeasts were grown at pH 6 . A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated.

Arch Microbiol, 1985 Sep, 142(4), 383 - 8
Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains; Braus G et al.; The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S . cerevisiae X2180-1A and in a Kluyveromyces marxianus strain . We could classify these strains into four groups, which did not correlate with their geographical distribution . In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes . Two strains were found which, in contrast to strain X2180-1A, show derepression of gene TRP1 . Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains.

Appl Environ Microbiol, 1985 Aug, 50(2), 257 - 60
Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp; Panchal CJ et al.; The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance . The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5 . Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source . Results indicated that the action of the K . lactis toxin was not mediated by catabolite repression in the sensitive strain . Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S . cerevisiae (RNA-plasmid-coded toxin) and K . lactis (DNA-plasmid-coded toxin) were tested . Three industrial polyploid yeasts sensitive to the S . cerevisiae killer yeast were resistant to the K . lactis killer yeast . The S . cerevisiae killer strain itself, however, was sensitive to the K . lactis killer yeast.

Ann Inst Pasteur Microbiol, 1985 Jul-Aug, 136B(1), 99 - 109
{Alcoholic fermentation of inulin by various strains of yeasts}; Poncet S et al.; Strains of fourteen species of yeasts able to ferment inulin without previous chemical or physical hydrolysis were studied on semi-synthetic medium by evaluation of CO2 production under anaerobic conditions . Among them, Kluyveromyces cicerisporus, Candida macedoniensis and Candida utilis showed the best kinetic characteristics of fermentation . Experiments were carried out to specify the action of different parameters such as temperature, pH and exogenous ethanol concentration . The results obtained on semi-synthetic medium were confirmed on Jerusalem artichoke juice . The optimal temperature for fermentation was 35 degrees C for the three strains, but K . cicerisporus alone conserved its fermentative capacity at 40 degrees C, at low pH (3.5) and with 6% (v/v) exogenous ethanol concentration . This strain appears to be a good yeast for industrial production of ethanol from inulin substrate.

EMBO J, 1985 Jul, 4(7), 1881 - 6
Hairpin plasmid--a novel linear DNA of perfect hairpin structure; Kikuchi Y et al.; The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed . The yeast Kluyveromyces lactis harbors two unique double-stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb . The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell . When the pGKL plasmids from K . lactis were transferred into Saccharomyces cerevisiae by transformation, non-killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained . F2 was shown to be a linear DNA arising from a 5-kb deletion of the right part of pGKL1 . F1 was an inverted dimer of F2 . Here we show that F2 has two different terminal structures: one end has a protein attached at the 5' terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure . This is a novel type of autonomously replicating DNA molecule.

EMBO J, 1985 Mar, 4(3), 793 - 8
A positive regulatory element is involved in the induction of the beta-galactosidase gene from Kluyveromyces lactis; Das S et al.; The regulation of the LAC4 gene encoding beta-galactosidase in the yeast Kluyveromyces lactis has been studied . The expression of cloned LAC4 gene present on autonomously replicating plasmids was normally regulated by lactose or galactose as inducers . The LAC4 transcription initiation sites were mapped on two plasmids, PTY75-LAC4 and pKL2 . The sites were found to be dependent on the level of gene expression and on the plasmid used . Under induced conditions, the normal cluster of initiation sites was used on both plasmids, whereas under non-induced conditions LAC4 on pKL2 showed additional sites . Deletion mapping of the 5' regulatory region of the LAC4 gene revealed a DNA element required for induction, presumably for the binding of a positive regulator.

Am J Clin Nutr, 1985 Feb, 41(2), 222 - 7
Effective in vivo hydrolysis of milk lactose by beta-galactosidases in the presence of solid foods; Solomons NW et al.; The feasibility of enzyme replacement therapy with exogenous, food-grade, microbial enzymes at mealtime to effect intragastrointestinal hydrolysis of the lactose from 360 ml of cow's milk consumed with a solid food meal (breakfast cereals) was investigated in adult Guatemalan lactose-malabsorbers using a hydrogen breath-analysis procedure to quantify the completeness of postprandial carbohydrate absorption . Adding 2 g of a commercial preparation of beta-galactosidase from Kluyveromyces lactis at mealtime to milk taken with a refined cereal (cornflakes) and an unrefined cereal (bran) reduced the production of excess breath H2 attributable to lactose maldigestion to a level not significantly different from that achieved with lactose-prehydrolyzed milk . Sucrase, as expected, had no effect on H2 production . A beta-galactosidase from Aspergillus niger was less effective that the K . lactis enzyme for in vivo hydrolysis . Thus, exogenous betagalactosidases can eliminate lactose malabsorption in lactase-deficient individuals even in the presence of solid foods, allowing lactose intolerant persons to consume milk and dairy products without gastrointestinal discomfort.

Am J Clin Nutr, 1985 Feb, 41(2), 209 - 21
Dietary manipulation of postprandial colonic lactose fermentation: II . Addition of exogenous, microbial beta-galactosidases at mealtime; Solomons NW et al.; The feasibility and efficacy of adding microbial beta-galactosidase enzymes directly to milk at the time of consumption was explored in adult lactose-malabsorbers . The hydrogen breath test, and on one occasion, the rise in blood glucose, were used as indices of the completeness of intraintestinal hydrolysis and absorption of milk lactose . When added to 360 ml of cow milk containing 18 g of lactose, empirical dosages of three beta-galactosidases--one from Kluyveromyces (yeast) and two from Aspergillus (fungal)--had some effectiveness in reducing postprandial H2 excretion, although no in vivo treatment at the dosages chosen was as effective as pre-incubation of the milk in vitro . The yeast enzyme also reduced symptom frequency as compared to intact milk and enhanced postprandial rises in blood glucose . The replacement therapy with exogenous, food-grade beta-galactosidases may provide a useful intervention to reduce lactose malabsorption and milk intolerance in individuals with primary lactase deficiency.

Antonie Van Leeuwenhoek, 1985, 51(1), 57 - 64
Antimycin A- and hydroxamate-insensitive respiration in yeasts; Lodi T et al.; In this paper evidence is presented for the mitochondrial localization of the antimycin A (AA) + salicylhydroxamate (SHAM)-insensitive respiration of the yeasts Kluyveromyces lactis, Endomycopsis capsularis and Hansenula saturnus . Such a respiration, which can be sustained by NADH and NADPH but not by succinate, is inhibited by high concentrations of azide . AA + SHAM-insensitive respiration is not phosphorylating and its postulated physiological role is to oxidize NADH.

Gene, 1985, 33(2), 207 - 13
Deletion analysis of a bacteriophage T4 late promoter; Volker TA et al.; We have conducted a BAL 31 unidirectional deletion analysis to determine whether the conserved consensus sequence found upstream of all sequenced phage T4 late genes represents the late T4 promoter or is only part of the promoter . The results confirm those of Elliott and Geiduschek {Cell 36 (1984) 211-219} that no sequences upstream from the consensus sequence are necessary for late transcription activity . In addition, they provide evidence that sequences downstream from the consensus sequence are important . We have also constructed a sequence that differs in several positions from the consensus but which still shows the properties of a late T4 promoter . Finally, we have noticed a remarkable homology between the consensus sequence for late T4 promoters and mitochondrial promoters from Saccharomyces cerevisiae and Kluyveromyces lactis.

Microbiol Sci, 1985, 2(4), 122 - 6
Current views on the yeast species; Lachance MA; The application of the biological species concept to the delineation of yeast species is becoming a reality . Studies centred on heterothallic Pichia and related species, and on homothallic species of Kluyveromyces have facilitated a better understanding of the concept of 'yeast species'.

Basic Life Sci, 1985, 30, 433 - 51
Linear plasmids with terminal inverted repeats obtained from Streptomyces rochei and Kluyveromyces lactis; Sakaguchi K et al.; Linear plasmids with inverted terminal repeats of 614 bp were obtained from Streptomyces rochei which produced lankacidin . The 5' ends were blocked by the association of a terminal protein . A DNA model of racket frame-like structure is presented implying the juxtaposition of 2 double-stranded DNAs of the same sequence through the binding of cohesive proteins which recognize and bind to the DNA . Two linear plasmids with the inverted terminal repeats of 202 and 184 bp were obtained from a yeast, Kluyveromyces lactis . A killer toxin was produced from the shorter plasmid . The protein toxin inhibited the adenylatecyclase activity of the yeast membrane.

Gastroenterology, 1984 Nov, 87(5), 1072 - 82
Enzyme replacement therapy for primary adult lactase deficiency . Effective reduction of lactose malabsorption and milk intolerance by direct addition of beta-galactosidase to milk at mealtime; Rosado JL et al.; The addition of microbial beta-galactosidases directly to milk at mealtime represents a potential "enzyme replacement therapy" for primary lactase deficiency . We used the hydrogen breath test as the index of incomplete carbohydrate absorption to assess the efficacy of two enzymes--one from yeast, Kluyveromyces lactis (LactAid), and the other from the fungus Aspergillus niger (Lactase N)--to assist in the hydrolysis of 18 g of lactose in 360 ml (12 oz) of whole milk when consumed by an adult lactose malabsorber . Graded amounts of Lactase N produced, at best, a 53% relative reduction in breath hydrogen excretion, whereas quantitative elimination of excess hydrogen excretion was produced by 1 and 1.5 g of LactAid . A double-blind, controlled, crossover trial was subsequently performed in 50 healthy, unselected Mexican adults, to whom 360 ml of cow's milk was presented in the three forms in a randomized order: intact milk, prehydrolyzed milk, and milk to which 1 g of LactAid was added immediately before consumption . Among the 25 subjects with incomplete carbohydrate absorption with intact milk, adding enzyme 5-min before consumption produced a 62% reduction in breath hydrogen excretion, and symptoms of intolerance were significantly reduced . The feasibility of effective enzyme replacement therapy with a beta-galactosidase from K . lactis is demonstrated.

Nucleic Acids Res, 1984 Oct 11, 12(19), 7581 - 97
Cloning and nucleotide sequences of the linear DNA killer plasmids from yeast; Hishinuma F et al.; The linear DNA killer plasmids (pGKL1 and pGKL2) isolated from a Kluyveromyces lactis killer strain are also maintained and expressed its killer character in Saccharomyces cerevisiae . After these killer plasmid DNAs isolated from S . cerevisiae were treated with alkali, four terminal fragments from each plasmid DNAs were cloned separately . Using these and other cloned DNA fragments, the terminal nucleotide sequences of pGKL2 and the complete nucleotide sequence of pGKL1 were determined . The inverted terminal repetitions of 202 bp and 182 bp were found in pGKL1 and pGKL2, respectively . The pGKL1 sequence showed an extremely high A + T content of 73.2% and it contained five large open reading frames . The largest of these open reading frame was suggested to code for a membrane-bound precursor of glycoprotein subunit of the killer toxin.

Nucleic Acids Res, 1984 Aug 10, 12(15), 6011 - 30
Nucleotide sequence and transcription analysis of a linear DNA plasmid associated with the killer character of the yeast Kluyveromyces lactis; Stark MJ et al.; In killer strains of the yeast Kluyveromyces lactis, production of a protein toxin which inhibits the growth of sensitive yeast cells is associated with the presence of two linear DNA plasmids, k1 and k2 . We have determined the nucleotide sequence of the smaller plasmid k1 (8.9kb) which is thought to carry the structural gene(s) encoding the toxin . The plasmid has a low G + C content (26.8%) and contains four long open reading frames which account for over 95% of the total sequence . The longest open reading frame (1146 amino acids) probably corresponds to a structural gene for the killer toxin . Transcripts from three of the putative genes have been detected in K.lactis by Northern hybridisation.

Appl Environ Microbiol, 1984 Aug, 48(2), 416 - 9
Survey of sensitivity of twelve yeast genera toward T-2 toxin; Sukroongreung S et al.; A survey was made to detect the sensitivity of 12 yeast genera to T-2 toxin . Seventy-five yeasts isolated from various sources were tested for their susceptibility to T-2 toxin . The MIC of T-2 for these yeasts varied from 1.0 to greater than 8.0 micrograms/ml . Of the yeasts studied, Kluyveromyces fragilis showed the greatest sensitivity, which ranged between 0.5 and 2.5 micrograms of T-2 toxin per ml of culture medium . The roles of incubation temperature, size of the inoculum, and incubation time on the MICs were determined . The results suggest that in comparison with other yeasts, K . fragilis is very sensitive to T-2 toxin.

J Bacteriol, 1984 Aug, 159(2), 533 - 9
Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae; Gunge N et al.; Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant {( rho0}) of Saccharomyces cerevisiae . However, when cytoplasmic components were introduced by cytoduction from a wild-type {( rho+}) strain of S . cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones . The phenomenon was ascribed to the incompatibility with the introduced S . cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by {rho0} mutations in the cytoductant cells . Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and {rho+} nonkiller cells lacking the plasmids . High-frequency transmission of the plasmids was observed in crosses lacking mtDNA {( rho0} by {rho0} crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome . In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids . Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in {rho0} cells of S . cerevisiae.

J Biol Chem, 1984 Jul 25, 259(14), 8718 - 23
Purification and phosphorylation of fructose-1,6-bisphosphatase from Kluyveromyces fragilis; Toyoda Y et al.; Fructose-1,6-bisphosphatase from the yeast Kluyveromyces fragilis was found to have an apparent Mr = 155,000 and to be composed of four Mr = 35,000 subunits . The extent and rate of phosphorylation of fructose-1,6-bisphosphatase (Fru-1,6-P2) by yeast cAMP-dependent protein kinase were dependent on fructose-1,6-bisphosphatase inhibitors, 5'-AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) . In the absence of inhibitor, the enzyme was slowly phosphorylated with a maximum incorporation of 1 mol of phosphate/mol of enzyme . The presence of both inhibitors greatly increased the phosphorylation rate with a maximum incorporation of 2 mol of phosphate/mol of enzyme . The presence of only one inhibitor led to an intermediate rate of phosphorylation with 2 mol of phosphate incorporated/mol of enzyme . There was no significant change in enzymatic activity after phosphorylation . The estimated sedimentation coefficient of fructose-1,6-bisphosphatase was lowered by 5'-AMP from 8.2 to 5.7 while Fru-2,6-P2 increased the S value to 8.5 . The presence of either Fru-1,6-P2 or Fru-2,6-P2 prevented the 5'-AMP lowering of S value . The susceptibility of enzyme to partial tryptic digestion was not changed by the presence of 5'-AMP . The presence of both Fru-2,6-P2 and 5'-AMP led to the protection of Mr = 35,000 subunit from tryptic digestion while Fru-2,6-P2 alone led to a protection of an Mr = 30,000 peptide fragment . This peptide fragment did not contain the phosphorylation sites . Our results suggest that the rapid regulation of fructose-1,6-bisphosphatase following glucose addition is controlled mainly by enzyme inhibitors.

Eur J Biochem, 1984 Jun 1, 141(2), 241 - 5
Characterization of a novel killer toxin encoded by a double-stranded linear DNA plasmid of Kluyveromyces lactis; Sugisaki Y et al.; A novel killer toxin, encoded by a double-stranded linear DNA plasmid pGK l-1 (5.4 MDa) in Kluyveromyces lactis IFO 1267 was purified 320 000-fold from the culture broth of yeast . The toxin was obtained in an electrophoretically homogeneous state with a yield of 24% by hydroxyapatite column chromatography, chromatofocusing and polyacrylamide gel electrophoresis . The purified toxin was dissociated into two subunits with molecular masses of 27 kDa and above 80 kDa, as estimated by Laemmli's sodium dodecylsulfate gel electrophoresis; the exact composition ratio of the two subunits remains unestablished . The isoelectric point was between 4.4 and 4.8 . As compared with the reported narrow pH range of action and instability of k1 killer toxin encoded by a double-stranded RNA plasmid of Saccharomyces cerevisiae, the K . Lactis toxin was effective with sensitive strains of S . cerevisiae in a relatively wider pH range between 4 and 8; it was stable for several months at pH 6.0 when stored below -20 degrees C . In contrast to the simple protein nature of the k1 killer toxin with a molecular mass of 11.47 kDa, the K . lactis toxin maintained a mannoprotein nature, as it was absorbed by a ConA-Sepharose column and eluted by methyl alpha-D-mannoside . The growth inhibitory activity of K . lactis toxin was enhanced 2-35-fold by the presence of 4-60% glycerol.

J Bacteriol, 1984 Jun, 158(3), 1165 - 7
Transformation of Kluyveromyces fragilis; Das S et al.; For the transformation of the yeast species Kluyveromyces fragilis, we have constructed a vector containing a bacterial kanamycin resistance (Kmr) gene, the TRP1 gene of Saccharomyces cerevisiae, and an autonomously replicating sequence of Kluyveromyces lactis called KARS2 . By utilizing the method based on treatment by alkali cations and with the Kmr gene as the selective marker, a wild-type strain of K . fragilis was transformed to resistance against the antibiotic G418 . In the transformed cell the plasmid replicates autonomously . The same plasmid could also be used to transform S . cerevisiae trp1 mutant to Trp+ . Thus, KARS2 of K . lactis enables the vector to replicate in K . fragilis, K . lactis, and S . cerevisiae, whereas ARS1 of S . cerevisiae allows autonomous replication only in S . cerevisiae.

Eur J Biochem, 1984 May 15, 141(1), 195 - 8
Catabolite repression in yeasts is not associated with low levels of cAMP; Eraso P et al.; relationship between levels of cAMP and catabolite repression in yeasts has been investigated . Strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Kluyveromyces fragilis were used . The yeasts were grown on different carbon sources to attain various degrees of repression . Galactose repressed as much as glucose, while maltose was less effective . Full derepression was achieved with ethanol . The enzymes tested were fructose-bisphosphatase, malate dehydrogenase, glutamate dehydrogenase (NAD dependent), cytochrome oxidase and isocitrate lyase (this last enzyme was found to be absent in Schizosaccharomyces) . The levels of cAMP were 2-3 times higher in the repressed conditions than in the derepressed ones . It is therefore concluded that in yeasts catabolite repression is not mediated by a lowering of the intracellular concentration of cAMP.

J Bacteriol, 1984 May, 158(2), 705 - 12
Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis; Riley MI et al.; We isolated and identified mutant strains of Kluyveromyces lactis that are defective for the Leloir pathway enzymes galactokinase, transferase, and epimerase, and we termed these loci GAL1 , GAL7 , and GAL10 , respectively . Genetic data indicate that these three genes are tightly linked, having an apparent order of GAL7 - GAL10 - GAL1 . This same gene order has been observed in Saccharomyces cerevisiae . Strains harboring gal7 mutations have elevated levels of beta-galactosidase, coded by an unlinked gene, galactokinase, and epimerase activities under uninduced conditions . We investigated the genetic basis of this constitutive gene expression and found no recombinants between the constitutive and Gal- phenotypes among 76 tetrads, suggesting that either GAL7 or a tightly linked gene codes for a regulatory function . This is the second gene that has been shown to specifically coregulate expression of the genes coding for beta-galactosidase and the Leloir pathway enzymes.

Gene, 1984 Apr, 28(1), 73 - 81
Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903; Sreekrishna K et al.; Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS) . To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol . Several ARS-containing plasmids were selected from a K . lactis recombinant DNA library in K . lactis and in Saccharomyces cerevisiae . Two of four ARS clones selected in K . lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA . This frequency of transformation was at least twice as high as with ARS clones selected in S . cerevisiae . The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug . In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype . Plasmids containing the ARS1 or 2 mu replicon of S . cerevisiae failed to transform K . lactis for G418 resistance . Inclusion of S . cerevisiae centromere, CEN4, in a K . lactis ARS recombinant plasmid did not increase the stability of the plasmid in K . lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis . We conclude that neither the ARS sequences or the centromere of S . cerevisiae was functioning in K . lactis.

Nucleic Acids Res, 1984 Mar 12, 12(5), 2327 - 41
Analysis of a eukaryotic beta-galactosidase gene: the N-terminal end of the yeast Kluyveromyces lactis protein shows homology to the Escherichia coli lacZ gene product; Breunig KD et al.; The LAC4 gene of Kluyveromyces lactis, encoding the enzyme beta-galactosidase was mapped on a cloned DNA fragment and the sequence of the 5' end was determined . This sequence includes the 5' regulatory region involved in the induction by lactose and the N-terminal end of the protein coding region . Comparison of the deduced amino acid sequence of this eukaryotic enzyme with the N-terminal end of the Escherichia coli beta-galactosidase revealed substantial homology . Two major RNA initiation sites were mapped at -115 and -105 . A number of structural peculiarities of the 5'non-coding region are discussed as in comparison to Saccharomyces cerevisiae genes.

Nucleic Acids Res, 1984 Jan 25, 12(2), 1137 - 48
A conserved sequence element is present around the transcription initiation site for RNA polymerase A in Saccharomycetoideae; Verbeet MP et al.; To identify DNA elements involved in the initiation of rRNA transcription in yeast we located the start site of the rRNA operon of Kluyveromyces lactis and Hansenula wingei, both members of the Saccharomycetoideae, by S1 nuclease analysis and determined the surrounding nucleotide sequences . Comparison of these sequences with those of Saccharomyces carlsbergensis, S . cerevisiae and S . rosei (all belonging to the same yeast subfamily) reveals an identical sequence at the site of transcription initiation from position +1 to +7 which is part of a larger conserved region extending from position -9 to +23; the conserved heptanucleotide sequence is supposed to constitute an important part of the promoter for yeast RNA polymerase A . The non-transcribed spacers (NTS) upstream of position -9 have diverged strongly with the exception of two short elements around positions -75 and -135 . The external transcribed spacer (ETS) downstream of position +23 is largely conserved between K . lactis, S . rosei and S . carlsbergensis except for a divergent region around position +75 . On the other hand, the ETS of H . wingei has diverged significantly.

Antonie Van Leeuwenhoek, 1984, 50(2), 167 - 75
Isolation of chloramphenicol-resistant mutants of Kluyveromyces lactis and characterization by mitotic segregation analysis of fused hybrids; Allmark BM; Chloramphenicol-resistant mutants of Kluyveromyces lactis were isolated following manganese mutagenesis . Mutants were characterized biochemically in order to determine the effect of CAP on respiration . The genetic basis of the resistance was determined using mitotic segregation analysis of fused hybrids where the transmission of mitochondrial genes from a parent in each parasexual cross was impaired by treatment with ethidium bromide.

Antonie Van Leeuwenhoek, 1984, 50(4), 349 - 53
The cell wall-associated inulinase of Kluyveromyces fragilis; Workman WE et al.; The yeast Kluyveromyces fragilis (ATCC 12424) was grown on a 2% inulin-1% yeast extract medium for 36 h and subsequently fixed with 0.5% glutaraldehyde . The glutaraldehyde treatment did not affect the beta-fructofuranosidase (inulinase, EC 3.2.1.7) activity of the cells but it did make the cells resistant to chemical and physical treatments that normally release beta-fructofuranosidase from untreated cells . The enzyme in the treated cells exhibited Km values for sucrose and raffinose identical to those obtained for the free enzyme . The cell wall of the treated cells exhibited the same diffusion properties for sucrose, raffinose, and inulin as those observed for untreated cells . The beta-fructofuranosidase was not bound covalently to the cell by the glutaraldehyde treatment . The results support the permeability barrier model for the enzyme retention in the yeast cell wall.

Mol Gen Genet, 1984, 195(1-2), 116 - 25
Evolution of yeast ribosomal DNA: molecular cloning of the rDNA units of Kluyveromyces lactis and Hansenula wingei and their comparison with the rDNA units of other Saccharomycetoideae; Verbeet MP et al.; We have studied the evolution of the yeast ribosomal DNA unit to search for regions outside the rRNA genes that exhibit evolutionary constraints and therefore might be involved in control of ribosome biosynthesis . We have cloned one complete rDNA unit of Kluyveromyces lactis and Hansenula wingei and established the physical and genetic organisation of both units . Both species belong to the subfamily of the Saccharomycetoideae . The lengths of the rDNA units of K . lactis and H . wingei are 8.6 and 11.1 kb respectively, and both comprise the 5S rRNA gene in addition to the large rRNA operon . Sequence conservation was monitored by restriction enzyme mapping as well as heteroduplex analysis of the two cloned rDNA units with S . carlsbergensis rDNA . These analyses showed that, phylogenetically, K . lactis is closer to S . carlsbergensis than H . wingei . The non-transcribed spacers (NTS) of both K . lactis and H . wingei have diverged completely from S . carlsbergensis; moreover in H . wingei the NTS are about double the length of these in the other two species . The transcribed spacers of both K . lactis and H . wingei contain conserved tracts . A homologous sequence of about 60 bp was found in the middle of the external transcribed spacer of H . wingei upon heteroduplexing with S . carlsbergensis rDNA, whereas the sequence at the transcription initiation site itself was insufficiently homologous to form a duplex . The sequence of the homologous region was determined both in H . wingei and K . lactis and compared with that of S . carlsbergensis . The function of this conserved element within the external transcribed spacer is discussed.

Mol Gen Genet, 1984, 195(1-2), 108 - 15
Cloning and expression of the structural gene for beta-glucosidase of Kluyveromyces fragilis in Escherichia coli and Saccharomyces cerevisiae; Raynal A et al.; Cellobiose, the last product in cellulose degradation, is converted into two molecules of glucose by a beta-glucosidase . S . cerevisiae does posses the structural gene for a beta-glucosidase, but it is very poorly expressed; we thus decided to isolate and characterize that of Kluyveromyces fragilis . We constructed in E . coli HB101 strain a genomic library of the Kluyveromyces fragilis Y610 strain (ATCC 12424), a yeast able to grow on cellobiose and which constitutively produces the beta-glucosidase . The structural gene for beta-glucosidase was identified by its expression in E . coli . The initial isolated cosmid KF1 contained an insert of 35 Kb and by successive subcloning the insert size was reduced to 3.5 Kb (KF4) . This cloned beta-glucosidase gene introduced in S . cerevisiae by transformation is expressed at a level of about 500 times that of K . fragilis . We checked by Southern hybridization that the high expression level was not due to a rearrangement of K . fragilis DNA during the cloning experiments . Nevertheless to obtain yeast transformants able to grow on cellobiose a yeast strain whose permeability to sugar is increased must be used and this last point is discussed.

Gene, 1983 Dec, 26(2-3), 243 - 52
Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903; Webster TD et al.; We have developed a new procedure for selecting yeast transformants without the need for complementing auxotrophic markers . The procedure is based on resistance to antibiotic G418 imparted to transformants by recombinant DNA vectors . We constructed several Escherichia coli-yeast shuttle vectors containing the kanamycin (G418)-resistance gene of Tn903, plus several yeast genes making dual selections possible . The efficiency for selecting G418-resistant transformants was dependent upon several factors including the composition of the growth medium and the time at which G418 selective pressure was administered . Media which contained levels of salts found in yeast nitrogen base rendered cells partially to completely resistant to G418 and could not be used for selecting G418-resistant transformants . On the other hand, untransformed cells remained sensitive to G418 when grown on YEPD medium thus allowing selection of G418-resistant transformants . A lag phase of 12 to 18 h, following growth at 30 degrees C, was required prior to administration of G418 to achieve maximal transformation frequency . Transformation frequencies ranged from 100 to 700 per micrograms of DNA and varied with the vector and strain used . The kanamycin gene imparted resistance to G418 in either the episomally or chromosomally integrated state . The gene was highly stable in the integrated state, even without selective pressure . The utility of the procedure was demonstrated by selecting transformants of four different strains of Saccharomyces cerevisiae and by cloning autonomous replication sequences (ARS) from the yeast Kluyveromyces lactis . We believe that this or related procedures could be used to develop transformation systems for many eukaryotic and prokaryotic cells for which no transformation procedure is available.

Can J Microbiol, 1983 Oct, 29(10), 1462 - 4
The occurrence of killer characters in yeasts; Rosini G; Strains of the genera Saccharomyces, Saccharomycodes, Schizosaccharomyces, Hanseniaspora, Kluyveromyces, Pichia, Kloeckera, and Torulopsis were examined for killer, sensitive, neutral, and killer-sensitive characteristics against a killer strain (NCYC738) and a sensitive strain (NYCC1006) of Saccharomyces cerevisiae . Thirty-one Saccharomyces cerevisiae strains out of 782 screened were able to kill the sensitive test strain; 707 appeared to be sensitive and 44 neutral . A high frequency of killer phenotypes (71.4%) was found in Hansenula anomala var . anomala strains . No killer strains were present in the remaining 17 species considered.

FEBS Lett, 1983 Aug 22, 160(1-2), 16 - 20
Purification and properties of the beta-fructofuranosidase from Kluyveromyces fragilis; Workman WE et al.; The beta-fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods . Two of the preparations were found to be impure by isoelectric focusing . This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme . The enzyme was found to be a glycoprotein, stable at 50 degrees C, with a pH optimum of 4.5 . The cations Hg2+, Ag+, Cu2+ and Cd2+ exhibited a marked inhibition of the enzyme . Competitive inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.

Nucleic Acids Res, 1983 Aug 11, 11(15), 5037 - 44
Inverted terminal repetitions of the two linear DNA associated with the killer character of the yeast Kluyveromyces lactis; Sor F et al.; The killer character of some Kluyveromyces lactis strains is associated with the presence of two linear double-stranded DNA, pGKl-1 (or k1) and pGKl-2 (or k2) . Nucleotide sequencing has revealed that each DNA has inverted terminal repetitions of about 200 base-pairs whose 5' ends seem to be blocked . The repetitions of the two DNA do not share extensive sequence homology . The role of these repetitions in the replication of killer DNA is discussed.

Nature, 1983 Aug 4-10, 304(5925), 464 - 6
Kluyveromyces lactis killer toxin inhibits adenylate cyclase of sensitive yeast cells; Sugisaki Y et al.; K1 killer toxin secreted by the K1 strain of Saccharomyces cerevisiae, has been well characterized . It is a simple protein of molecular weight (MW) 11,470 (ref . 3), encoded by a double-stranded, linear RNA plasmid, called M RNA, of MW 1.1-1.7 x 10(6) (refs 4-6) . It is lethal to sensitive Saccharomyces cerevisiae which does not carry M RNA . Leakage of K+ and ATP is the first distinct response in sensitive cells, and the toxic action is thought to be due to its action as a protonophore or K+ ionophore . Recently, a further killer toxin has been found in Kluyveromyces lactis IFO 1267, and it is associated with the presence of the double-stranded linear DNA plasmids, pGK1-1 (MW 5.4 x 10(6)) and pGK1-2 (MW 8.4 x 10(6)) . It has been shown, by curing pGK1-1 or deletion mapping, that the structural gene for the killer toxin and immunity-determining gene reside on the smaller plasmid . Moreover, the plasmids could be transferred from K . lactis to S . cerevisiae by protoplast fusion and protoplast transformation . As the K . lactis toxin is encoded by a DNA plasmid and has a relatively wider action spectrum than K1 killer toxin, the mode of action of the toxin is highly interesting . Here we report that K . lactis toxin inhibits adenylate cyclase in sensitive yeast cells and brings about arrest of the cells at the G1 stage.

J Bacteriol, 1983 Jun, 154(3), 1245 - 51
Characterization of lactose transport in Kluyveromyces lactis; Dickson RC et al.; We have determined that lactose uptake in Kluyveromyces lactis is mediated by an inducible transport system . Induction, elicited by lactose or galactose, of the transporter required protein synthesis . Transport of lactose required an energy-generating system and occurred by an active process, since an intracellular lactose concentration 175 times greater than the extracellular concentration could be obtained . The Km for lactose transport was about 2.8 mM in uninduced and lactose- or galactose-induced cells . The lactose transporters in K . lactis and Escherichia coli appear to be different since they respond uniquely to inhibition by substrate analogs.

J Bacteriol, 1983 May, 154(2), 737 - 42
Transformation of Kluyveromyces lactis by killer plasmid DNA; de Louvencourt L et al.; Some strains of Kluyveromyces lactis contain two linear double-stranded DNA plasmids, k1 and k2 . The presence of the two plasmids confer on the cell a "killer" character, due to the production of a toxin that kills the sensitive cells . We have used one of these linear DNA molecules as a gene vector to transform K . lactis cells . Hybrid plasmids containing parts of the k1 plasmid and the URA3 gene of Saccharomyces cerevisiae have been constructed . We have found that the hybrid plasmids were able to transform a uracil-requiring strain of K . lactis (uraA mutant) to a prototrophic form . The transformed phenotype cosegregated with the hybrid plasmids . The transforming plasmids contained the sequence of one or both ends of the linear k1 DNA, but they were integrated into a circular molecule.

J Cell Sci, 1983 May, 61, 339 - 49
Absence of step changes in activity of certain enzymes during the cell cycle of budding and fission yeasts in synchronous cultures; Creanor J et al.; Synchronous cultures prepared by selection from an elutriating rotor were used to measure activity changes during the cell cycle of the following enzymes: acid phosphatase in Schizosaccharomyces pombe and Saccharomyces cerevisiae, alpha-glucosidase in S . cerevisiae and beta-galactosidase in Kluyveromyces lactis . There was no sign of step rises in activity in acid phosphatase but there were indications in S . cerevisiae of the linear pattern with rate doublings once per cycle that had been found previously in S . pombe . There was also no sign of step rises in the other two enzymes, in contrast to earlier results using different techniques . Asynchronous control cultures showed little or no perturbations after the first hour.

Biochim Biophys Acta, 1983 Mar 9, 728(3), 363 - 70
Asymmetry of glucose transport in the yeast, Kluyveromyces lactis; Royt PW; Uptake and efflux of 6-deoxy-D-{3H}glucose and of 2-deoxy-D-{14C}glucose by the yeast Kluyveromyces lactis was studied . The tritiated, nonphosphorylatable hexose analogue leaves the cell in the absence and presence of intracellular 2-deoxy-D-glucose 6-phosphate . In energy-rich cells containing pools of hexose 6-phosphate, 2-deoxy-D-glucose is trapped in the cells, for it neither effluxes into glucose-free medium nor exchanges with external, free sugar . In starved, poisoned cells containing negligible amounts of 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucose does leave the cells upon transfer to glucose-free medium . An involvement of analogue structure and availability of metabolites of energy-rich cells in hexose retention is suggested . An internal pool of 6-deoxy-D-glucose does not affect the rate of uptake of 6-deoxy-D-{3H}glucose, nor does internal 2-deoxy-D-{14C}glucose 6-phosphate influence that rate . Hence, transport of glucose by this yeast is probably not regulated by internal pools of glucose 6-phosphate.

Mol Gen Genet, 1983, 192(3), 487 - 99
The intron of the mitochondrial 21S rRNA gene: distribution in different yeast species and sequence comparison between Kluyveromyces thermotolerans and Saccharomyces cerevisiae; Jacquier A et al.; We have screened numerous different yeast species for the presence of sequences homologous to the intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (intron r1) and found them in all Kluyveromyces species, some of the Saccharomyces species and none of the other yeasts tested . We have determined the nucleotide sequence of the r1-intron in K . thermotolerans and compared it with that of S . cerevisiae . The two introns are inserted at the same position within the 21S rRNA gene . They contain homologous internal open reading frames (ORFs) initiated at the same AUG codon which can be aligned over their entire length . Several silent multi-substitutions indicate that these intronic ORFs represent selectively conserved functional genes . Other intron segments, on the contrary, reveal short blocks of extensive homology separated by non-homologous stretches and/or additions-deletions . Comparison of our two yeast r1-introns with equivalent introns of N . crassa and A . nidulans mitochondria reveals that introns with very similar RNA secondary structures can accommodate different types of ORFs.

Annu Rev Microbiol, 1983, 37, 253 - 76
Yeast DNA plasmids; Gunge N; The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae . This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication . Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes . Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts . The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype) . This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes . The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector . The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

Nucleic Acids Res, 1982 Dec 20, 10(24), 7993 - 8006
A nonanucleotide sequence involved in promotion of ribosomal RNA synthesis and RNA priming of DNA replication in yeast mitochondria; Osinga KA et al.; We have examined the initiation of transcription of the mitochondrial genes for ribosomal RNA (rRNA) in the yeast Kluyveromyces lactis and show that these are transcribed independently from individual promoters . The mature large rRNA contains a 5' di- or triphosphate end which can be labelled in vitro with {alpha-32P}GTP using guanylyltransferase and this enabled us to determine the nucleotide sequence of its 5' terminus . For the small rRNA, a minor in vitro capped RNA species hybridizes in the region where--as judged from S1 nuclease protection experiments--the precursor of this RNA starts . We have determined the DNA sequence around the beginning of both rRNA genes and this reveals the existence of an identical nonanucleotide sequence (5' -ATATAAGTA- 3') just preceding the positions where the rRNAs start . This sequence is identical to the one preceding the rRNA genes in the mtDNA of the distantly related yeast Saccharomyces cerevisiae (Osinga, K.A . and Tabak, H.F . (1982) Nucl.Acids Res . 10, 3617-3626) and supports our proposal that this sequence motif is part of a yeast mitochondrial promoter . We have noticed that the same sequence is located in the putative origin of replication present in hypersuppressive petite mutants of S . cerevisiae and consider the possibility that this sequence is involved in RNA priming of DNA replication.

Biochimie, 1982 Oct, 64(10), 867 - 81
Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure; Michel F et al.; The complete sequences of nine Saccharomyces cerevisiae mitochondrial introns, six of which carry long open reading frames, have already been published . We have recently determined the sequence of an intron in the large ribosomal mitochondrial RNA of Kluyveromyces thermotolerans (Jacquier et al., in preparation), which we found to be closely related to its S . cerevisiae counterpart . This latter result prompted us to undertake a systematic search for possible homologous elements in the other, available sequences with the help of an original computer program . A previously unsuspected wealth of evolutionarily conserved sequences and secondary structures was thus uncovered . Seven at least of the available sequences may be folded up into elaborate secondary structure models, the cores of which are nearly identical . These models result in bringing together the exon-intron junctions into relatively close spatial proximity and looping out either all or most of the sequences in open reading frame, when present . These results and their possible implications with respect to the mechanism of splicing are discussed in the light of available genetic and biochemical data.

Appl Environ Microbiol, 1982 Sep, 44(3), 570 - 5
Isolation of fungi from bats of the Amazon basin; Mok WY et al.; A total of 2,886 bats captured in the Amazon Basin of Brazil were processed for the isolation of fungi . From the livers, spleens, and lungs of 155 bats (5.4%), 186 fungal isolates of the genera Candida (123 isolates), Trichosporon (26 isolates), Torulopsis (25 isolates), Kluyveromyces (11 isolates), and Geotrichum (1 isolate) were recovered . Seven known pathogenic species were present: Candida parapsilosis, C . guilliermondii, C . albicans, C . stellatoidea, C . pseudotropicalis, Trichosporon beigelii, and Torulopsis glabrata . Twenty-three culture-positive bats showed identical fungal colonization in multiple organs or mixed colonization in a single organ . The fungal isolation rates for individual bat species varied from 1 fungus per 87 bats to 3 fungi per 13 bats, and the mycoflora diversity for members of an individual fungus-bearing bat species varied from 16 fungi per 40 bats to 7 fungi per 6 bats . Of the 38 fungal species isolated, 36 had not been previously described as in vivo bat isolates . Of the 27 culture-positive bat species, 21 had not been previously described as mammalian hosts for medically or nonmedically important fungi.

J Bacteriol, 1982 Jul, 151(1), 462 - 4
Transformation of Saccharomyces cerevisiae with linear DNA killer plasmids from Kluyveromyces lactis; Gunge N et al.; Protoplasts of Saccharomyces cerevisiae were mixed with linear DNA plasmids, pGKl1 and pGKl2, isolated from a Kluyveromyces lactis killer strain and treated with polyethylene glycol . Out of 2,000 colonies regenerated on a nonselective medium, two killer transformants were obtained . The pGKl plasmids and the killer character were stably maintained in one (Pdh-1) of them . Another transformant, Pdl-1, was a weak killer, and the subclones consisted of a mixture of weak and nonkiller cells . The weak killers were characterized by the presence of pGKl1 in a decreased amount, and nonkillers were characterized by the absence of pGKl1 . The occurrence of two new plasmids which migrated faster than pGKl1 in an agarose gel was observed in Pdl-1 and its subclones, whether weak or nonkillers . Staining with 4',6-diamidino-2-phenylindole revealed that the pGKl plasmids exist in the cytosol of transformant cells with numerous copy numbers.

Biochim Biophys Acta, 1982 May 7, 687(2), 226 - 30
Glucose transport in nongrowing yeast; Royt PW; The inducible, nonenergy-requiring glucose transport system of the yeast Kluyveromyces lactis is inactivated upon starving cells of glucose by (1) transferring logarithmic phase glucose-grown cells to synthetic medium containing a nonglycolytic carbon source, and (2) upon transition of logarithmic phase glucose-grown cells to stationary phase . The steady-state accumulation of nonmetabolizeable 6-deoxyglucose and the apparent Km of transport of 6-deoxyglucose is the same in stationary phase cells and in logarithmic phase cells . The rate of transport is lower in the nongrowing cells . Restoration of activity requires energy and protein synthesis as well as inducer.

Biochemistry, 1982 Mar 30, 21(7), 1561 - 70
Purification of an alpha-N-acetylglucosaminyltransferase from the yeast Kluyveromyces lactis and a study of mutants defective in this enzyme activity; Douglas RH et al.; An enzyme activity in Kluyveromyces lactis that catalyzes the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine to alpha Man(1 leads to 3) alpha Man ( 1 leads to 2) alpha Man (1 leads to 2)Man to yield alpha Man(1 leads to 3) {alpha GlcNAc(1 leads to 2)} alpha Man(1 leads to 2) alpha Man (1 leads to 2)Man, a mannoprotein side-chain unit, has been solubilized by Triton X-100 and purified 18000-fold by a combination of ion-exchange chromatography, gel filtration, hydrophobic chromatography, and adsorption to a lectin column . The enzyme activity from a K . lactis mutant (mnn2-2) that made mannoprotein lacking N-acetylglucosamine in its side chains, but that possessed a normal level of transferase activity in cell extracts, was purified and compared with the enzyme from the wild-type strain . Both transferase activities are integral membrane proteins found in particles associated with endoplasmic reticulum . The two purified enzymes had the same apparent size, heat stability, Mn2+ requirement, and Km for donor and acceptor and a similar Vmax . Wild-type and mutant cells had similar pool sizes of sugar nucleotide donor, and they incorporated labeled N-acetylglucosamine into chitin at similar rates . No evidence was obtained for an inactive enzyme precursor in mutant cells that was activated upon breaking the cells, nor did the mutant cells contain a transferase inhibitor or a hexosaminidase that could remove the sugar from the mannoprotein during processing and secretion . The mnn2-2 locus appears to be allelic with a second mutant, mnn2-1, that has the same phenotype but that lacks transferase activity in cell extracts . This suggests that the two mutations affect the structural gene for the transferase, and we conclude that the mnn2-2 mutant could contain an altered enzyme that fails to function because it is improperly localized or oriented in the membrane.

J Bacteriol, 1981 Dec, 148(3), 988 - 90
Curing of the killer deoxyribonucleic acid plasmids of Kluyveromyces lactis; Niwa O et al.; Ultraviolet irradiation gave rise to frequent curing of killer plasmids pGKl1 and pGK12 of Kluyveromyces lactis . Almost all of the nonkillers obtained lost both plasmids, but one of them lost only pGKl1 . The disappearance of pGKl1 was accompanied by the simultaneous loss of the killer activity and of the resistance to the killer factor . A new plasmid, pGKl1S, was obtained, which arose from a deletion in the central region of pGKl1 . Genetic analysis suggested that pGKl1S has the killer gene lost by the deletion and the resistance gene intact and that pGKl1S shares the same replication control with pGKl1.

Mol Cell Biol, 1981 Nov, 1(11), 1048 - 56
Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid; Dickson RC et al.; Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness . These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene . In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type . The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation . Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation . Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely . Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription . Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport . It would appear that LAC10 regulates lactose-inducible proteins.

Genetics, 1981 Aug, 98(4), 729 - 45
Lac4 is the structural gene for beta-galactosidase in Kluyveromyces lactis; Sheetz RM et al.; Using genetic and biochemical techniques, we have determined that beta-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus . The following data support this conclusion: (1) mutations in this locus result in levels of beta-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac+ revertant, bearing lac4--14 and this unlinked suppressor, has subnormal levels of beta-galactosidase activity, and the Km for hydrolysis of o-nitrophenyl-beta, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of beta-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acrylamide gel electrophoresis shows that strains bearing lac4--23 and lac4--30 contain an inactive beta-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is observable in lac4--8, lac14--14 or lac4--29 mutants; (6) of all lac4 mutants, only those bearing lac4--23 or lac4--30 contain a protein that cross-reacts with anti beta-galactosidase antibody, a finding consistent with the previous result; and (7) beta-galactosidase activity in several Lac+ revertants of strains carrying lac4--23 or lac4--30 has greatly decreased thermostability.

Mol Cell Biol, 1981 Jul, 1(7), 629 - 34
Transcriptional regulation of the Kluyveromyces lactis beta-galactosidase gene; Lacy LR et al.; We examined the molecular basis for beta-D-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis . The protein synthesis inhibitor anisomycin effectively blocked both protein synthesis and enzyme induction by lactose . Further, hybridization analysis with the cloned beta-galactosidase gene indicated coordinate increases in the concentration of beta-galactosidase messenger ribonucleic acid and enzyme activity . The half-life of beta-galactosidase messenger ribonucleic acid was the same (4.8 +/- 0.4 min) when measured both before and at succeeding times during enzyme induction . These results strongly support the hypothesis that expression of the yeast beta-galactosidase gene is subject to transcriptional regulation.

Mikrobiologiia, 1981 Jul-Aug, 50(4), 598 - 602
{Assimilation of glucuronic and gluconic acids and ketogluconates by Torulopsis species}; Blagodatskaia VM et al.; Yeast strains (75) of the Torulopsis genus belonging to 42 species and varieties were studied . Most of them readily assimilated glucono-delta-lactone, with the exception of species that were imperfect analogues of the species Saccharomyces and Kluyveromyces . Only five of the species were capable of assimilating 5-keto-D-gluconate, and merely two of them, namely, T.ingeniosa and T . fragaria, assimilated D-glucuronic acid . These species have a basidiomycetic affinity in chemotaxonomic features.

J Bacteriol, 1981 Jul, 147(1), 155 - 60
Intergeneric transfer of deoxyribonucleic acid killer plasmids, pGKl1 and pGKl2, from Kluyveromyces lactis into Saccharomyces cerevisiae by cell fusion; Gunge N et al.; Two novel linear deoxyribonucleic acid plasmids, pGKl1 and pGKl2, were isolated from the yeast Kluyveromyces lactis . K . lactis strains harboring the pGK1 plasmids killed a certain group of yeasts, including Saccharomyces cerevisiae, Saccharomyces italicus, Saccharomyces rouxii, K . lactis, Kluyveromyces thermotolerans, Kluyvermyces vanudenii, Torulopsis glabrata, Candida utilis, and Candida intermedia . In this experiment, the pGKl1 and pGKl2 plasmids were intergenerically transferred from a K . lactis killer strain into a non-killer (killer-sensitive) strain of S . cerevisiae by the use of a protoplast fusion technique . Both of the pGKl plasmids replicated autonomously and stably in the new host cells of S . cerevisiae and could coexist with the resident 2-micrometers deoxyribonucleic acid plasmid . The S . cerevisiae cells which accepted the pGKl plasmids expressed the same killer phenotype as that of the donor K . lactis killer and became resistant to the K . lactis killer . The pGKl plasmids existing in the S . cerevisiae cells were cured by treatment with ethidium bromide, and the killer and resistance characters were simultaneously lost . From there results, it was concluded that both the killer and the resistance genes are located on the pGKl plasmids.

Can J Microbiol, 1981 May, 27(5), 550 - 3
Production of protoplasts in different yeasts by mutanase; Stephen ER et al.; Mutanase (Mutanase Novo) affects the high frequency production of protoplasts in the following strains of yeast: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, and Schwanniomyces alluvius . Regeneration frequencies varied with the strain used and ranged between 10 and 18% . This enzyme preparation appears to be a very useful means of obtaining protoplasts from a wide variety of yeasts currently being used for experimental purposes.

Antonie Van Leeuwenhoek, 1981 Mar, 47(1), 11 - 24
Induction by glucose of an antimycin-insensitive, azide-sensitive respiration in the yeast Kluyveromyces lactis; Ferrero I et al.; Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (QO2 of 80 microliter O2 . h-1 . mg-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 microliter O2 . h-2 . mg-1 dry weight . Antimycin A inhibits the growth of K . lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced . It can be concluded that a minimal antimycin-insensitive QO2 is necessary for cellular growth when the normal respiratory pathway is not functional . The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 microM azide or 1 mM cyanide . These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements . A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells . The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.

Microbios, 1981, 30(119), 37 - 45
The respiration pathways of wild-type and petite mutants of Kluyveromyces lactis; Heritage J et al.; The existence of two alternative respiration pathways in Kluyveromyces lactis has been confirmed . Both pathways may be demonstrated in petite mutants of this yeast, suggesting that the major components in these pathways have an extramitochondrial origin . The antimycin A insensitive/sodium azide insensitive pathway (pathway I) is abolished by glucose repression of this yeast, whereas the antimycin A insensitive/sodium azide sensitive pathway (pathway II) is unaffected by this treatment . In K . lactis wild-type cells, the antimycin A insensitive/sodium azide sensitive respiration pathway (pathway II) is inhibited by salicyl hydroxamic acid, and this inhibition may be overcome upon addition of exogenous Fe3+ ions . This pathway in petite cells is unaffected by the presence of this inhibitor . The possible reasons for this are discussed.

Folia Microbiol (Praha), 1981, 26(2), 147 - 50
Selection of yeast strains for ethanol production from inulin; Guiraud JP et al.; Of the many yeast species capable of fermenting inulin, some can produce sufficient amounts of ethanol from the substrate, in particular Kluyveromyces fragilis and Torulopsis colliculosa . The results indicate the feasibility of producing ethanol from inulin-rich plants, such as Jerusalem artichoke.

Folia Microbiol (Praha), 1981, 26(5), 370 - 6
Production of beta-galactosidase from Kluyveromyces fragilis grown on whey; Sonawat HM et al.; Optimum conditions for beta-galactosidase production by K . fragilis were studied . Enzyme production has a maximum after 8-12 h of incubation . Composition of whey (from different sources) did not affect enzyme production . Different heart treatments also had no effect . Whey reconstituted to 8-12% total solids and adjusted to pH 4.0 afforded maximum enzyme production . Whereas inorganic nitrogen sources (specially ammonium salts) only slightly stimulated enzyme production, organic nitrogen sources (specially partially digested proteins) provided a nearly four-fold increase in enzyme production . Yeast extract and beef extract and industrial by-products like corn-steep liquor significantly stimulated enzyme production . Manganese and magnesium salts had a very little stimulation effect.

J Bacteriol, 1981 Jan, 145(1), 382 - 90
Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character; Gunge N et al.; Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267 . pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively . Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids . A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI . pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K . lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K . lactis, Kluyveromyces thermotolerans, and K . vanudenii . All K . lactis strains lacking the pGK1 plasmids were nonkillers . A hybrid was constructed between K . lactis IFO 1267 and a nonkiller K . lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation . The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids . The double-stranded ribonucleic acid killer plasmid could not be detected in any K . lactis killer strains . It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids . A single chromosomal gene was found which was responsible for the resistance to the K . lactis killer.

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