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