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R C Dickson

Publications and source records attributed to R C Dickson.

At least 91 records · Page 5Linked to original sources
PubMed1987

Isolation and characterization of mutants of Kluyveromyces lactis defective in lactose transport.

Mutants of Kluyveromyces lactis defective in lactose transport were identified among lactose-resistant revertants of lactose-sensitive strains. The mutations are closely linked to the beta-galactosidase gene, LAC4, and they are located in a previously identified gene, LAC12, which has been shown to code for a lactose permease. Our data establish that LAC12 is the only lactose permease gene in K. lactis. The lactose permease also transports galactose. LAC12 is transcribed in a direction opposite to that of LAC4, there being about 2.5 kb between their transcription start sites. Transcription of LAC12 is inducible as is that of all other structural genes in the lactose-galactose regulon of K. lactis.

Biological Transport↗
PubMed1987

Cloning and characterization of Saccharomyces cerevisiae genes that confer L-methionine sulfoximine and tabtoxin resistance.

Pseudomonas tabaci produces a toxin, tabtoxin, that causes wildfire disease in tobacco. The primary target of tabtoxin is presumed to be glutamine synthetase. Some effects of tabtoxin in tobacco can be mimicked by methionine sulfoximine (MSO), a compound that is known to inactivate glutamine synthetase. To understand how organisms can be made resistant to tabtoxin and MSO, we used Saccharomyces cerevisiae. We demonstrate that yeast strains carrying the glutamine synthetase gene, GLN1, on a multicopy plasmid overproduced glutamine synthetase and showed increased drug resistance. These and other data indicate that glutamine synthetase is the primary target of tabtoxin and MSO in S. cerevisiae. We also isolated three S. cerevisiae DNA inserts of 2.1, 2.3, and 2.8 kilobases that conferred tabtoxin and MSO resistance when the inserts were present on a multicopy plasmid. These plasmids conferred resistance to MSO by blocking intracellular transport of the drug. Transport appeared to occur by one or more methionine permeases. Resistance to tabtoxin could also occur by blockage of intracellular transport, but the drug was transported by some permease other than a methionine permease. These drug resistance plasmids did not block transport of citrulline, indicating that they did not affect the general amino acid permease.

Biological Transport↗
PubMed1987

Identification of upstream activator sequences that regulate induction of the beta-galactosidase gene in Kluyveromyces lactis.

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

DNA, Fungal↗
PubMed1987

GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon.

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

Enzyme Induction↗
PubMed1987

Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae.

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

Base Sequence↗
PubMed1985

Construction of strains of Saccharomyces cerevisiae that grow on lactose.

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

Biological Transport, Active↗
PubMed1984

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903.

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

DNA Transposable Elements↗
PubMed1984

Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis.

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

Ascomycota↗
PubMed1983

Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903.

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

DNA, Recombinant↗
PubMed1983

Characterization of lactose transport in Kluyveromyces lactis.

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

2,4-Dinitrophenol↗
PubMed1981

Lac4 is the structural gene for beta-galactosidase in Kluyveromyces lactis.

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

Enzyme Induction↗
PubMed1981

Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid.

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

Chromosome Mapping↗
PubMed1981

Transcriptional regulation of the Kluyveromyces lactis beta-galactosidase gene.

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

Anisomycin↗
PubMed1980

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: beta-galactosidase from Kluyveromyces lactis.

Three recombinant DNA vectors carrying the beta-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the beta-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded beta-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However S. cerevisiae transformed with LAC4 cannot grow on lactose, probably because lactose does not enter the cell.

Cloning, Molecular↗
PubMed1980

Mutations affecting synthesis of beta-galactosidase activity in the yeast Kluyveromyces lactis.

Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac-) were isolated and characterized. All the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9. Strains bearing mutations in lac3, lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal-). Since the Gal- and Lac- phenotype co-segregate, they are probably due to a single mutation. Strains bearing mutations in any of the seven complementation groups grow normally on glucose. However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium. Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose. Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate chromosome 2. The remaining five groups are unlinked. Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.--To further characterize the nature of the Lac- phenotype, the basal and inducible level of beta-galactosidase activity were measured. All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels. Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups. In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells. Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of beta-galactosidase. In summary, these data indicate that several genes are necessary for synthesis of beta-galactosidase activity.

Enzyme Activation↗
PubMed1980

Physiological studies of beta-galactosidase induction in Kluyveromyces lactis.

We examined the kinetics of beta-galactosidase (EC 3.2.1.23) induction in the yeast Kluyveromyces lactis. Enzyme activity began to increase 10 to 15 min, about 1/10 of a cell generation, after the addition of inducer and continued to increase linearly for from 7 to 9 cell generations before reaching a maximum, some 125- to 150-fold above the basal level of uninduced cells. Thereafter, as long as logarithmic growth was maintained, enzyme levels remained high, but enzyme levels dropped to a value only 5- to 10-fold above the basal level if cells entered stationary phase. Enzyme induction required the constant presence of inducer, since removal of inducer caused a reduction in enzyme level. Three nongratuitous inducers of beta-galactosidase activity, lactose, galactose, and lactobionic acid, were identified. Several inducers of the lac operon of Escherichia coli, including methyl-, isopropyl- and phenyl-1-thio-beta-d-galactoside, and thioallolactose did not induce beta-galactosidase in K. lactis even though they entered the cell. The maximum rate of enzyme induction was only achieved with lactose concentrations of greater than 1 to 2 mM. The initial differential rate of beta-galactosidase appearance after induction was reduced in medium containing glucose, indicating transient carbon catabolite repression. However, glucose did not exclude lactose from K. lactis, it did not cause permanent carbon catabolite repression of beta-galactosidase synthesis, and it did not prevent lactose utilization. These three results are in direct contrast to those observed for lactose utilization in E. coli. Furthermore, these results, along with our observation that K. lactis grew slightly faster on lactose than on glucose, indicate that this organism has evolved an efficient system for utilizing lactose.

Ascomycota↗
PubMed1979

Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis.

beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose. When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed. The enzyme has a sedimentation coefficient of 9.6S. This beta-galactosidase and the one from Escherichia coli are not antigenically related. Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent. beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively. The hydrolase and transgalactosylase activities of the enzyme are similar to those of E. coli beta-galactosidase.

Antigens, Fungal↗