Biomedical subjects

J Marmur

Publications and source records attributed to J Marmur.

At least 37 records · Page 2Linked to original sources

PubMed1988

Localization of yeast glucoamylase genes by PFGE and OFAGE.

Chromosomes of two closely related yeast strains, the amylolytic Saccharomyces diastaticus and the non-amylolytic Saccharomyces cerevisiae, were resolved by pulsed field gel electrophoresis (PFGE) and orthological field alteration gel electrophoresis (OFAGE). Electrophoretic karyotypes of these two strains are identical. Sixteen cloned Saccharomyces genes of known chromosomal location were used to identify individual chromosomes by Southern hybridization analyses. The Southern blots were reprobed with a cloned fragment of the STA2 glucoamylase gene of S. diastaticus. STA2 exhibits homology to STA1 and STA3 as well as the sporulation-specific glucoamylase (SGA) gene from both Saccharomyces strains. The three unlinked, homologous genes, STA1 (DEX2, MAL5), STA2 (DEX1) and STA3 (DEX3) encoding the extracellular glucoamylase isozymes GAI, GAII and GAIII in S. diastaticus were then assigned to chromosomes IV, II and XIV, respectively. The SGA gene, encoding an intracellular glucoamylase in both S. diastaticus and S. cerevisiae, was assigned to chromosome IX. Electrophoretic mapping of the STA and SGA genes is at present the only way to localize these genes, since glucoamylase repressor gene(s) (STA10, INH1 and/or IST2) are present in most laboratory strains of S. cerevisiae and the SGA phenotype is only detectable during sporulation.

Chromosome Mapping↗

PubMed1987

Regulation of MAL gene expression in yeast: gene dosage effects.

Both the MAL1 and MAL6 loci in Saccharomyces strains have been shown by functional and structural studies to comprise a cluster of at least three genes necessary for maltose utilization. They include regulatory, maltose transport and maltase genes designated MALR, MALT and MALS, respectively. Subclones of each gene derived from the MAL6 locus were inserted into the multicopy shuttle plasmid YEp13, introduced into MAL1 and mal1 strains and the effects of altered gene dosage of each gene, or a combination of them, on MAL gene expression investigated. MAL1 strains transformed with a plasmid carrying the MAL6S gene showed coordinate four to five fold increases in both maltase enzyme activity and its mRNA, whereas no increase in maltose transport activity or of MALT mRNA was observed when MAL6T was present on multicopy plasmids. The presence of the MAL6R gene on a multicopy plasmid led to greatly increased transcription of both inducible and constitutive mRNAs with homology to the regulatory gene; it also gave rise to two fold increases in both induced maltase mRNA levels and enzyme activity, but only in the presence of maltose. However, it had no apparent effect on the accumulation of MALT mRNA. Finally, the induction kinetics of plasmid-borne and chromosomal MALS and MALT gene expression were examined under conditions of altered gene dosage of the MAL6 regulatory and structural genes. The results of these experiments indicate that MALR encodes a trans-acting positive activator that requires maltose for induction of MALS and MALT transcription even when the regulatory gene is present on a multicopy plasmid. Maltose transport can be a rate-limiting factor in MAL gene expression, at least in the early stages of induction. The regulation of the MALS and MALT genes, whose activities are coordinately induced in MAL1 strains by maltose, may in fact exhibit some important differences.

Escherichia coli↗

PubMed1987

Upstream regulatory regions controlling the expression of the yeast maltase gene.

The expression of the maltase (MALS) and the maltose permease (MALT) genes in Saccharomyces species is coregulated at the transcriptional level; they are coordinately induced by maltose in the presence of a positively acting regulatory (MALR) gene and carbon catabolite repressed by glucose. We generated a series of deletions in the upstream region of the MAL6S gene to examine the regulatory elements in detail. The results showed that inducible expression by maltose was lost when the region between 320 and 380 base pairs upstream of the translation initiation codon was deleted. This region contained an imperfect inverted repeat sequence (-361 to -327) or four copies of short direct repeats that might serve as components of the upstream activation site (UASM) for the maltase gene, or both. When a stretch of T-rich sequence (-253 to -237) was deleted, the susceptibility of the maltase gene to carbon catabolite repression was affected.

Chromosome Deletion↗

PubMed1986

Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus.

The Saccharomyces diastaticus structural gene STA2, encoding an exracellular glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of a stao strain. A genomic library was initially constructed from a STA2 yeast strain in the yeast Escherichia coli shuttle cosmid vector pYCl. The Sta+ complementing function was further delimited to an 8.3 kb BglII fragment whose restriction map was found to be similar to related genomic regions of STA1 and STA3. Fusions of several DNA fragments derived from the 8.3 kb BglII fragment with a truncated E. coli beta-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins in E. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+ complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of the STA1, STA2 and STA3 RNA transcripts by RNA-DNA hybridization using an internal fragment of the cloned STA2 gene as the probe indicated that a common transcript of 2.5 kb is produced by each of the STA genes. Integrative disruption of the STA2 gene through homologous recombination was achieved by transforming a STA2 yeast strain to Sta- using an in vitro constructed donor DNA fragment that has the URA3 gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disrupted STA2 and a functional STA2. Southern blot analysis using BamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta- phenotype with STA2::URA3. These data further confirm that the cloned DNA that showed Sta+ complementing activity carries a functional STA2 gene that encodes the yeast extracellular glucoamylase II.

Cloning, Molecular↗

PubMed1986

Identification and physical characterization of yeast glucoamylase structural genes.

Each one of at least three unlinked STA loci (STA1, STA2 and STA3), in the genome of Saccharomyces diastaticus controls starch hydrolysis by coding for an extracellular glucoamylase. Cloned STA2 sequences were used as hybridization probes to investigate the physical structure of the family of STA genes in the genomes of different Saccharomyces strains. Sta+ strains, each carrying a single genetically defined STA locus, were crossed with a Sta- strain and the segregation behavior of the functional locus (i.e. Sta+) and sequences homologous to a cloned STA2 glucoamylase structural gene at that locus were analyzed. The results indicate that in all strains examined there is a multiplicity of sequences that are homologous to STA2 DNA but that only the functional STA loci contain extensive 5' and 3' homology to each other and can be identified as residing on unique fragments of DNA; that all laboratory yeast strains examined contain extensive regions of the glucoamylase gene sequences at or closely linked to the STA1 chromosomal position; that the STA1 locus contains two distinct glucoamylase gene sequences that are closely linked to each other; and that all laboratory strains examined also contain another ubiquitous sequence that is not allelic to STA1 and is nonfunctional (Sta-), but has retained extensive sequence homology to the 5' end of the cloned STA2 gene. It was also determined that the DEX genes (which control dextrin hydrolysis in S. diastaticus), MAL5 (a gene once thought to control maltose metabolism in yeast) and the STA genes are allelic to each other in the following manner: STA1 and DEX2, STA1 and MAL5, and STA2 and DEX1 and STA3 and DEX3.

Cloning, Molecular↗

PubMed1986

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus.

In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.

Antibodies, Monoclonal↗

PubMed1986

Primary structure of the maltase gene of the MAL6 locus of Saccharomyces carlsbergensis.

We have determined the complete nucleotide (nt) sequence of a 2937-bp DNA fragment containing the yeast maltase (EC 3.2.1.20) gene (MAL6S) as well as part of the contiguous maltose permease gene (MAL6T) from the MAL6 locus of Saccharomyces carlsbergensis. The MAL6S gene encodes an alpha-glucosidase that is required for the utilization of maltose as a carbon source by yeast. The 5' transcription initiation sites for both MAL6S and MAL6T were determined by primer extension experiments using reverse transcriptase. The sequence data show one major open reading frame (ORF) of 584 amino acids (aa) for maltase with a calculated Mr of 68 107, somewhat larger than the value of 63 000 previously determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis. The nucleotide sequences upstream of both the MAL6S and MAL6T genes, which are divergently transcribed, show common structural features for the transcription initiation of yeast genes as well as signals required for their translation. The codon bias index shows that the MAL6S gene is moderately expressed. The possible significance of two 17-bp dyad symmetric sequences, found in the intergenic region of MAL6S and MAL6T, for the control of expression of these genes is also discussed.

Amino Acid Sequence↗

PubMed1986

Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species.

Three unlinked, homologous genes, STA1, STA2, and STA3, encode the extracellular glycosylated glucoamylase isozymes I, II, and III, respectively, in Saccharomyces species. S. cerevisiae, which is sta0 (absence of functional STA genes in haploids), does carry a glucoamylase gene, delta sta, expressed only during sporulation (W. J. Colonna and P. T. Magee, J. Bacteriol. 134:844-853, 1978; I. Yamashita and S. Fukui, Mol. Cell. Biol. 5:3069-3073, 1985). In this study we examined some of the physiological and genetic factors that affect glucoamylase expression. It was found that STA2 strains grown in synthetic medium produce glucoamylase only in the presence of either Maltrin M365 (a mixture of maltooligosaccharides) or starch. Maximal levels of glucoamylase activity were found in cells grown in rich medium supplemented with glycerol plus ethanol, starch, or Maltrin. When various sugars served as carbon sources they all supported glucoamylase synthesis, although at reduced levels. In any given growth medium glucoamylase isozyme II synthesis was modulated by functionality of the mitochondria. Synthesis of glucoamylase is continuous throughout the growth phases, with maximal secretion taking place in the early stationary phase. In the various regimens, the differences in enzyme accumulation are accounted for by differences in the levels of glucoamylase mRNA. Both glucoamylase mRNA and enzyme activity were drastically and coordinately inhibited in MATa/MAT alpha diploids and by the presence of the regulatory gene STA10. Both effects were partially overcome when the STA2 gene was present on a multicopy plasmid. The STA2 mRNA and glucoamylase were coinduced in sporulating STA2/STA2 diploids. A smaller, coinduced RNA species was also detected by Northern blotting with a STA2 probe. The same mRNA species was detected in sporulating sta0 diploids and is likely to encode the sporulation-specific glucoamylase.

Culture Media↗

PubMed1985

Organization of the MAL loci of Saccharomyces. Physical identification and functional characterization of three genes at the MAL6 locus.

We have physically and functionally identified three genes at the MAL6 locus of Saccharomyces carlsbergensis. Using multicopy yeast plasmid vectors, we have subcloned various segments of the entire MAL6 locus. The functional characterization of the MAL6 subcloned regions was determined by (1) analyzing biochemically the levels of MAL-encoded proteins (maltase [alpha-D-glucosidase, E.C. 3.2.1.20] and maltose transport protein) in cells transformed with various MAL6 subclones, and (2) testing the ability of the subclones to complement the maltose fermentation defects of well characterized Mal- mutants in the highly homologous MAL1 locus. The physical homology between MAL6 and MAL1 is in part demonstrated by the gene disruption of MAL1 using subcloned MAL6 DNA sequences. The results demonstrate that the MAL6 locus is a complex of at least three genes: MAL6R, MAL6T and MAL6S. These genes specify, respectively, a regulatory function, a maltose transport activity (presumably the maltose permease) and the structural gene for maltase. The functional organization of the MAL6 locus is thus identical to that which we had previously determined by mutational analysis for the MAL1 locus.

ATP-Binding Cassette Transporters↗

PubMed1984

Mutational analysis of the MAL1 locus of Saccharomyces: identification and functional characterization of three genes.

Fermentation of maltose by Saccharomyces strains depends on the presence of any one of five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 or MAL6). Earlier mutational analyses of MAL2 and MAL6 containing strains have identified a single complementation group at each of these two loci. However complementation analysis between naturally occurring Mal- Saccharomyces strains isolated from the wild demonstrated the presence of two complementation groups (designated MALp and MALg) at the MAL1, MAL3 and MAL6 loci. The available evidence suggests that the MALp gene is functionally equivalent to the complementation group identified by mutational analysis at the MAL6 locus and that this gene encodes a protein involved in the regulation of the coordinate induction of both maltase and maltose permease synthesis. In this paper we report the isolation, in a well characterized MAL1 strain, of 47 mutants unable to ferment maltose. All the mutants, with one exception, map at the MAL1 locus. These mal1 mutants, except for one, are recessive to MAL1 and fall into two major complementation groups. Evidence is presented that these two classes of mutants identify both a gene involved in the regulation of maltose identify both a gene involved in the regulation of maltose fermentation (MAL1R) and a gene involved in maltose transport (MAL1T). We also report here the isolation of a temperature sensitive maltose nonfermenting mutant mapping at the MAL1 locus identifying a third gene (MAL1S) at this locus. The maltase synthesized by this mutant, when assayed in cell-free extracts, is significantly more thermolabile than the wild type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Fermentation↗

PubMed1983

Identification and physical characterization of yeast maltase structural genes.

Each of at least five unlinked MAL loci (MAL1 through MAL4 and MAL6) on the yeast genome controls the ability to synthesize an inducible alpha-D-glucosidase (maltase). A subcloned fragment of the coding sequence of the MAL6 maltase structural gene was used as a hybridization probe to investigate the physical structure of the family of MAL structural genes in the genomes of different Saccharomyces strains. MAL+ strains, each carrying a genetically defined MAL locus, were crossed with a MAL- strain and the segregation behavior of the functional locus and of sequences complementary to the maltase structural gene at that locus analyzed. The maltase structural gene sequences of each MaL locus were detected by Southern blot hybridization using BamH1 digests of genomic DNA of the meiotic products. This restriction enzyme was previously shown to cleave outside the confines of the MAL 6 locus. The results of such experiments indicate that each MAL locus encompasses at least one maltase structural gene sequence homologous to that of MAL6, that yeast strains that lack functional MAL loci may or may not contain the corresponding maltase structural gene sequence, that the MAL1 maltase structural gene sequence or one of its alleles can be detected in all laboratory yeast strains examined and that each MAL locus can be identified as a characteristic BamH1 fragment of genomic DNA which includes a maltase structural gene. Yeast strains vary in the number of maltase structural gene sequences that they carry. By using the approach described in this report, the ones corresponding to the different functional MAL loci and residing within a BamH1 generated restriction fragment can be identified.

DNA, Fungal↗

PubMed1983

Expression of a prokaryotic gene in yeast: isolation and characterization of mutants with increased expression.

The Escherichia coli Tn9 derived chloramphenicol resistance gene (camr) is functionally expressed in the yeast Saccharomyces cerevisiae. This gene was introduced into yeast cells as part of a hybrid yeast/E. coli shuttle plasmid. A number of plasmid associated yeast mutants overproducing the camr gene product, chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-0-acetyltransferase, E.C. 2.3.1.28) were isolated. One of the plasmid mutants was analyzed in some detail. Even though this mutant showed a 1,000 fold overproduction of chloramphenicol acetyltransferase in the yeast host the level of RNA complementary to the camr gene was not increased. A deletion of 127 base pairs in the region immediately upstream from the 5' end of the camr gene appeared to be responsible for the "up" phenotype of this mutant. This mutation affected the expression of the camr gene in E. coli in a "down" fashion, in contrast to its effect in yeast.

Acetyltransferases↗

PubMed1983

Regulation of maltase synthesis in Saccharomyces carlsbergensis.

The induction of maltase (EC 3.2.1.20) by its inducer maltose in a strain of the yeast Saccharomyces carlsbergensis carrying a functional MAL locus is regulated at the level of transcription. Preceding the synthesis of increased levels of maltase is the de novo synthesis of maltase-specific RNA sequences. This was detected by determining the level of maltase mRNA by DNA-RNA hybridizations by using a maltase structural gene DNA sequence probe and by assaying functional maltase mRNA by in vitro RNA-directed synthesis of immunologically reactive maltase. Once maltase has accumulated, late in induction, further synthesis of the enzyme is inhibited, as reflected by reduced levels of the mRNA that encodes maltase.

Enzyme Induction↗

PubMed1983

Carbon catabolite repression of maltase synthesis in Saccharomyces carlsbergensis.

Carbon catabolite repression of maltase gene expression is brought about by the addition of glucose, resulting in a drastic inhibition of the induction of maltase. When added to induced cells, glucose leads to the inhibition of maltase synthesis within 30 min, which can be accounted for by the disappearance of hybridizable maltase RNA sequences. The loss of maltase-specific RNA due to catabolite repression can be traced to the combined effects of a 15-fold decrease in the rate of transcription of the maltase structural gene 15 to 20 min after the addition of glucose and a change in the half-life of maltase mRNA. However, the stability of maltase, once induced, is not affected by the addition of glucose.

Enzyme Repression↗

PubMed1982

Isolation of a maltase structural gene from Saccharomyces carlsbergensis.

The maltase structural gene MAL6 of the yeast Saccharomyces carlsbergensis has been cloned by transformation of a maltose nonfermenting recipient strain with autonomously replicating chimeric recombinant plasmids. One recombinant plasmid, pMAL26, was shown by positive hybridization translation, as well as by Southern and Northern blot experiments, to carry the MAL6 structural gene.

Cloning, Molecular↗

PubMed1980

Functional expression in yeast of the Escherichia coli plasmid gene coding for chloramphenicol acetyltransferase.

The Escherichia coli R factor-derived chloramphenicol resistance (camr) gene is functionally expressed in the yeast Saccharomyces cerevisiae. the gene was introduced by transformation into yeast cells as part of a chimeric plasmid, pYT11-LEU2, constructed in vitro. The plasmide vector consists of the E. coli plasmid pBR325 (carrying the camr gene), the yeast 2-micron DNA plasmid, and the yeast LEU2 structural gene. Yeast cells harboring pYT11-LEU2 acquire resistance to chloramphenicol and cell-free extracts prepared from such cells contain chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), the enzyme specified by the camr gene in E. coli. Resistance to chloramphenicol and the presence of chloramphenicol acetyltransferase activity segregate with the yeast marker LEU2, carried by the transforming plasmid, during both mitotic growth and meiotic division.

Acetyltransferases↗

PubMed1979

Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.

Adenosine Triphosphate↗

PubMed1978

Purification and characterization of an alpha-glucosidase from Saccharomyces carlsbergensis.

alpha-Glucosidase (EC 3.2.1.20) was purified to homogeneity from logarithmically growing cells of Saccharomyces carlsbergensis. The purification involved the following steps: (a) ammonium sulfate fractionation; (b) Sephadex G-100 chromatography; (c) DEAE-cellulose chromatography; and (d) hydroxylapatite chromatography. This procedure gave a preparation judged to be greater than 98% pure by Na-DodSO4-polyacrylamide gel electrophoresis. The enzyme was shown to be a monomer of 63 000 daltons by gel filtration on Sephacryl S-200 under native conditions and by polyacrylamide gel electrophoresis under denaturing conditions. The Km values of the enzyme for the substrates maltose and p-nitrophenyl alpha-D-glucoside were found to be 1.66 X 10(-2) and 3.1 X 10(-4) M, respectively. The corresponding Vmax value for maltose was 44.8 X 10(-6) mol min(-1) mg(-1) and that for p-nitrophenyl alpha-D-glucoside was 134 X 10(-6) mol min-1 mg-1. The pH optimum for the purified enzyme was found to be between pH 6.7 and 6.8. The enzyme has an absolute anomeric specificity for alpha-glycosidic linkages and appears to recognize a glucosyl residue in alpha linkage on the nonreducing end of its substrate. For the strain used in this study, which carries the MAL 6 locus, only a single form of the enzyme was detected.

Amino Acids↗