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L V Wray

Publications and source records attributed to L V Wray.

33 records · Page 2Linked to original sources
PubMed1991

Identification and cloning of the glnR locus, which is required for transcription of the glnA gene in Streptomyces coelicolor A3(2).

Six Streptomyces coelicolor mutants that required glutamine for growth at the wild-type rate on all nitrogen sources (Gln-) were isolated. The phenotypes of all six mutants were similar. The glutamine synthetase (GS) levels were 20- to 100-fold lower in extracts of the Gln- mutants than in extracts of their parents. The reduced levels of GS activity in the Gln- mutants were not due to adenylylation of the GS protein, because GS activity in Gln- extracts did not increase after snake venom phosphodiesterase treatment. No transcripts of the GS structural gene (glnA) could be detected in RNA isolated from the Gln- mutants in primer extension experiments. All six gln mutations mapped adjacent to adeA. S. coelicolor chromosomal DNA complementing the Gln- mutants was isolated from a library of S. coelicolor chromosomal DNA constructed in the low-copy-number S. coelicolor plasmid pIJ922. Subcloning experiments showed that a 1.45-kb DNA fragment could complement all six Gln- mutants. This DNA fragment did not hybridize with either the cloned S. coelicolor glnA gene or the cloned S. viridochromogenes GSII gene in Southern blots. Since glnA transcription was restored in the Gln- mutants containing the complementing DNA, the gln mutations appear to lie in one or more closely linked genes that are required for glnA transcription in S. coelicolor.

Cloning, Molecular↗
PubMed1990

Regulation of histidine and proline degradation enzymes by amino acid availability in Bacillus subtilis.

The first enzymes of the histidine (hut) and proline degradative pathways, histidase and proline oxidase, could not be induced in Bacillus subtilis cells growing in glucose minimal medium containing a mixture of 16 amino acids. Addition of the 16-amino-acid mixture to induced wild-type cells growing in citrate minimal medium repressed histidase synthesis 25- to 250-fold and proline oxidase synthesis 16-fold. A strain containing a transcriptional fusion of the hut promoter to the beta-galactosidase gene was isolated from a library of Tn917-lacZ transpositions. Examination of histidase and beta-galactosidase expression in extracts of a hut-lacZ fusion strain grown in various media showed that induction, catabolite repression, and amino acid repression of the hut operon were mediated at the level of transcription. This result was confirmed by measurement of the steady-state level of hut RNA in cells grown in various media. Since amino acid repression was not defective in B. subtilis mutants deficient in nitrogen regulation of glutamine synthetase and catabolite repression, amino acid repression appears to be mediated by a system that functions independently of these regulatory systems.

Ammonia-Lyases↗
PubMed1989

Regulation of glutamine synthetase in Streptomyces coelicolor.

Glutamine synthetase (GS) in Streptomyces coelicolor was shown to be regulated at two levels. First, the S. coelicolor GS protein is subject to a posttranslational covalent modification which is likely to involve adenylylation. Adenylylation is important in regulating GS activity both after sudden changes in ammonium availability and during steady-state growth. Since higher levels of adenylylated GS were seen in S. coelicolor mutants deficient in glutamate synthase than in wild-type cells, glutamine or a metabolite derived from glutamine is likely to be involved in the metabolic signal that regulates GS adenylylation. Second, the GS structural gene (glnA) is transcriptionally regulated in response to nitrogen availability during steady-state growth. Transcription of the glnA gene occurred from the same promoter during vegetative growth, stationary phase, and sporulation. The nucleotide sequence of this promoter has significant homology with the -10, but not the -35, region of the consensus sequence of Streptomyces vegetative promoters. The glnA gene is transcribed as a monocistronic mRNA.

Adenosine Monophosphate↗
PubMed1988

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase.

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.

Amino Acid Sequence↗
PubMed1988

Differential regulation of the Tn10-encoded tetracycline resistance genes tetA and tetR by the tandem tet operators O1 and O2.

The Tn10-encoded tet transcriptional control sequence consists of bidirectional, overlapping promoters which are superimposed by a tandem operator arrangement. Three mutations have been constructed by oligonucleotide-directed mutagenesis which reduce binding of Tet repressor to either one or both of the tandem tet operators 1000-fold as determined by DNAseI footprinting in vitro. The affinity of Tet repressor for mutant tet operators is not affected by the presence of an already occupied neighbouring wild-type operator, indicating little or no cooperativity. The regulation of the divergently oriented tet promoters PA and PR by the tet operators O1 and O2 and Tet repressor provided in trans is determined using transcriptional fusions of the promoters to lacZ and galK indicator genes located with different polarity on the same plasmid. The results demonstrate that expression of the resistance gene tetA is regulated by Tet repressor bound to either O1 or O2. Expression of the repressor gene tetR is only marginally reduced when Tet repressor is bound to O2. This result is discussed with respect to the double promoter structure found for PR. Occupation of O1 with Tet repressor turns off transcription from PR completely. The implications of these findings on the establishment of tetracycline resistance upon induction are discussed.

Base Sequence↗
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↗
PubMed1986

Tn10 tet operator mutations affecting Tet repressor recognition.

The effect of single base pair alterations of the Tn10 encoded tet operator on recognition of Tet repressor was studied in vivo using a repressor titration system and in vitro by dissociation rate determinations of the respective complexes. Both methods reveal that the two operators, O1 and O2, which are in a tandem arrangement in the wild type, are recognized with a two-fold different affinity when separated. Studies on synthetic operator sequences indicate that the Tet repressor binds with higher affinity to the non-palindromic O2 wildtype than to the respective palindromic sequences. The in vivo repressor titration system links the expression of lacZ to the affinity of tet operator to Tet repressor. It was used to isolate tet operator mutations with reduced affinity to the repressor. The in vivo and in vitro obtained results with these mutants agree quantitatively and indicate, that the GC base pairs at positions 2, 6, and 8 are involved in interaction with the Tet repressor. Their importance for recognition decreases in that order. Transitions at position 7 of the tet operator show smaller effects on recognition than transversions.

Drug Resistance, Microbial↗
PubMed1984

Plasmid vectors based on Tn10 DNA: gene expression regulated by tetracycline.

The regulatory region of the tetracycline resistance determinant from transposon Tn10 has been used to construct plasmid vectors for gene expression regulated by tetracycline. Plasmids pRS tetBam-8 and pRS tetBam-16 include the tet regulatory region, the segment coding for the first four amino acids of the tetracycline resistance protein (tetA protein), and a linker region with SalI, HpaII, and BamHI restriction sites for gene fusions. Plasmid pTB-1, a derivative of pRS tetBam-8 and of the beta-galactosidase gene-containing plasmid pMC1403, constitutively expresses a tetA fragment-beta-galactosidase fusion protein. If a multicopy runaway replication plasmid, pMOBglII-16 that includes a 2.7-kb BglII DNA fragment from Tnl10 that provides tetR protein is present along with pTB-1, the expression of beta-galactosidase is reduced eightfold. Tetracycline acts as an inducer of the system and restores the level of beta-galactosidase activity measured in transformants containing pTB-1 alone. Plasmid mutants unable to produce active tetR protein are ineffective in reducing expression. Escherichia coli carrying plasmids that express both tetA protein and tetR protein show an increase in the tetracycline resistance level after incubation with the drug. The observations are consistent with the previously proposed mechanism of regulation of tetracycline resistance in Tn10.

DNA Transposable Elements↗
PubMed1984

Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant.

The construction and characterization of a promoter expression vector, lambda RS205 , is described. lambda RS205 can be used for the in vitro construction of transcriptional (operon) fusions to the lacZ gene of Escherichia coli K-12. The level of beta-galactosidase activity in lysogens of lambda RS205 fusion phages provides a quantitative measure of promoter function under single-copy conditions. The regulation of the Tn10 tetracycline resistance gene ( tetA ) and the Tn10 tet repressor gene (tetR) was examined by inserting DNA fragments that span the tetR- tetA promoter-operator region into lambda RS205 . Levels of beta-galactosidase in tetA -lacZ and tetR-lacZ fusion strains indicate that the tetA and tetR promoters are strong promoters; the tetA promoter is fourfold more active than the tetR promoter. Introduction of tetR+ plasmids into tetA -lacZ and tetR-lacZ fusion strains represses beta-galactosidase synthesis 15- to 60-fold and 6- to 15-fold, respectively. The concentration of tetracycline required to induce half-maximal beta-galactosidase synthesis in these tetR+ tet-lac strains depends on both the tetracycline resistance phenotype and the level of tetR repressor in the fusion strain. However, the induction of beta-galactosidase in isogenic tetA -lacZ and tetR-lacZ strains is coordinate. The data presented here support the current model of Tn10 tet gene organization and regulation and provide quantitative information about the regulation of tetA and tetR in vivo.

Amino Acid Sequence↗
PubMed1983

Overlapping divergent promoters control expression of Tn10 tetracycline resistance.

We have previously examined the genetic organization and regulation of the Tn10 tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for tetA, the Tn10 tetracycline-resistance function, and for tetR, the Tn10 tet repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both tetA and tetR is induced by tetracycline. Here we report the DNA sequence of the Tn10 tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by S1 nuclease mapping of the 5' ends of in vivo tet RNA. The tetA and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the Tn10 tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby inhibiting transcription initiation at the tetA and tetR promoters. The Tn10 tet regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).

Base Sequence↗
PubMed1983

Identification of repressor binding sites controlling expression of tetracycline resistance encoded by Tn10.

The regulatory region controlling the expression of tetracycline resistance and repressor genes contains two nearly identical regions of dyad symmetry. Deletions of this control region were isolated by digestion with S1 nuclease. The ability of these deletions to bind the tet repressor was determined by an in vivo repressor titration assay. The results indicate that repressor specifically binds both regions of dyad symmetry.

Base Sequence↗
PubMed1981

Identification of the tetracycline resistance promoter and repressor in transposon Tn10.

The structural and regulatory functions encoding tetracycline resistance in transposon Tn10 lie within a 2,700-base pair region. Using recombinant plasmids with different deoxyribonucleic acid sequences adjacent to a HincII site in this region, we located the promoter controlling the expression of tetracycline resistance. These various sequences conferred altered levels of tetracycline resistance. Plasmids containing deletions of a 695-base pair HincII fragment were constitutive and showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thus identifying the repressor and the location of its gene.

Bacterial Proteins↗