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D Vapnek

Publications and source records attributed to D Vapnek.

31 records · Page 2Linked to original sources
PubMed1977

Expression in Escherichia coli K-12 of the structural gene for catabolic dehydroquinase of Neurospora crassa.

The inducible quinic acid catabolic pathway of Neurospora crassa is controlled by four genes, the qa cluster which includes structural genes qa-2, qa-3, qa-4 for three enzymes and a regulatory gene, qa-1. In this paper we report the molecular cloning of at least the qa-2 gene which encodes the catabolic dehydroquinase (5-dehydroquinate hydro-lyase, EC 4.2.1.10). Endo.R.HindIII restriction endonuclease fragments of N. crassa DNA from a qa-1(c) (constitutive) mutant and of Escherichia coli plasmid pBR322 DNA were ligated in vitro and used to transform an aroD6 (5-dehydroquinate hydrolyase deficient) strain of E. coli K12. The recombinant plasmid (pVK55) isolated from one AroD(+) transformant (SK1518) contained, in addition to pBR322, two N. crassa HindIII fragments with molecular weights of 2.3 x 10(6) and 1.9 x 10(6). Derivatives of SK1518 cured of plasmid DNA were phenotypically Amp(s) and AroD(-). These cured strains, retransformed with pVK55, were phenotypically Amp(R) and AroD(+). Strains transformed with pVK55 possessed 5-dehydroquinate hydrolyase activity but no activity was present in any AroD(-) strain. The enzyme extracted from strains containing the recombinant plasmid was identical to N. crassa catabolic dehydroquinase by the criteria of heat stability, ammonium sulfate fractionation, immunological crossreactivity, molecular weight, and purification characteristics. This identity demonstrates that the N. crassa qa-2(+) gene is carried by the recombinant plasmid and is apparently transcribed and translated with complete fidelity. Furthermore, subunit assembly of the N. crassa polypeptides also occurs in E. coli, because the catabolic dehydroquinase is a multimer composed of approximately 20 identical subunits.

DNA Restriction Enzymes↗
PubMed1976

Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I.

Endo-R-HindIII restriction endonuclease fragments obtained from F30 and pMB9 plasmid DNAs were ligated in vitro and used to transform a recB21 recC22 sbcB15 strain of E. coli K-12. The inability of this strain to stably maintain pMB9 alone permitted the isolation of transformants that carried hybrid plasmids containing the sbcB+ allele. These transformants became sensitive to ultraviolet light and recombination defieient and showed a 25-fold increase in the level of exonuclease I activity. The stability of the sbcB hybrid plasmids and their effects on exonuclease I activity have also been determined in wild-type and recA1 genetic backgrounds. The presence of the plasmids results in a 7-fold increase in the level of exonuclease I in a wild-type strain and 15-fold increase in a recA1 strain. The increased activity in the recA1 mutant appears to be a result of increased plasmid stability in this genetic background.

Base Sequence↗
PubMed1976

In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency.

The amounts of plasmid deoxyribonucleic acid (DNA) and the levels of the in vivo transcription of the Escherichia coli plasmids R538-1 (repressed for conjugal transfer) and R538-1drd (derepressed for transfer) were determined by DNA-DNA hybridization and DNA-ribonucleic acid hybridization, respectively. The results demonstrate that the level of plasmid transcription is increased by two-fold in the strain carrying the derepressed plasmid, compared to an isogenic strain carrying the repressed plasmid, whereas the amount of plasmid DNA is approximately the same, suggesting that the transfer genes are under transcriptional control. Levels of plasmid DNA, plasmid DNA transcription, and chloramphenicol acetyltransferase activity were also compared in a mutant strain that carried the R538-1drd plasmid and was resistant to high levels of antibiotics. This strain produces about 13 copies of plasmid DNA per chromosome compared to five copies for the parent strain. The level of transcription of plasmid DNA was found to be twofold higher in the high-level resistant strain, whereas the level of chloramphenition, acetyltransferase activity was increased by 10-fold. In addition the levels of plasmid DNA transcription and chloramphenicol acetyltransferase activity in the high-level resistant strain were found to be further increased by the presence of high levels of chloramphenicol in the growth medium. The amount of plasmid DNA remained constant under these conditions, indicating that high levels of chloramphenicol can stimulate the expression of plasmid genes at the level of transcription in this strain.

Acetyltransferases↗
PubMed1974

Asymmetry and extent of in vivo transcripition of R-plasmid deoxyribonucleic acid in Escherichia coli.

Deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization studies have been performed with R-plasmid DNA (R538-1drd) and in vivo-synthesized RNA. R-plasmid DNA was isolated from Escherichia coli K-12, and the complementary strands were separated in cesium chloride-polyuridylic acid-polyguanylic acid gradients. DNA-RNA hybridization was performed with the separated DNA strands and RNA purified from R-plasmid-carrying cells. The results demonstrated that an asymmetric transcription of the R-plasmid DNA occurs in vivo. Hybridization was only detected with the H strand (denser strand in cesium chloride-polyuridylic acid-polyguanylic acid). By determining the density of the RNA-DNA hybrid in CsCl gradients, it was estimated that greater than 60% of the nucleotide sequences in the R-plasmid DNA are transcribed in logarithmically growing E. coli cells. No R-plasmid-specific RNA was detected in E. coli cells that did not carry the plasmid.

Base Sequence↗
PubMed1971

Suppression by derepression in threonine dehydratase-deficient mutants of Bacillus subtilis.

Partial suppressors of isoleucine-requiring mutants (Ile(-)), deficient in threonine dehydratase (TD), were studied. The suppression enables these auxotrophs to grow on the precursors homoserine or threonine as well as isoleucine. There are two genetically distinct classes of these suppressors: sprB, linked to threonine mutational sites, and sprA which is not linked to the Ile or Thr genetic region. SprA leads to the appearance of a low level of TD activity (2 to 4% of wild type) compared to <0.2% in Ile(-) mutants. This new TD activity is not activated by glutathione or adenosine monophosphate. Unlike the major TD, it is insensitive to inhibition by isoleucine and it is not coordinately controlled with dihydroxy acid dehydratase. Another class of auxotrophs, containing no suppressor mutation, can grow on isoleucine and homoserine or threonine because they possess structural gene mutations located near the terminus of the TD gene that result in partial impairment of TD. This TD activity (2% of wild type) is end product inhibited by isoleucine to a lesser extent than wild type TD. In this and a following paper, evidence is presented that sprA acts by derepressing a minor TD activity encoded within the threonine synthetic region. The major TD gene is separated from the region encoding the enzymes of threonine biosynthesis by 73% of the chromosome. Like the major TD, the minor TD catalyzes the first reaction in the conversion of threonine to isoleucine and is therefore closely related biochemically and genetically to the enzymes of the threonine synthetic region.

Adenine Nucleotides↗
PubMed1971

Minor threonine dehydratase encoded within the threonine synthetic region of Bacillus subtilis.

Challenging auxotrophs on metabolites that are precursors of a biosynthetic step involving a mutated enzyme has revealed a new class of suppressor mutations which act by derepressing a minor enzyme activity not normally detected in the wild-type strain. These indirect, partial suppressor mutations which allow isoleucine auxotrophs to grow on homoserine or threonine have been analyzed to determine their effect on enzymes involved in the biosynthesis of these amino acids. It has been found that one class of these suppressor mutations (sprA) leads to the derepression of homoserine kinase, homoserine dehydrogenase, and a minor threonine dehydratase that is not sufficiently active to be detected in the wild-type strain. The gene encoding this second threonine dehydratase activity has been found to be located between the structural genes for homoserine kinase and homoserine dehydrogenase. The results of these experiments indicate that plating of auxotrophs on precursors of a biosynthetic step involving mutated enzymes could prove to be a valuable method for the detection of regulatory mutants as well as a possible tool in studying the evolution of biochemical pathways.

Bacillus subtilis↗
PubMed1971

Physical properties and mechanism of transfer of R factors in Escherichia coli.

The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.

Centrifugation, Density Gradient↗
PubMedDate not supplied

Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9.

The transposable genetic element Tn9 consists of two direct repeats of the insertion sequence IS1 flanking a region of 1,102 base pairs which determines chloramphenicol resistance. Transposition of Tn9 leads to the duplication of a 9-base pair sequence which preexists at the site of insertion. One copy of this sequence is found at each end of the inserted element. The chloramphenicol resistance determined by Tn9, and by various other R plasmids, is due to the synthesis of the enzyme chloramphenicol acetyl transferase (CAT). This enzyme catalyses the formation of acetylated derivatives of chloramphenicol which are inactive as inhibitors of protein synthesis. By using the chain termination technique of DNA sequencing, we have now determined the nucleotide sequence of the 1,102 base pair region between the directly repeated IS1 sequence in the bacterial transposon Tn9 (encoding chloramphenicol resistance). The amino acid sequence of CAT predicted from the nucleotide sequence is identical to that determined by Shaw and coworkers. An analysis of the sequence suggests that the internal 1,102 base pair region is not directly involved in transposition.

Acetyltransferases↗