The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR 322

Widera, Georg; Gautier, Fritz; Lindenmaier, Werner; Collins, John

doi:10.1007/bf00271959

Cited by 51 publications

(15 citation statements)

References 10 publications

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“…The low level of expression of the bphC gene in the plasmid pBS312 could be explained by the ineffective transcription from a promoter which is not a natural promoter of the bphC gene. These data are in agreement with findings by other authors showing that Pseudomonas genes are poorly expressed in E. coli [11, [23][24][25], because its promoter sequences are significantly different from the E. coli promoter consensus sequence [22,23,26,27]. Reintroduction of the bph genes into a pseudomonad cellular environment would be expected to improve transcription from the Pseudomonas promoter.…”

Section: 5-kb Tn5 Region Located At the Left End Of Thesupporting

confidence: 92%

“…It might be assumed that the transcription of the fragment containing gene bphC in plasmid pBS312 proceeds from the BgllI site to the EcoRI site, from right to left. Since the cloned fragment is inserted into the BamHI site of the tetracycline gene, expression of gene bphC is not the result of the readthrough transcription initiated from the promoter of the tetracycline gene because the tet gene is transcribed from the EcoRI site to the BamHI site of pBR322 [22]. The low level of expression of the bphC gene in the plasmid pBS312 could be explained by the ineffective transcription from a promoter which is not a natural promoter of the bphC gene.…”

Section: 5-kb Tn5 Region Located At the Left End Of Thementioning

confidence: 99%

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Increased expression of the plasmid-determined 2,3-dihydroxybiphenyl dioxygenase gene in strains of Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa

Andreyeva

1993

FEMS Microbiology Letters

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A 6.5-kb EcoRI fragment containing the gene encoding 2,3-dihydroxybiphenyl dioxygenase from the plasmid pBS312 was cloned into broad host range plasmid RSF1010 and expressed in Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa strains. The increased expression of the gene was orientation-dependent and probably due to the transcription read through from the streptomycin promoter of the vector. Subcloning experiments of the PstI fragments of pBS312 plasmid using vector pBR322 revealed that the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase is localized on the 2.1-kb fragment. In Escherichia coli JM109, transformed by the plasmid pBS314 carrying the 2.1-kb insert in orientation which allowed expression of the bphC gene from the ampicillin promoter of pBR322, the enzyme activity of 2,3-dihydroxybiphenyl dioxygenase was ten times higher than that in parental strain Pseudomonas putida SU83. The results presented show the first case of the increased expression of Pseudomonas degradative gene in Escherichia coli.

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Section: 5-kb Tn5 Region Located At the Left End Of Thesupporting

confidence: 92%

Section: 5-kb Tn5 Region Located At the Left End Of Thementioning

confidence: 99%

Increased expression of the plasmid-determined 2,3-dihydroxybiphenyl dioxygenase gene in strains of Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa

Andreyeva

1993

FEMS Microbiology Letters

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“…Ligation of target DNA upstream from a promoterless marker gene is an established strategy for the identification of promoters. The tetracycline resistance gene of pBR322 has been used as a promoter probe (22)(23)(24). In these experiments the promoter typically was inactivated by deletion within the recognition site.…”

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confidence: 99%

Promoters selected from random DNA sequences.

Horwitz¹,

Loeb²

1986

Proc. Natl. Acad. Sci. U.S.A.

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We have selected a group of Escherichia coli promoters from random DNA sequences by replacing 19 base pairs at the -35 promoter region of the tetracycline resistance gene te" of the plasmid pBR322. Substitution of 19 base pairs with chemically synthesized random sequences results in a maximum of 419 (about 3 x 1011) possible replacement sequences. From a population of about 1000 bacteria harboring plasmids with these random substitutions, tetracycline selection has revealed several functional -35 promoter sequences. These promoters have retained only partial. homology to the -35 promoter consensus sequence. In three ofthese promoters, the consensus agent shifts 10 nucleotides downstream, allowing the RNA polymerase to recognize another Pribnow box from within the original pBR322 sequence. Two of the sequences promote transcription more strongly than the native promoter. This technique may have application for the selection ofadditional DNA sequences with varied biological activity.Comparison of known RNA polymerase binding sites of different genes of Escherichia coli reveals two highly conserved promoter elements centered at about -10 and -35 base pairs (bp) from the start of transcription (1-5). A consensus sequence of the nontemplate strand includes "TATAAT" from positions -13 to -8, the "Pribnow box," and "TTGACA" from positions -36 to -31, the "recognition" site, with 17 bp between the two (6). The involvement of each nucleotide in the initiation of transcription has been inferred largely from an analysis of mutations. Rare mutations that increase transcription, "up mutations," usually increase homology with the consensus sequence and spacing, while the more common mutations that decrease transcription, "down mutations," usually decrease homology with the consensus sequence and spacing (7). Targeted random mutagenesis has been used to define prokaryotic (8) and eukaryotic (9) translation initiation signals. In these experiments base substitutions were limited in number to no more than three and were identified before activity was manually assayed. This strategy limits the search for functional sequences to derivatives of those already known.We report here a technique that has allowed us to create several unusual promoter recognition sequences. We have substituted for the promoter recognition site of a plasmidborne selectable marker chemically synthesized random sequences of 19 MATERIALS AND METHODSOligonucleotides. Oligonucleotides were synthesized by the phosphoramidite method with an Applied Biosystems 380A DNA synthesizer and purified by thin layer chromatography (10). Random sequences were synthesized with equimolar mixtures of phosphoramidites.Plasmid Constructions. Restriction endonucleases and enzymes of nucleic acid metabolism were obtained commercially and use followed the supplier's instructions. Standard molecular cloning methods were employed (11).A plasmid with a deletion in the promoter recognition site, pBdEC, was constructed by digestion of pBR322 with EcoRI and Cla I, by extension o...

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“…Tetracyclines (TCs) are known to repress protein synthesis by preventing the binding of ribosomes with t-RNA (4,11). TC is also mutagenic in bacterial plasmids (14,16) and inhibits DNA replication (10) and the binding of metals to nucleic acids (7). Recently, we reported studies on the structural change of purified DNA caused by CTC (17).…”

mentioning

confidence: 99%

Biparametric fluorometry of chlorotetracycline and ethidium bromide in DNA structure.

Miyata

Takasugi

Yamada

1984

Acta Histochem. Cytochem

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When chlorotetracycline (CTC) was introduced into the DNA-ethidium bromide (EB) complex, the CTC fluorescence was interpolated into the EB fluorescence of DNA. The fluorescence parameters of EB and CTC could be measured biparametrically in relation to DNA structures. The EB fluorescence enhanced by intercalation into DNA was decreased by addition of CTC in vitro.Similar decrease of fluorescence occurred in cell nuclei isolated from rat liver cells and their chromatin.On CsCl density gradient centrifugation, the peak position of the CTCtreated DNA was at a lower density than that of untreated DNA. On the sucrose density gradient centrifugation, CTC-treated chromatin was also at a lower density than untreated chromatin. When rats were treated with CTC in vivo, the fluorescence response of their liver DNA appeared to be similar to that in vitro.Thus, biparametric fluoro-assay could be used for following structural change of DNA caused by CTC in vitro or in vivo.Ethidium bromide (EB) is known to bind with DNA by intercalation between DNA base pairs. When the molar ratio of DNA-phosphate (DNA-P) to EB is more than 10, EB fully intercalates into double-stranded DNA and its fluorescence is remarkably enhanced (8, 13). That fluorescence can be used as a marker of the structure of DNA. If the DNA structure is modified by another fluorescent probe, the original DNA-EB fluorescence will be changed in intensity and/or polarity. Thus, for fluorometric examination of structural change of DNA, it is necessary to use DNA fully intercalated with EB, Chlorotetracycline (CTC) was used as a second fluorescent probe in the present examination. Tetracyclines (TCs) are known to repress protein synthesis by preventing the binding of ribosomes with t-RNA (4, 11). TC is also mutagenic in bacterial plasmids (14, 16) and inhibits DNA replication (10) and the binding of metals to nucleic acids (7). Recently, we reported studies on the structural change of purified DNA caused by CTC (17). In the present work we examined the effects of CTC on rat liver cell nuclei and its chromatin to confirm that CTC has the same effect on the structure of native DNA. With our micro-assay, biparametric fluorometry system, change of DNA can be detected even in a single cell.Details of the method used and application of the method to studies on DNA structure are reported here.

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The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR 322

Cited by 51 publications

References 10 publications

Increased expression of the plasmid-determined 2,3-dihydroxybiphenyl dioxygenase gene in strains of Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa

Increased expression of the plasmid-determined 2,3-dihydroxybiphenyl dioxygenase gene in strains of Escherichia coli, Pseudomonas putida and Pseudomonas aeruginosa

Promoters selected from random DNA sequences.

Biparametric fluorometry of chlorotetracycline and ethidium bromide in DNA structure.

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