Plasmid vector pBR322 and its special-purpose derivatives — a review

Balbás, Paulina; Soberón, Xavier; Merino, Enrique; Zurita, Mario; Lomelı́, Hilda; Valle, Fernando; Flores, Noemí; Bolívar, Francisco

doi:10.1016/0378-1119(86)90307-0

Cited by 290 publications

(84 citation statements)

References 171 publications

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“…1), in which the prc orientation matched the direction of transcription from the promoter of the tet repressor gene (tetRp, originated from the ancestral plasmid [2]). A remarkable overproduction of the 80-kDa protein was observed with pHR140.…”

Section: Resultsmentioning

confidence: 99%

“…The 3.5-kb fragment between the EcoRI1 site and the EcoRP site within the multiple cloning site fragment of pHR76 was inserted into the EcoRI site of pACYC184 (11) in either orientation to construct pHR87 and pHR88. Plasmid pHR140 was constructed by ligating the 2.6kb EcoRI1-PstI3 fragment with the larger EcoRI-PstI fragment of pBR322 (2). In pHR126 a synthetic translation terminator (39) was sandwiched between two BamHI-HindIII regions of the pUC18/19 multiple cloning site in an inverted orientation.…”

Section: Methodsmentioning

confidence: 99%

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Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

et al. 1991

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The prc gene, which is involved in cleavage of the C-terminal peptide from the precursor form of penicillin-binding protein 3 (PBP 3) of Escherichia coli, was cloned and mapped at 40.4 min on the chromosome. The gene product was identified as a protein of about 80 kDa in maxicell and in vitro systems. Fractionation of the maxicells producing the product suggested that the product was associated with the periplasmic side of the cytoplasmic membrane. This was consistent with the notion that the C-terminal processing of PBP 3 probably occurs outside the cytoplasmic membrane: the processing was found to be dependent on the secY and secA functions, indicating that the prc product or PBP 3 or both share the translocation machinery with other extracytoplasmic proteins. DNA sequencing analysis of the prc gene region identified an open reading frame, with two possible translational starts 6 bp apart from each other, that could code for a product with a calculated molecular weight of 76,667 or 76,432. The prc mutant was sensitive to thermal and osmotic stresses. Southern analysis of the chromosomal DNA of the mutant unexpectedly revealed that the mutation was a deletion of the entire prc gene and thus that the prc gene is conditionally dispensable. The mutation resulted in greatly reduced heat shock response at low osmolarity and in leakage of periplasmic proteins.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

et al. 1991

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“…Additionally, this strain possesses biochemical and nutritional properties that fit the typical phenotype of P. marginalis as described by Lelliott et al (21) and the typical phenotype of biotype B (or biovar II) of P. fluorescens as described by Stanier et al (36). Escherichia coli HB101 and cloning vectors (pBR322 [1], pBR325 [1], and pUC18 [41]) were obtained from Bethesda Research Laboratories (Gaithersburg, Md.). NaOCl for 10 min, rinsed three times with sterile water, and dried.…”

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confidence: 96%

Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida

Liao

1991

J Bacteriol

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Pectate lyase (PL) depolymerizes pectin and other polygalacturonates (PGAs) and is thought to play a role in bacterial invasion of plants. Production of PL by the soft-rotting pathogen Pseudomonasfluorescens CY091 is regulated by Ca2+. In the presence of Ca2+, this bacterium constitutively synthesizes PL in media containing glucose, glycerol, or PGA and excretes over 87% of total PL into culture fluids. In the absence of Ca2 , the organism fails to use PGA as a carbon source and produces very low levels of PL in media containing glucose or glycerol. Of the small amount of PL produced by the bacterium in Ca2+-deficient media, over 78% was detected within the cells, indicating that Ca2' is critical not only for the production but also for the secretion of PL. The pel gene, encoding an alkaline PL (p1 10.0, Mr 41,000) was cloned and located on the overlapping region of a 4.3-kb Sall and a 7.1-kb EcoRI fragment. Pseudomonas fluorescens is a heterogenous species and consists of a diversity of ecologic and physiological groups (36). Certain strains of this species are postharvest pathogens of plants, which cause soft rot of fruits and vegetables in storage and at markets (25). Soft-rotting P. fluorescens (often referred to as P. marginalis) is capable of degrading pectic components of plant cell walls by producing a wide variety of pectolytic enzymes, including pectin methylesterase (30), pectin lyase (33, 35), polygalacturonase (10,30,42), and pectate lyase (PL) (9,10,12,22,30,42). Production of methylesterase, pectin lyase, and polygalacturonase is rare and has been detected only in a few strains (30,33,35,42). With the exception of one strain (30), all of the soft-rotting pseudomonads so far studied produce PL. Thus, it is generally believed that PL is the principal or sole enzyme responsible for tissue maceration caused by most strains of P. fluorescens and P. viridiflava (22, 24).The PL system of P. fluorescens is considerably different from that of Erwinia spp., although soft-rot symptoms caused by the two organisms are similar. In Erwinia spp., all strains so far studied produce three to five PL isozymes (pl 4.5 to 10.0) (18), whereas in P. fluorescens, all eight strains recently examined in our laboratory produce one or possibly two PLs (22). Furthermore, production of PLs in Erwinia spp. is induced by pectic substrates and subjected to catabolite or self-catabolite repression (18). In P. fluorescens, however, the mode of PL production varies considerably among strains and can be constitutive (30,42) or inducible (9,30,43). In some strains, PL production is induced by pectic substances in a mechanism resembling that previously demonstrated in Erwinia spp. (18). In others, the regulatory factors or mechanisms have not been clearly defined. For example, Zucker and Hankin (43) showed that a nonpectolytic isolate of P. fluorescens can be converted to pectolytic by a series of subcultures in media containing pectin or plant tissue extracts. Hildebrand (10) reported that expression of a pectolytic phenotype in fluo...

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“…Because TnStacl at site 1 is between the end of the tet promoter at pBR322 position 39 and the normal transcriptional start at position 45 (13,24) (Tn5tacl sequence begins at position 40), transcription from the tet promoter probably both starts and stops within Tn5tacl when it is at this site. Because inserts at site 2 are between the start of tet transcription and the start of tet translation (at position 86), the Tets phenotype of the site 2 mutants grown without IPTG shows that Tn5tacl is polar.…”

Section: Resultsmentioning

confidence: 99%

Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations.

Chow

Berg

1988

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

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Conditional lethal mutations are valuable for analyzing essential genes. We MATERIALS AND METHODSThe bacterial strains used are derivatives of E. coli K-12 (Table 1). Bacteria were grown in LN (complex) medium or in M9 (minimal) glucose medium solidified with 1.5% agar as appropriate (13,25). Antibiotics were used at the following concentrations: ampicillin, 250pg/ml; kanamycin, 60;Lg/ml; and tetracycline, 12 ;kg/ml (or 6 ;g/ml where noted). IPTG was used at 0.5 mM. Standard procedures were used for bacterial growth, plasmid DNA extraction, restriction endonuclease digestion, electrophoresis, and transformation (13). DNA sequencing was carried out by a modification of the chain-termination method (12,26). All enzymes were obtained from commercial suppliers (New England Biolabs, Boehringer Mannheim, or Bethesda Research Laboratories) and used as directed.The oligonucleotides used as sequencing primers are: A, 5'-GTATCACGAGGCCCT (pBR322 positions 4334-4348); B, 5'-GCAATTTAACTGTGAT (pBR322 positions 64-49); C, 5'-GATAAGCTGTCAAAC (pfR322 positions 22-8); and D, 5'-CAGAATTCCCGGGGATCCCC. Oligonucleotides A, B, and C were from New England Biolabs; oligonucleotide D was made on an Applied Biosystems 380A DNA synthesizer. Oligonucleotides A and B are specific for DNAs that flank Tn5tacl in the plasmid in which it was constructed and were used as primers in reactions that confirmed the sequences at the transposon ehds. Oligonucleotides C and D match sequences within TnStacl near its ends (Fig. 1 legend). Oligonucleotide C also matches the HindIII-EcoRI segment of pBR322 and was used as a sequencing primer for inserts in pBR322 targets only after this segment was removed from the target DNA.Abbreviations: IPTG, isopropyl P-D-thiogalactoside; Amp5 and Tets, ampicillin-and tetracycline-sensitive phenotypes; TetR and KanR, tetracycline-and kanamycin-resistant phenotypes; IS, insertion sequence.

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Plasmid vector pBR322 and its special-purpose derivatives — a review

Cited by 290 publications

References 171 publications

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3

Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida

Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations.

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