Improved single and multicopy lac-based cloning vectors for protein and operon fusions

Simons, Robert W.; Houman, Fariba; Kleckner, Nancy

doi:10.1016/0378-1119(87)90095-3

Cited by 1,533 publications

(1,474 citation statements)

References 24 publications

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“…To generate the isogenic arcA fnr double mutant, GLC3, the arcA allele was transferred from strain PC35 into PC2 (fnr ) by phage P1 transduction (Miller, 1972). High-titre lysates of JS2, JS7, JS8, JS9 and JS14 were used to introduce the individual sdhC -lacZ fusions into the PC2 (fnr ), PC35 (arcA ) and GLC3 (arcA fnr ) strains, as described previously (Simons et al, 1987).…”

Section: Methodsmentioning

confidence: 99%

“…Using plasmid pSDHJT as the template, the 651 bp DNA fragment was PCR amplified and then digested with both BamHI and EcoRI. The resulting fragment was cloned into pRS415, a promoterless lacZ operon fusion vector (Simons et al, 1987) to give the sdhC -lacZ operon fusion plasmid, pJS8. This fusion contains DNA sequences from position ¹331 bp to þ 321 bp relative to the transcriptional start site of sdhC.…”

Section: Construction Of Sdhc-lacz Operon Fusion Plasmidsmentioning

confidence: 99%

“…The entire cloned sequence in each of the five fusion plasmids, pJS2, pJS7, pJS8, pJS9 and pJS14, was confirmed by double-stranded DNA sequencing (Sanger et al, 1977) before transferring the sdh-lacZ fusions into RS45 to generate JS2, JS7, JS8, JS9 and JS14 respectively. High-titre lysates were then used to introduce each phage into MC4100, PC2 (fnr ), PC35 (arcA ) and GLC3 (arcA fnr ), as described previously (Simons et al, 1987).…”

Section: Construction Of Sdhc-lacz Operon Fusion Plasmidsmentioning

confidence: 99%

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Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Shen

Gunsalus

1997

Molecular Microbiology

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SummarySuccinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli is negatively regulated by the arcA and fnr gene products during anaerobic cell growth conditions. The controlled synthesis of this sole membrane-bound enzyme of the tricarboxylic acid cycle allows optimal participation in the aerobic electron transport pathway for the generation of energy via oxidative phosphorylation reactions. To understand how ArcA participates in the anaerobic repression of sdhCDAB expression, a family of sdhC-lacZ fusions was constructed and analysed in vivo. DNase I footprint and gel shift assays using purified ArcA protein revealed the location of four distinct and independent ArcA binding sites in the sdhC promoter region. ArcA sites, designated sites 1 and 2, are centred at ¹205 bp and ¹119 bp upstream of the sdhC promoter, respectively, whereas ArcA site 3 overlaps the ¹35 and ¹10 regions of the sdhC promoter. A fourth ArcA site is centred at þ 257 bp downstream of the sdhC promoter. They are bound with differing affinity by ArcA and ArcA phosphate. The in vivo studies, in combination with the in vitro studies, indicate that ArcA site 3 is necessary and sufficient for the ArcA-dependent repression of sdhC gene expression, while the DNA region containing ArcA site 2 contributes to maximal gene expression. The DNA-containing ArcA sites 1 and 4 provide minor roles in the ArcA regulation of sdhC expression. Lastly, the Fnr-dependent control of sdhCDAB gene expression was shown to occur independently of the ArcA and to require DNA sequences near the start of sdhC transcription.

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Section: Methodsmentioning

confidence: 99%

Section: Construction Of Sdhc-lacz Operon Fusion Plasmidsmentioning

confidence: 99%

Section: Construction Of Sdhc-lacz Operon Fusion Plasmidsmentioning

confidence: 99%

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Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Shen

Gunsalus

1997

Molecular Microbiology

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“…In each case, the Bgl II-EcoRI fragments were cloned into BamHI-EcoRI-cut pRS1274. Plasmid pPC45 was generated by exonuclease III digestion of the 3Ј end of pPC64 DNA followed by insertion of the resulting DNA into pRS1274 (Simons et al, 1987). The cyd-lacZ fusion junction of each plasmid was confirmed by DNA sequence analysis.…”

Section: ␤-Galactosidase Assaymentioning

confidence: 99%

“…The cyd-lacZ fusion junction of each plasmid was confirmed by DNA sequence analysis. Each cyd-lacZ fusion was crossed onto RS45 and introduced into the indicated strains at the lambda attachment site so that gene expression could be measured in single copy (Simons et al, 1987).…”

Section: ␤-Galactosidase Assaymentioning

confidence: 99%

Aerobic regulation of cytochrome d oxidase (cydAB ) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation

Cotter

Melville

Albrecht

et al. 1997

Molecular Microbiology

126

120

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A Set of Vectors with a Tetracycline-Regulatable Promoter System for Modulated Gene Expression inSaccharomyces cerevisiae

Garí

Piedrafita

Aldea

et al. 1997

Yeast

580

435

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A set of Saccharomyces cerevisiae expression vectors has been developed in which transcription is driven by a hybrid tetO‐CYC1 promoter through the action of a tetR‐VP16 (tTA) activator. Expression from the promoter is regulated by tetracycline or derivatives. Various modalities of promoter and activator are used in order to achieve different levels of maximal expression. In the presence of antibiotic in the growth medium at concentrations that do not affect cell growth, expression from the tetO promoter is negligible, and upon antibiotic removal induction ratios of up to 1000‐fold are observed with a lacZ reporter system. With the strongest system, overexpression levels comparable with those observed with GAL1‐driven promoters are reached. For each particular promoter/tTA combination, expression can be modulated by changing the tetracycline concentration in the growth medium. These vectors may be useful for the study of the function of essential genes in yeast, as well as for phenotypic analysis of genes in overexpression conditions, without restrictions imposed by growth medium composition. © 1997 by John Wiley & Sons, Ltd.

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Improved single and multicopy lac-based cloning vectors for protein and operon fusions

Cited by 1,533 publications

References 24 publications

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB ) gene expression in Escherichia coli

Aerobic regulation of cytochrome d oxidase (cydAB ) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation

A Set of Vectors with a Tetracycline-Regulatable Promoter System for Modulated Gene Expression inSaccharomyces cerevisiae

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