The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r: Directed mutagenesis; single-stranded DNA cloning; hybridization screening; protein-binding sites; positive gene regulation

doi:10.1016/0378-1119(82)90070-1

Gene

Volume 17, Issue 2, February 1982, Pages 167-177

https://doi.org/10.1016/0378-1119(82)90070-1 Get rights and content

Abstract

Two oligodeoxyribonucleotides were chemically synthesized and used to specifically mutate the regulatory region of the araBAD operon in Escherichia coli B/r. One oligodeoxyribonucleotide introduced a 3-bp deletion in the araC activator binding site, the other a 3-bp deletion in the CRP-cAMP binding site. The mutations were introduced onto an ara insert cloned in an M13 vector using the synthetic oligodeoxyribonucleotides as primers and the (+) strand of an M13 mp2::ara hybrid phage as a template in an in vitro polymerization reaction. Hybridizations using the original synthetic oligodeoxyribonucleotide as a radioactive probe identified phage containing the desired deletion. The mutant ara inserts were subcloned into a stable plasmid for functional analysis. Transcription studies performed on strains containing the mutant ara plasmids demonstrated that both mutations reduced the amount of araBA mRNA synthesized in the presence of l-arabinose.

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^∗: Present address: Departamento de Biología Molecular, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apdo. postal 70228, México 20, D.F.

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The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r: Directed mutagenesis; single-stranded DNA cloning; hybridization screening; protein-binding sites; positive gene regulation

Abstract

Gene

Gene

Gene

Gene

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Screening λgt recombinant clones by hybridization to single plaques in situ

Science

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Nucl. Acids Res.

The araC regulatory gene mRNA contains a leader sequence

Mol. Gen. Genet.

Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event

Biochemistry

Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA

Regulation: positive control

Annu. Rev. Genet.

DNA sequence of the araBAD promoter in Escherichia coli B/r