Construction of pBR322-ara hybrid plasmids by in vivo recombination

Horwitz, Arnold H.; Heffernan, L; Cass, Laura G.; Miyada, C G; Wilcox, Gary

doi:10.1007/bf00271752

Cited by 10 publications

(12 citation statements)

References 18 publications

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“…(ii) The ara regulatory region from strain LA2000 or LA2001 was cloned into plasmid pAH15 by in vivo recombination (28). Subsequent DNA sequence determinations showed that the correct mutation was present on the bacterial chromosome with no other nucleotide changes in the ara regulatory region.…”

Section: Resultsmentioning

confidence: 99%

Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.

Miyada¹,

Stoltzfus²,

Wilcox³

1984

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

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The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation). This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression. A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wildtype promoter and is unresponsive to various concentrations of intracellular cAMP in vivo. The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro. However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression. The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon. The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed.L-Arabinose utilization in the bacterium Escherichia coli B/r requires the activation of three unlinked genetic loci by a single regulatory gene, araC (1, 2). The araBAD operon encodes three enzymes that are responsible for the initial catabolism of L-arabinose; the araE and araF genes encode proteins that are responsible for the transport of L-arabinose into the bacterium. In the presence of L-arabinose, araC protein is an activator of transcription for the araBAD operon and the araE and araF genes (1, 2). In the presence or absence of L-arabinose, araC protein is a repressor of its own synthesis (3, 4). In addition to regulation by its own protein product, araC gene expression is under positive control of the cAMP receptor protein (CRP) (3).The regulatory region for the araC gene is adjacent to the regulatory region for the araBAD operon. Their respective promoters are transcribed in opposite directions (5) and their transcriptional initiation sites are separated by 147 base pairs (bp) (6). The nucleotide sequence of this region of DNA has been determined (7,8) and it will be referred to as the ara regulatory region. Binding sites for regulatory proteins have been identified between the two transcriptional initiation sites (refs. 4 and 9; see Fig. 1). The localization of the regulatory protein binding sites in the promoter region has been the basis for several proposed models of the regulation of the araBAD operon and the araC gene (4, 9, 10). The portions of these models that are relevant to this work are the following. The DNase I protection studies form the basis for the most recent models for araBAD and araC expression (4, 9). Although consistent with physiological data, these models have...

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Section: Resultsmentioning

confidence: 99%

Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.

Miyada¹,

Stoltzfus²,

Wilcox³

1984

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

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“…In the transformation of Saccharomyces cerevisiae, in which plasmid DNA apparently enters in a CC form, evidence for Campbell-like integration and excision has been reported (8). In Escherichia coli transfer of markers from the chromosome to an established plasmid has also been observed (9,16) and presumably occurred by such a mechanism.…”

Section: Tit1iiimentioning

confidence: 99%

Facilitation of Plasmid Transfer in Streptococcus pneumoniae by Chromosomal Homology

et al. 1982

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The frequency of plasmid establishment in the transformation of Streptococcus pneumoniae by plasmid DNA was increased more than 10-fold when the plasmid carried DNA homologous to the host chromosome. Perfect homology was not necessary for such facilitation; small additions or deletions were tolerated, but extensive deletions in the homologous segment of either plasmid or chromosome reduced or eliminated facilitation. The facilitated plasmid transfer showed a linear dependence on monomeric plasmid concentration rather than the quadratic dependence found in the absence of homology, which indicated that entering plasmid fragments interacted with the chromosome rather than with each other to establish a plasmid replicon. Restriction enzyme cleavage of the plasmid in the nonhomologous segment destroyed its activity, but cleavage in the homologous segment or even enzymatic removal of part of that segment did not prevent plasmid transfer, and plasmids of the original size were established. In facilitated transfer, chromosomal markers (additions and deletions as well as single-site mutations) entered the plasmid with a frequency ranging from 10 to 90% depending on the marker location. Several possible mechanisms for the establishment of plasmids in the presence of chromosomal homology and for the transfer of chromosomal information are considered. They depend on synapsis of the newly entered single-strand plasmid fragment with the host chromosome and subsequent copying of, donation from, or integration into the homologous chromosomal segment. After plasmid establishment, equilibration of donor and chromosomal markers between the chromosome and the plasmid pool, presumably by homologous recombination events, was observed.

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“…Cloning of the ara mutations. Horwitz et al (14) have described a rapid method for cloning chromosomal mutations onto recombinant plasmids. The method can be applied to the araBAD promoter by introducing plasmid pAH15, which contains A(araB-C)747, into a strain containing a mutation near the promoter (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In vivo cloning of mutations mapping near the ara-BAD promoter. Cloning of the ara mutations mapping near the promoter was done by using strains DS4020, DS4021, DS4022, and DS4024 as previously described for ara-1027, except that the X lysogens were grown at 30°C (14). The major steps are summarized in Fig.…”

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Five mutations in the promoter region of the araBAD operon of Escherichia coli B/r

Miyada¹,

Sheppard²,

Wilcox³

1983

J Bacteriol

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Five mutations that result in reduced expression of the araBAD operon were cloned onto the plasmid pBR322. The position of each mutation was determined by DNA sequence analysis. Three of the mutations were located in the RNA polymerase binding site of the araBAD promoter. The first, ara-1016, was a onebase-pair deletion at position-35; the second, ara-1036, was a transversion at position-13; the third, ara-1027, was a nine-base-pair deletion from +5 to +13. S1 nuclease mapping showed that mutations ara-1016 and ara-1036 greatly reduced transcription and that mutation ara-1027 had little, if any, effect on transcription. Two other mutations resulted from the transposition of the insertion element, IS], downstream from the transcriptional start site of the operon. Molecular mechanisms for all of the mutations are discussed.

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Construction of pBR322-ara hybrid plasmids by in vivo recombination

Cited by 10 publications

References 18 publications

Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.

Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.

Facilitation of Plasmid Transfer in Streptococcus pneumoniae by Chromosomal Homology

Five mutations in the promoter region of the araBAD operon of Escherichia coli B/r

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