The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r

Miyada, C G; Soberón, Xavier; Itakura, Keiichi; Wilcox, Gary

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

Cited by 40 publications

(30 citation statements)

References 22 publications

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“…The primary sequence of this peptide is identical to that of cecropin A, but it lacks the C-terminal amide present in the naturally occurring peptide (CA-COOH). For the production of a cecropin-like peptide with a reactive C terminus (for C-terminal modification), a primerdirected mutagenesis technique (22) A linear salt gradient (0 to 0.5 M NaCl) in the same phosphate buffer was used to elute the peptides. Fractions were analyzed for antimicrobial activity against E. coli D21 cells, and they were also analyzed with acetate-urea gels (15% acrylamide, pH 4.5) and then concentrated and desalted with an Amicon concentrator.…”

Section: Methodsmentioning

confidence: 99%

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity

Callaway

Lai

Haselbeck

et al. 1993

Antimicrob Agents Chemother

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Cecropin A is a naturally occurring peptide with bactericidal activity against gram-negative and grampositive bacteria. Production of large quantities of bactericidal peptides that are similar in structure and activity to cecropin A has been achieved by combining recombinant DNA techniques and chemical modification. Expression of the bactericidal peptide in Escherichia coli was accomplished through the formation of a fusion protein. The 5' end of the L-ribulokinase gene was fused to a single copy of a synthetic gene encoding cecropin A. A methionine codon was engineered between the two genes, and a methionylglycine extension was introduced at the C terminus of cecropin A. Cyanogen bromide treatment of the fusion protein yielded cecropin A with a C-terminal homoserine. The recombinant cecropin A with a homoserine at the C terminus did not kill most gram-positive bacteria tested. However, recombinant cecropin A with a chemically modified C terminus has antimicrobial activity similar to that of cecropin produced by cecropia pupae.

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Section: Methodsmentioning

confidence: 99%

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity

Callaway

Lai

Haselbeck

et al. 1993

Antimicrob Agents Chemother

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“…2). These sequences were complementary to sequences in the single-stranded bacteriophage template used, M13mp2::aral (33). This phage contains a 2.5-kilobase (kb) EcoRI restriction fragment containing the entire ara regulatory region, part of araA, part of araC, and all of araB.…”

Section: Methodsmentioning

confidence: 99%

“…This phage contains a 2.5-kilobase (kb) EcoRI restriction fragment containing the entire ara regulatory region, part of araA, part of araC, and all of araB. The in vitro polymerization reaction and ligation were performed as described previously (33) and used to transform E. coli 71-18 (31). Phage containing the desired mutation was identified by plaque hybridization, using the radioactively labeled oligonucleotide as a probe.…”

Section: Methodsmentioning

confidence: 99%

Effect of mutations in the cyclic AMP receptor protein-binding site on araBAD and araC expression

Stoltzfus

Wilcox

1989

J Bacteriol

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Maximum expression of the adjacent but divergently transcribed araBAD operon and araC gene requires the presence of cyclic AMP (cAMP) and the cAMP receptor protein (CRP). DNase I protection studies have previously revealed a high-affinity CRP-binding site in the ara regulatory region. Deletion mutations introduced into this site resulted in reduced expression of araBAD and araC. However, other experiments have demonstrated that spacing changes in the ara regulatory region may have multiple effects due to disruption of a DNA loop. Thus, the deletions could have destroyed the CRP-binding site, the ability to form a loop, or both. In the present study, substitution mutations were introduced into the CRP site in order to avoid creating spacing changes. We found that a 3-base-pair substitution resulted in a 30% reduction in araBAD expression, whereas a 6-base-pair substitution resulted in an 80% reduction. Both of these substitution mutations reduced araC expression threefold. We conclude that CRP bound to this site regulates expression in both directions. We found that a spacing change in the CRP site does not alter araBAD expression any more than does a substitution mutation.Utilization of L-arabinose in Escherichia coli requires the expression of four transcription units (14,24). The araE gene and araFGH operon encode proteins responsible for lowaffinity and high-affinity transport of L-arabinose into the cell (4,20,23,24,37). The araBAD operon encodes three enzymes that are responsible for the initial reactions in the catabolism of L-arabinose (14, 24). The regulatory gene, araC, encodes a protein that controls expression of each of the four transcription units. In the presence of L-arabinose, AraC activates expression of araBAD, araE, and araFGH (4,14,20,23,24,37). In the absence of L-arabinose, AraC represses transcription of araBAD (13). In the presence or absence of L-arabinose, AraC represses its own transcription (6,18,19), yielding a constant level of AraC protein in the cell. In addition, maximum expression of the araBAD operon and araC gene requires the presence of cyclic AMP (cAMP) and the pleiotropic regulator cAMP receptor protein (CRP) (1,6,27). Cells containing mutations in either the adenyl cyclase gene (cya) or the CRP gene (crp) are Ara-(45). The promoters for the araBAD operon and araC gene are adjacent to one another and are transcribed in opposite directions (43). The transcription initiation sites are separated by 147 base pairs (41). The region between the araC and araB genes is referred to as the ara regulatory region. Binding sites for the proteins in the ara regulatory region have been determined by methylation protection and DNase I protection studies (Fig. 1) (9,25,26,35 to the proposed model, the cAMP-CRP complex binds to this single site in the ara regulatory region to activate transcription of both the araC gene and araBAD operon. In order to test this model, a 3-base-pair deletion was introduced into this site, and CRP dependence of the araBAD and araC promoters was tested (34). I...

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“…The pellets were re-suspended in 40ml X-buffer (lmM MgSO4, 0.002% gelatine, 2AsM sodium phosphate buffer pH 7.2) and the phage suspension centrifuged for 16h at 80,000 x g. The resulting pellets were re-suspended in 1-2ml X-buffer. DNA was then isolated by phenol extraction and extensive dialysis against TES-buffer (5C1M NaCl, with filamentous phage (typically less than 10%) 13,17). It seemed to us that a possible explanation for this behaviour could be offered by the study of Radman et al ) on mismatch repair acting on duplex DNA molecules of the X genome containing one or two mismatch positions.…”

Section: Methodsmentioning

confidence: 99%

Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments

1982

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Gapped duplex DNA molecules of recombinant genomes of filamentous phage are constructed in vitro. Denatured restriction fragments covering (part of) the precisely constructed gap are hybridized to the gapped duplex DNA molecules to form ternary duplices. The two strands of the ternary duplex molecules carry different genetic markers within the region spanned by the restriction fragment leading to a one base pair mismatch or to an insertion loop of 93 nucleotides, respectively. The two strands also vary with respect to A-methylation in GATC sites. In cases of asymmetrical methylation, transfection of E.coli with these heteroduplex molecules leads to marker recoveries with a pronounced bias in favour of the marker encoded by the methylated strand. This effect at least partly explains the comparably low marker yields achieved in previous directed mutagenesis experiments using filamentous phage as the vector. The results suggest how these procedures can be optimized. Precise construction of a 93 bp insertion in 9.5% marker yield is described.

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The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r

Cited by 40 publications

References 22 publications

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity

Effect of mutations in the cyclic AMP receptor protein-binding site on araBAD and araC expression

Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments

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