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G Wilcox

Publications and source records attributed to G Wilcox.

At least 73 records · Page 4Linked to original sources
PubMed1980

DNA sequence of the araC regulatory gene from Escherichia coli B/r.

The DNA sequence of the araC regulatory gene from Escherichia coli B/r has been determined by the base-specific chemical cleavage reactions of Maxam and Gilbert. An open reading frame is found which codes for a protein of 292 amino acids. A nonsense mutation, araC5, is shown to result from a G to A transition at nucleotide 429 converting the tryptophan codon TGG to the amber codon TAG. A deletion which does not recombine with any known point mutation in araC, delta(araCO)719, removes all but the last 22 codons of the gene.

Amino Acid Sequence↗
PubMed1980

The araC regulatory gene mRNA contains a leader sequence.

An estimation of the size of the araC gene in Escherichila coli B/r was made by sub-cloning restriction fragments of the araC-containing hybrid plasmid pTB1 into the plasmid pBR322. Plasmids which contained a functional araC gene were identified by genetic complementation tests. DNA sequence analysis of the promoter-proximal region of the araC gene revealed that araC mRNA contains a 150 nucleotide leader.

Arabinose↗
PubMed1980

Construction of pBR322-ara hybrid plasmids by in vivo recombination.

In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analyses demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%--3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araC mutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.

Arabinose↗
PubMed1980

Deoxyribonucleic acid sequence of araBAD promoter mutants of Escherichia coli.

The controlling site region for the araBAD operon is defined, in part, by two classes of cis-acting constitutive mutations. The aralc mutations allow low-level constitutive expression of ara-BAD in the absence of the positive regulatory protein coded for by the araC gene, whereas the araXc mutations allow expression of araBAD in the absence of the cyclic adenosine monophosphate receptor protein. Six independently isolated aralc mutations and three independently isolated araXc mutations were cloned onto the plasmid pBR322 using in vitro recombinant deoxyribonucleic acid techniques and in vivo recombination between plasmid and chromosomal deoxyribonucleic acid. The location of these mutations was determined by deoxyribonucleic acid sequence analysis. All of the aralc mutations occurred at position -35 within the araBAD promoter (+1 = messenger ribonucleic acid start for araBAD) and resulted from an AT leads to GC transition. All of the araXc mutations occurred at position -10 within the araBAD promoter and resulted from a GC leads to AT transition. Models are presented to explain the mode of action of the aralc and araXc mutations.

Arabinose↗
PubMed1980

Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mu d (Apr lac).

A specialized Mu transducing phage containing a gene encoding ampicillin resistance and the lac structural genes without the lac promotor [Mu d(apr lac)] has been constructed and used to create gene fusions in Escherichia coli (M. J. Cadadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530--4533, 1979). Transposition of the Mu d(Apr lac) phage to chromosomal sites can result in lac expression being controlled by a chromosomal promoter. We have constructed an Escherichia coli K-12 strain in which the Mu d(Apr lac) phage is integrated into an F factor. The F+::Mu d(Apr lac) was then transferred by conjugation into a Salmonella typhimurium strain that was sensitive to L-arabinose. Strains containing gene fusions were selected as L-arabinose-resistant colonies after partial induction of the phage. Two classes of ara-lac fusion strains were isolated: (i) araC-lac fusions in which the expression of beta-galactosidase synthesis was constitutuve and not inducible by L-arabinose; and ((ii) fusion of the lac genes to the ara structural genes in which the expression of beta-galatosidase synthesis was induced 263-fold by L-arabinose.

Arabinose↗
PubMed1979

Regulation of the L-arabinose operon in strains of Escherichia coli containing ColE1-ara hybrid plasmids.

Hybrid plasmids were constructed from fragments of F'ara episomes formed by the restriction endonuclease EcoRI and a linear form of the plasmid ColE1 created by cleavage with EcoRI. Hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted. E. coli K12 host strains were constructed which contained different deletions of the ara region. The hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid. The initial differential rates of synthesis of L-arabinose isomerase, the product of the araA gene, were determined for the Ara+, plasmid containing strains. These studies demonstrated that strains containing delta(araOIBA)718 produce elevated levels of araC protein, suggesting the araC promoter has been altered by this deletion. Evidence is also presented which suggests that araC protein activates the ara-BAD operon to higher levels when it is present in cis rather than trans. Amplification of the products of the cloned genes is observed when compared to haploid levels in some cases.

Arabinose↗
PubMed1978

The shape of L-arabinose isomerase from Escherichia coli.

L-Arabinose isomerase, EC 5.3.1.4, catalyzes the conversion of L-arabinose to L-ribulose, the first step in the catabolism of L-arabinose by Escherichia coli B/r. Patrick and Lee (1969) J. Biol. Chem. 244, 4277--4283) demonstrated that native L-arabinose isomerase is composed of six identical subunits of approximately Mr = 60,000. In this paper we describe an electron microscopy study of the arrangement of the six identical subunits. The isomerase is seen in two distinctly different orientations. The first has three subunits visible, with a 3-fold axis of symmetry, corresponding to a face-on view of two stacked, eclipsed trimers. The second orientation is rectangular in shape with 2-fold symmetry; suggesting a side-on view of the stacked trimers. The six identical subunits are thus arranged with D3 symmetry as in a trigonal prism. Measurements were made on the maximum profile of the three 2-fold axes of symmetry of the face-on orientations, and of both the long and short dimensions of the side-on orientation. The best estimate for the maximum profile of the 2-fold axes of symmetry of the face-on view is 106 +/- 8 A, using glutamine synthetase as an internal size standard. Measurements from micrographs of the isomerase alone, using an external magnification calibration, give the following results: for the maximum profile of the three 2-fold axes of symmetry of the face-on view, 132 +/- 7 A; for the long axis of the side-on view, 136 +/- 10 A; and for the short axis, 105 +/- 6 A. These measurements are consisting with the interpretation of the profiles as representing two different orientations of the L-arabinose isomerase.

Aldose-Ketose Isomerases↗
PubMed1978

Hybrid plasmids containing the araBAD genes of Escherichia coli B/r.

The DNA fragments generated by restriction endonuclease BamI which contain the araCBAD genes from E.coli B/r have been cloned. The DNA fragments containing ara genes were idenified by a compairson of the BamI fragments of lambdah80dara phages containing different ara deletion mutations. The ara genes were cloned into the plasmid pBR317, a derivative of ColE1. The cloned DNA fragments were analyzed by digestion with pairs of restriction endonucleases to determine the molecular weight of the chimeras and to identify the cloned ara DNA fragments. The cloned ara fragments were also identified by genetic complementation and recombination tests.

Arabinose↗
PubMed1978

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

The L-arabinose operon in Escherichia coli is a model system for the study of the control of gene expression. Maximal expression of the araBAD operon requires two positive control components: the araC protein-L-arabinose complex and the cyclic AMP receptor protein-cyclic AMP complex. Both araC protein and cyclic AMP receptor protein are required for the initiation of transcription of araBAD mRNA. We have used the plasmid pBR322 as a vector for cloning DNA fragments that contain the araBAD promoter. The cloned ara fragments were identified by both physical and genetic tests. A restriction map was constructed and the DNA sequence of the promoter was determined. The promoter contains a site that is similar to the RNA polymerase recognition sites in the galactose and lactose operons. It also contains a region similar to the known cyclic AMP receptor protein binding sites in the galactose and lactose operons.

Arabinose↗
PubMed1977

Constitutive mutations in the controlling site region of the araBAD operon of Escherichia coli B/r that decrease sensitivity to catabolite repression.

Strains of Escherichia coli B/r containing a deletion of the regulatory gene araC are Ara-. Slow-growing revertants of these strains were isolated and designated aralc because they contain a second mutation in a controlling site, aral, that allows for a low level of constitutive expression of the araBAD operon (Englesbert et al., 1969). We mutagenized aralc delta C strains and selected mutants that grow faster in mineral L-arabinose medium. The new mutations, called araXc, map very close to the original aralc mutations and are in the controlling site region between araB and araC. The aralcXc delta C strains have a higher constitutive level of expression of the araBAD operon than the aralc delta C parents. The araXc mutations are cis acting and decrease the araBAD operon's sensitivity to catabolite repression. The araBAD operon is expressed equally well in ara delta C and ara C cya crp backgrounds. The repressor form of ara C protein is able to repress the constitutive synthesis due to the ara Xc allele.

Arabinose↗
PubMed1977

Bromothymol blue and carbohydrate-sensitive plating media.

A new plating medium using bromothymol blue (BTB) indicator is described and compared with eosin-methylene blue (EMB), MacConkey, and Endo media. These media were tested with L-arabinose by plating fermenting and nonfermenting mutant strains of Escherichia coli. The minimum concentrations of L-arabinose that permitted differentiation of these strains were determined. Different concentrations were required for differentiating confluent patches of cells, isolated colonies, and closely spaced or adjacent colonies. L-Arabinose, L-rhamnose, D-lactose, and D-galactose were tested with modified enteric media and with BTB medium, again to determine minimum usable concentrations. BTB media and reformulated conventional media allowed detection of acidification, aerobically, at one-fifth to one-hundredth the (1%, wt/vol) concentration of carbohydrate used in standard indicator plates.

Aerobiosis↗
PubMed1976

Stabilization and size of araC protein.

AraC protein from Escherichia coli has been further stabilized and characterized. pH is a critical variable in conferring stability. araC protein has a sedimentation coefficient of 4.0 +/- 0.2s on standardized 5%-20% glycerol gradients. Its isoelectric point is at a pH of 7.1.

Arabinose↗