Oestrogenic effects of plant foods in postmenopausal women.
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Aged↗
Biomedical subjects
G Wilcox
Publications and source records attributed to G Wilcox.
Discrepancies in hemoglobin levels.
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Female↗
Improved patient selection for angioplasty utilizing color Doppler imaging.
The accepted approach to patients with intermittent calf claudication is nonoperative unless the symptoms are truly incapacitating, in which case arteriography and either percutaneous angioplasty or operation is indicated. Arteriography is considered a preoperative procedure. However, with improvements in percutaneous angioplasty, we have altered our approach to those patients with limiting, but not incapacitating, claudication. These patients are given the choice of conservative treatment or percutaneous angioplasty, but not operation. In an attempt to limit arteriography to only those patients who would benefit from angioplasty, we have employed color Doppler imaging as a screening technique. Over a 2-year period, 62 patients with limiting claudication were evaluated with color Doppler imaging. Thirty-six patients had long occlusions and did not undergo arteriography since they were not believed to be candidates for percutaneous angioplasty. Three short (less than 5 cm) occlusions and 23 localized stenoses were identified in the superficial femoral and popliteal arteries. Angiography confirmed the Doppler findings in all 26 cases. In 24 patients, dilatation was successful, whereas 2 patients' arteries could not be dilated. Two stenoses recurred early and were redilated. There were no complications from the angioplasties. We conclude that in the subset of patients with limiting, but not incapacitating, claudication, color Doppler imaging can accurately select those patients who will benefit from angioplasty. In this way, patients can be spared unnecessary angiograms.
Angiography↗
Effect of mutations in the cyclic AMP receptor protein-binding site on araBAD and araC expression.
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.
Arabinose↗
Characterization of the Erwinia carotovora pelA gene and its product pectate lyase A.
The pelA gene from Erwinia carotovora strain EC encodes pectate lyase A (PLa). The gene was cloned and sequenced. The PLa was purified from Escherichia coli containing the pelA gene on a recombinant plasmid. The optimum pH for the enzyme reaction is at pH 8.5; the optimum temperature for the enzyme reaction is at 40 degrees C; and the purified PLa alone can macerate potato slices. Nucleotide sequence of pelA and the deduced polypeptide sequence of PLa were compared with the pelB gene of E. carotovora and the deduced amino acid sequence of PLb. The nucleotide sequences are 82% homologous and the amino acid sequences are 88% homologous.
Amino Acid Sequence↗
Novel activation of araC expression and a DNA site required for araC autoregulation in Escherichia coli B/r.
Mutations in the araC gene have been isolated which alter both the activator and autoregulatory functions of AraC protein (L.G. Cass and G. Wilcox, J. Bacteriol. 166:892-900, 1986). In this study, the effect of each araC mutation on autoregulation was characterized in vivo and in vitro in the presence of L-arabinose. The effect of L-arabinose in some of these araC mutants revealed a novel activation of araC expression which was not observed in the araC+ cell. Experiments were therefore focused on understanding the mechanism of this novel activation. We describe a systematic analysis of the effect of mutations within the known regulatory binding sites for araBAD and araC transcription on araC expression. Our results suggest that the novel activation of araC expression requires the AraC activator-binding site, araI, and the cyclic AMP receptor protein-cyclic AMP complex-binding site. We also found that in the absence of L-arabinose, the araI site was required for maximal autoregulation by the wild-type AraC protein.
AraC Transcription Factor↗
Expression of foreign proteins in microorganisms.
The various alternative strategies for the expression of heterologous proteins in microorganisms are reviewed. To illustrate how these general considerations can be addressed in particular cases, the expression of chimeric human-mouse antibodies in Escherichia coli and the production of thaumatin, an intensely sweet plant protein, in yeast, are described.
Gene Expression Regulation↗
The araC gene of Citrobacter freundii.
The araC gene of Citrobacter freundii was cloned into plasmid pBR322 and expressed in Escherichia coli and Salmonella typhimurium. The nucleotide sequence and the predicted translational product were determined and compared to those of E. coli, S. typhimurium and Erwinia carotovora. The predicted translational product is 281 amino acids (aa) long, identical in size to that of S. typhimurium, and is 11 and 29 aa shorter than that of E. coli and E. carotovora, respectively. The nucleotide sequence of the araC gene of C. freundii is 83% homologous to the araC genes of both E. coli and S. typhimurium, but only 60% homologous to that of E. carotovora with respect to the regions they share. The predicted amino acid sequence is highly conserved and shows 96% and 94% homology to S. typhimurium and E. coli, respectively. E. carotovora shows only a 58% aa homology. The activator and autoregulatory activities of each plasmid encoded AraC protein in a S. typhimurium araC::lacZ protein fusion strain were examined.
Amino Acid Sequence↗
Characterization of the Erwinia carotovora pelB gene and its product pectate lyase.
The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.
Amino Acid Sequence↗
Mutations resulting in promoter-like sequences which enhance the expression of araC in Salmonella typhimurium.
The araC gene in Salmonella typhimurium is autogeneously regulated. Nine non-self-regulated mutants were isolated by selecting for increased expression of an araC-lacZ fusion in the presence of a repressing AraC protein. S1 mapping experiments demonstrated that the effect of the mutations was to increase the amount of araC mRNA in the cell. The 5'-end of the major araC transcript was the same in the wild-type (wt) and mutant strains. DNA sequence analysis showed that all nine mutations occurred in the araC promoter. Two mutations were a G-to-T transversion at position -25, six were a G-to-T transversion at position -47 and one was a single bp deletion at position -83. The data suggest that the mutations have created a new RNA polymerase binding site which enhances transcription from the wt start point.
Arabinose↗
Mutations in the araC regulatory gene of Escherichia coli B/r that affect repressor and activator functions of AraC protein.
Mutations in the araC gene of Escherichia coli B/r were isolated which alter both activation of the araBAD operon expression and autoregulation. The mutations were isolated on an araC-containing plasmid by hydroxylamine mutagenesis of plasmid DNA. The mutant phenotype selected was the inability to autoregulate. The DNA sequence of 16 mutants was determined and found to consist of seven different missense mutations located within the distal third of the araC gene. Enzyme activities revealed that each araC mutation had altered both autoregulatory and activator functions of AraC protein. The mutational analysis presented in this paper suggests that both autoregulatory and activator functions are localized to the same determinants of the AraC protein and that the amino acid sequence within the carboxy-terminal region of AraC protein is important for site-specific DNA binding.
Arabinose↗
An improved DNA sequencing strategy.
A modification of Hong's systematic DNA sequencing strategy is described. The original procedure has been simplified and transfectant yield increased. After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel. After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site. The original intact DNA is linearized whereas the deleted subclone is not. The background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora. A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol.
Bacteriophages↗
Cloning of the pectate lyase genes from Erwinia carotovora and their expression in Escherichia coli.
A hybrid cosmid coding for pectate lyase (PL) activity was identified from an Erwinia carotovora genomic library by an immunological screening method. A 7-kb DNA fragment was identified which codes for three proteins identical in size to proteins with PL activity purified from E. carotovora culture supernatants. The three proteins had apparent Mrs of 41, 44 and 44 X 10(3) as estimated by SDS-PAGE. None of the PLs were exported from Escherichia coli strain HB101 but all were found in the periplasmic space. Plant tissue was macerated by the PLs made in E. coli.
Bacterial Proteins↗
The araBAD operon of Salmonella typhimurium LT2. I. Nucleotide sequence of araB and primary structure of its product, ribulokinase.
Hybrid plasmids containing the araBAD operon of Salmonella typhimurium LT2 were characterized by Southern blot and genetic analyses. The nucleotide sequence of araB was determined. The araB gene product, ribulokinase (EC 2.7.1.16), was purified and the results of amino acid composition analysis and partial amino acid sequence are in agreement with predictions from the DNA sequence. Ribulokinase is 569 amino acid residues long and has a calculated Mr of 61 793. Ribulokinase shares significant homology with xylulose kinase from Escherichia coli. Codon usage in the araB gene does not favor those codons which have intermediate codon-anticodon binding energy.
Amino Acid Sequence↗
The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase.
The nucleotide sequence of gene araA of Salmonella typhimurium LT2 has been determined. The gene encodes an L-arabinose isomerase (EC 5.3.1.4) of 500 amino acid residues with a calculated Mr of 55814. The ATG start codon of araA is 10 bp distal to the TAA termination codon of araB. A presumed ribosome-binding site (RBS) "TAAGGA" 7 bp from the ATG codon overlaps the stop codon of araB. L-Arabinose isomerase was purified and the amino acid composition is in agreement with that predicted from the DNA sequence. The NH2-terminus of the protein is modified as the sequence cannot be analyzed by the automated Edman degradation. Amino acid composition analyses of both NH2-terminal and C-terminal cyanogen bromide (CNBr) cleaved peptides and partial amino acid sequence of the C-terminal peptide are consistent with the deduced amino acid sequence.
Aldose-Ketose Isomerases↗
The araBAD operon of Salmonella typhimurium LT2. III. Nucleotide sequence of araD and its flanking regions, and primary structure of its product, L-ribulose-5-phosphate 4-epimerase.
The araD gene of Salmonella typhimurium LT2 consists of 744 nucleotides and has an ORF coding for a protein of 248 amino acid residues with a calculated Mr of 27059. The product of araD, L-ribulose-5-phosphate 4-epimerase, was purified and the amino terminal sequence was determined. It is identical to that predicted by the ORF. A 143-bp intercistronic region was found between the araA and araD genes which can form stem-loop structures with calculated free energies of up to -76.7 kcal/mol. Three sets of sequences within this 143-bp region are comparable to a consensus sequence deduced from several known intercistronic regions. Approximately equimolar amounts of the araBAD operon products were present in L-arabinose-induced cells. Sequences similar to both rho-independent and rho-dependent transcription terminators are present after the araD gene.
Amino Acid Sequence↗
araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora.
The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.
Amino Acid Sequence↗