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W J Craigen

Publications and source records attributed to W J Craigen.

47 records · Page 3Linked to original sources
PubMed1991

Induction of the Cyp1a-1 dioxin-responsive enhancer in transgenic mice.

Cyp1a-1, whose product, aryl hydrocarbon hydroxylase, assists in detoxification of polycyclic aromatic hydrocarbons, is the best characterized of the murine cytochrome P450 genes. The Cyp1a-1 dioxin-responsive enhancer region has been previously analyzed in vitro and found to induce expression of heterologous genes upon exposure of transfected cells to various aromatic hydrocarbons. A 2.58 kbp DNA fragment containing the Cyp1a-1 enhancer elements and promoter region was coupled to the chloramphenicol acetyltransferase (CAT) reporter gene and used to create transgenic mice. CAT assays were performed on tissues harvested from three different lines of transgenic mice following mock-induction or induction using the aromatic hydrocarbon, 3-methylcholanthrene. Basal levels of expression were detected in the spleen and small bowel of non-induced mice, with little or no expression detected in the liver. Treatment with 3-methylcholanthrene increased hepatic expression levels by as much as 10,000-fold. More modest levels of induction were also recorded in the spleen, small bowel, kidney, and lung. The results indicate that the dioxin-responsive enhancer region functions as a strongly inducible promoter in vivo. Differences in the response to induction between male and female mice suggest that Cyp1a-1 expression may be governed in a gender related manner.

Animals↗
PubMed1990

Ectopic correction of ornithine transcarbamylase deficiency in sparse fur mice.

The sparse fur (spf) mutant mouse is a model for human X-linked ornithine transcarbamylase (OTC) deficiency. Human OTC cDNA placed under transcriptional control of the mouse OTC promoter was microinjected into fertilized oocytes of spf mice. Two founder lines of transgenic mice were phenotypically and biochemically corrected for OTC deficiency by the expression of the human gene at high levels in the small intestine with little or no expression occurring in the liver. The tissue pattern of expression of transgenic mice bearing the chloramphenicol acetyltransferase gene placed under the control of the mouse OTC promoter parallels these results. These experiments demonstrate that human OTC cDNA is selectively expressed in small bowel by a truncated OTC promoter, and such ectopic expression corrects the spf phenotypic and metabolic features of this inborn error. These data suggest that somatic gene therapy of OTC deficiency can be achieved by intestine-targeted gene transfer.

Animals↗
PubMed1990

Cloning and expression of a mammalian peptide chain release factor with sequence similarity to tryptophanyl-tRNA synthetases.

The termination of protein synthesis is encoded by in-frame nonsense (stop) codons. Most organisms use three nonsense codons: UGA, UAG, and UAA. In contrast to sense codons, which are decoded by specific tRNAs, nonsense codons are decoded by proteins called release factors (RFs). Here we report the cloning of a mammalian RF cDNA by the use of monoclonal antibodies specific for rabbit RF. Functional studies showed that, when expressed in Escherichia coli, the protein encoded by this cDNA has in vitro biochemical characteristics similar to those of previously characterized mammalian RFs. DNA sequencing of this eukaryotic RF cDNA revealed a remarkable sequence similarity to bacterial and mitochondrial tryptophanyl-tRNA synthetases, with the greatest similarity confined to the synthetase active site, and no obvious similarity to bacterial RFs.

Amino Acid Sequence↗
PubMed1990

Recent advances in peptide chain termination.

Peptide chain termination occurs when a stop codon is decoded by a release factor. In Escherichia coli two codon-specific release factors (RF1 and RF2) direct the termination of protein synthesis, while in eukaryotes a single factor is required. The E. coli factors have been purified and their genes isolated. A combination of protein and DNA sequence data reveal that the RFs are structurally similar and that RF2 is encoded in two reading frames. Frame-shifting from one reading frame to the next occurs at a rate of 50%, is regulated by the RF2-specific stop codon UGA, and involves the direct interaction of the RF2 mRNA with the 3' end of the 16S rRNA. The RF genes are located in two separate operons, with the RF1 gene located at 26.7 min and the RF2 gene at 62.3 min on the chromosome map. Ribosomal binding studies place the RF-binding region at the interface between the ribosomal subunits. A possible mechanism of stop-codon recognition is reviewed.

Codon↗
PubMed1990

3-Hydroxy-3-methylglutaryl-CoA lyase deficiency as detected by radiochemical assay in cell extracts by thin-layer chromatography, and identification of three new cases.

In this rapid radiochemical assay for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (I) activity in cell extracts, DL-3[glutaryl-3-14C]hydroxy-3-methylglutaryl-coenzyme A is used as substrate and the radiochemical product, [3-14C]acetoacetic acid, is converted to the more stable [3-14C]-3-hydroxybutyric acid in the presence of added NADH and 3-hydroxybutyrate dehydrogenase. Substrate and product are separated and quantified by thin-layer chromatography on cellulose (solvent system: butanol/water/formic acid, 77:13:10 by vol). All reagents for the assay are commercially available. No detailed column chromatography or spectrophotometry is required. Thus the assay is suited for any clinical laboratory. Using this procedure, we studied cultured fibroblasts or lymphocytes isolated from whole blood from five patients in whom the urinary organic acid profile was suggestive of deficiency of I. Three patients had less than or equal to 18% of control I activity in fibroblast or lymphocyte extracts. The other two had activity within the normal range. In one of the latter cases, urinary excretion of three of the characteristic acids disappeared with age, and 3-hydroxyisovaleric acid excretion was within normal limits. The other case presented with urinary excretion of moderate amounts of all four metabolites and the characteristic absence of urinary ketone bodies. Evidently, confirmatory enzyme studies should be undertaken, even when the profile of urinary organic acids appears definitive for this deficiency.

Cells, Cultured↗
PubMed1988

Rapid and precise mapping of the Escherichia coli release factor genes by two physical approaches.

The termination of protein synthesis in Escherichia coli requires two codon-specific factors termed RF1 and RF2. RF1 mediates UAA- and UAG-directed termination, while RF2 mediates UAA- and UGA-directed termination. The genes encoding these factors have been isolated and sequenced, and RF2 was found to be encoded in two separate reading frames. The map position of RF1 has been reported as 27 min on the E. coli chromosome, while the RF2 map position has not yet been identified. In this study, two new and independent methods for gene mapping, using pulsed field gel electrophoresis and an ordered bacteriophage library spanning the entire chromosome, were used to localize the map position of the RF2 gene. In addition, the location of the RF1 gene was more precisely defined. The RF2 gene is located at 62.3 min on the chromosome, while the RF1 gene is located at 26.7 min. This approach to mapping cloned genes promises to be a rapid and simple means for determining the gene order of the genome.

Amino Acid Sequence↗
PubMed1987

The function, structure and regulation of E. coli peptide chain release factors.

The termination of protein synthesis in Escherichia coli depends upon the soluble protein factors RF1 or RF2. RF1 catalyzes UAG and UAA dependent termination, while RF2 catalyzes UGA and UAA dependent termination. The proteins have been purified to homogeneity, their respective genes isolated, and their primary structures deduced from the DNA sequences. The sequences reveal considerable conserved homology, presumably reflecting functional similarities and a common ancestral origin. The RFs are encoded as single copy genes on the bacterial chromosome. RF2 exhibits autogenous regulation in an in vitro translation system. The mechanism of autoregulation appears to be an in-frame UGA stop codon that requires a 1+ frameshift for the continued synthesis of the protein. Frameshifting prior to the inframe stop codon occurs at a remarkably high frequency by an unknown mechanism. Future studies will be directed at understanding how RFs interact with the ribosomal components, and further defining the mechanism of RF2 frameshifting.

Amino Acid Sequence↗
PubMed1986

The 5' flanking region of the ornithine transcarbamylase gene contains DNA sequences regulating tissue-specific expression.

Ornithine transcarbamylase (OTCase) is a mitochondrial matrix enzyme that catalyzes the 2nd step in the mammalian urea cycle. The gene encoding OTCase is located on the X chromosome and expression of OTCase is limited almost exclusively to hepatocytes. We have characterized a lambda phage recombinant, isolated from a mouse genomic library, that spans the first two exons of the mouse OTCase gene. Nuclease S1 mapping and primer extension analysis of this clone allowed us to determine that the transcription start site is 136 base pairs (bp) upstream from the translation initiation codon. Two TATA-like sequences were found 25 and 153 bp from the transcription initiation point. An 800-bp fragment containing the 5' flanking region of the OTCase gene was fused upstream to the coding sequence of the chloramphenicol acetyltransferase gene to assay promoter activity. This plasmid was introduced into mouse fibroblast NIH 3T3 cells and human hepatoma Hep G2 cells by the calcium phosphate co-precipitation method. After DNA transfection chloramphenicol acetyltransferase activity was observed only in Hep G2 cells. We conclude that this 800-bp fragment contains sufficient information to control OTCase gene expression in a tissue-specific manner, probably by interacting with trans-acting factor(s) which are not present in the other cell line.

Acetyltransferases↗
PubMed1985

Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2.

Escherichia coli peptide chain release factors are proteins that direct the termination of translation in response to specific peptide chain termination codons. The mechanisms of codon recognition and peptidyl-tRNA hydrolysis are unknown. We have characterized the genes encoding release factor 1 (RF-1) and release factor 2 (RF-2) to study the structure-function relationships of the proteins and their regulation in the bacterium. In this report, we present the gene structure of RF-1 and RF-2, and a partial peptide sequence of RF-2. RF-1 and RF-2 are highly homologous in their primary structure. In addition, an in-frame premature opal (UGA) termination codon is located within the RF-2 coding region at amino acid position 26. This region of the protein was sequenced by automated Edman degradation to confirm the predicted reading frame, and a second independent isolate of the RF-2 gene was identified and sequenced to confirm the DNA sequence. These results imply that a frameshift occurs prior to the premature termination codon, thus allowing for translation of RF-2 to be completed. This may represent a mechanism of translational control of RF-2 expression. An alternative possible means of translational regulation is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Expression of peptide chain release factor 2 requires high-efficiency frameshift.

Peptide chain release factors are soluble proteins that participate in the stop codon-dependent termination of polypeptide biosynthesis. In Escherichia coli, two release factors are necessary for peptide chain termination: release factor 1 (RF1) specifies UAG- and UAA-dependent termination whereas release factor 2 (RF2) specifies UGA- and UAA-dependent termination. Release factors are found in low concentrations relative to other translation factors, suggesting that their expression is tightly regulated and, accordingly, making the study of their structure-function relationship difficult. RF1 and RF2 exhibit significant sequence homology, probably reflecting their similar functions and perhaps a common evolutionary origin. DNA and peptide sequencing have suggested the existence of a unique mechanism for the autogenous regulation of RF2 in which an in-frame UGA stop codon requires an obligatory +1 frameshift within the coding region of the RF2 gene. In this report we present in vitro experimental results consistent with the autogenous regulation of RF2. Additionally, we used RF2-lacZ gene fusions to demonstrate that autogenous regulation occurs, at least in part, by premature termination at the in-frame stop codon, since deletion of this stop codon leads to overproduction of the RF2-LacZ fusion protein. Frameshifting at this premature termination codon occurs at the remarkably high rate of 50%.

Codon↗