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Biomedical subjects

R G Fenwick

Publications and source records attributed to R G Fenwick.

At least 37 records · Page 2Linked to original sources
PubMed1991

Improved molecular diagnostics for ornithine transcarbamylase deficiency.

Since the cloning of the cDNA for X-linked ornithine transcarbamylase (OTC) in 1984, diagnostic accuracy of OTC deficiency for prenatal and carrier detection has been greatly improved by the use of linkage analysis. However, the use of RFLP-based diagnosis is limited in this and in other new mutation diseases. Here we report both the use of direct mutation detection by new PCR-based techniques and our experience with linkage-based diagnosis in 18 families. We have previously reported the use of chemical mismatch cleavage to detect mutations first in amplified mRNA and then in genomic DNA of patients. This technique has now been utilized for prenatal diagnosis. Primers for specific amplification of OTC exons 1, 3, 5, 9, and 10 have been developed and been employed to map deletions of the OTC gene in two families. These primers also have been used to detect alterations in the TaqI sites found in exons 1, 3, 5, and 9. Four novel mutations of the OTC gene leading to abolition of a TaqI site in the OTC cDNA were discovered. One of these mutations is in exon 1; two lie in exon 3; and one is in exon 9. In addition, we have used the PCR products as probes to identify the exon-specific bands seen on Southern blots and to map the polymorphic BamHI and MspI sites, which are commonly used for linkage analysis. This information will facilitate the interpretation of altered band patterns seen in deletion cases and in cases of point mutations affecting restriction sites. Utilization of the appropriate combination of these molecular techniques permitted accurate diagnostic evaluations in 17 of 18 families.

Base Sequence↗
PubMed1991

Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms.

To improve carrier detection and prenatal diagnosis for Duchenne and Becker muscular dystrophy families, we determined allele frequencies and measures of variation for four (dC-dA)n.(dG-dT)n loci identified within a deletion-prone region of the human dystrophin gene. The loci are highly polymorphic, with predicted heterozygosities of 71.6%-93.3%. Direct DNA sequence analysis of the (dC-dA)n.(dG-dT)n locus in intron 49 revealed an additional length polymorphism which varies by single-basepair increments, is adjacent to the dinucleotide repeat block, and enhances the polymorphic content of this marker. The four (dC-dA)n.(dG-dT)n loci are each easily amplified by PCR in two diplex reactions. The variability of allele lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing diagnostic information when RFLP analysis is uninformative. These markers have aided in identification of deletion mutations, exclusion of maternal cell contamination of chorionic villus samples, confirmation of paternity, and mapping of gene recombinations. The allele identification of these loci can be performed either with a radiolabel or with an automated, nonradioactive, fluorescent gel detection system.

Alleles↗
PubMed1990

A transforming Kirsten ras oncogene in an oral squamous carcinoma.

This investigation has employed the NIH 3T3 cell transfection assay in an effort to detect transforming genes in DNA from squamous carcinomas of the head and neck. Of 11 tumor DNAs tested, 1 DNA sample from a gingival squamous carcinoma was able to produce primary and secondary transformants containing the human K-ras oncogene. This is the first report of an activated ras oncogene derived from a carcinoma of the head and neck. Head and neck cancers may possess activated ras oncogenes more often than is indicated by this study because of the relative inefficiency of transfection assays in detecting large transforming genes such as K-ras.

Animals↗
PubMed1989

The apolipoprotein A-I gene is actively expressed in the rapidly myelinating avian peripheral nerve.

The expression of the apolipoprotein A-I (apo A-I) gene was investigated in the myelinating sciatic nerve. Hybridization analysis with an apo A-I cDNA probe obtained from a cDNA library of mRNA isolated from rapidly myelinating chick sciatic nerve indicated that apo A-I coding transcripts increase during development in the chick sciatic nerve in parallel with the increase of myelin lamellae. Substantial apo A-I-like immunoreactivity in chick sciatic nerve homogenates was detected by Western blotting. The amount of antigen increased from the 15-d embryonic stage to 1 d posthatch and then decreased. Two subcellular fractions corresponding to the cytoplasmic compartments were particularly enriched in apo A-I. apo A-I immunoreactivity was also found in highly purified myelin preparations. Immunohistochemical staining provided further evidence for the presence of apo A-I in the endoneurial compartment of the sciatic nerve. Electron microscopic examination of these fractions after negative staining showed the presence of spherical and disc-shaped particles resembling high density lipoproteins. The presence of apo A-I, cholesterol esters, phospholipids, and triacylglycerols in ultracentrifugal fractions corresponding to serum lipoproteins and the behavior of apo A-I on nondenaturing gradient gels implied that apo A-I was associated with lipid. Studies with short-term organ cultures of sciatic nerves from 1-d chicks strengthened the evidence for local synthesis and secretion of apo A-I and apo A-I-containing lipoproteins by this tissue. These results establish that the apo A-I gene is actively expressed in developing sciatic nerve during the period of rapid myelination. These findings support the hypothesis that apo A-I synthesized within the nerve participates in the local transport of lipids used in myelin biosynthesis.

Aging↗
PubMed1989

Loss of Harvey ras heterozygosity in oral squamous carcinoma.

This investigation of oral squamous carcinoma in five individuals revealed that four of the patients were constitutionally heterozygous at the c-Ha-ras-1 locus and that the tumour from one patient had lost that heterozygosity. The loss of c-Ha-ras-1 alleles provides a useful marker for detecting deletions of genetic material located on the short arm of chromosome 11 (11p) and has been found in association with a number of malignant tumours but has not been previously described in carcinoma of the head and neck. The repeated association of 11p deletions with malignancies has led to the postulation of a recessive cancer gene or tumour suppressor gene at this location involved in carcinogenesis and tumour progression. This study indicates that such a mechanism may contribute to the development of oral squamous carcinoma.

Carcinoma, Squamous Cell↗
PubMed1986

Characterization of transferable plasmids from Shigella flexneri 2a that confer resistance to trimethoprim, streptomycin, and sulfonamides.

A set of plasmids conferring resistance to several antibiotics, including the combination of trimethoprim and sulfamethoxazole, has been isolated from Escherichia coli following conjugative cotransfer from a clinical isolate of Shigella flexneri 2a. One of the plasmids, pCN1, was shown by subcloning and DNA sequencing to carry a gene encoding a trimethoprim-insensitive dihydrofolate reductase identical to that found in E. coli transposon 7. This plasmid was also shown to confer resistance to both streptomycin and spectinomycin by production of an adenylyltransferase that inactivated the drugs and the gene encoding this enzyme has also been sequenced. A second plasmid from the set, pCN2, was shown to inactivate streptomycin by a phosphotransferase mechanism and also to confer resistance to sulfonamides. The third plasmid from the set could not be correlated with a drug-resistance phenotype, but does appear to play a crucial role in plasmid mobilization.

Base Sequence↗
PubMed1985

Quantitative measurement of single- and double-strand breakage of DNA in Escherichia coli by the antitumor antibiotics bleomycin and talisomycin.

We developed an assay in which single-strand breakage (ssb) and double-strand breakage (dsb) of intracellular DNA by chemical agents can be accurately quantitated and differentiated. Escherichia coli cells containing plasmid pBR322 DNA were incubated with the antitumor antibiotics bleomycin A2 (BLM A2) or talisomycin A (TLM A). The plasmid DNA was isolated and then analyzed by electrophoresis on 1% agarose gels to separate the following conformational forms of plasmid DNA: (i) native, covalently closed, super helical, form I; (ii) nicked, relaxed circular, form II; and (iii) double-strand broken, linear, form III. Quantitation by densitometric analysis of the gels showed that BLM A2 and TLM A were equally active in terms of the concentrations of drug necessary to reduce equivalent amounts of form I DNA in the cells, whereas in vitro (using isolated pBR322 DNA as a drug substrate) twofold more TLM A than BLM A2 was required to produce an equivalent amount of reduction in form I DNA. TLM A produced more intracellular dsb than did BLM A2. The intracellular dsb activities (dsb/ssb ratio) measured from BLM A2 and TLM A were equivalent to those measured for the respective agents when isolated pBR322 DNA was used as the substrate. In E. coli both ssb and dsb were repaired, but TLM A damage was repaired more slowly and to a lesser extent, which may reflect the relative frequency of dsb.

Antibiotics, Antineoplastic↗
PubMed1985

Analysis of mobilization elements in plasmids from Shigella flexneri.

The mobilization properties of three plasmids were examined after cotransfer from Shigella flexneri to Escherichia coli. The largest plasmid, pCN1, was shown to be a conjugative R factor that could promote its own transfer and allow cotransfer of a 4.1-kilobase plasmid, pCN3; mobilization of the third plasmid, pCN2 (6.3 kilobases), required the presence of both pCN1 and pCN3. Sequences from pCN2 and pCN3 homologous to the bom (basis of mobilization) sites of ColE1 and pBR322 were localized by analysis of site-specific deletion derivatives generated in vivo during the transfer of composite plasmids and were characterized by DNA sequencing.

Base Sequence↗
PubMed1984

Amplification versus mutation as a mechanism for reversion of an HGPRT mutation.

We have used a cloned cDNA for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) to analyze the HGPRT gene and mRNA in an HGPRT-deficient mutant of Chinese hamster cells (RJK10) and its HGPRT-positive revertants. By Southern blot analysis, no DNA rearrangements were detected within the genes from any of the cell lines examined. However, four of five spontaneous revertants each contained 10- to 20-fold more copies of the HGPRT gene than did RJK10 or wild-type cells. In contrast, the gene was not amplified in four mutagen-induced revertants. The RJK10 mutation did not alter the size or concentration of HGPRT mRNA and representatives of the revertants contained the mRNA in amounts proportional to the number of genes they carried. Examples of clones with either stable or unstable gene amplification were identified and their HGPRT-positive phenotypes were shown to be dependent on the gene amplification. In a stably amplified revertant, the extra genes were found to be syntenic with the X chromosome marker glucose-6-phosphate dehydrogenase. In an unstable revertant only one of the 10 to 20 copies of the gene could be shown to be X linked. Thus, we found that RJK10 can revert by at least two distinct mechanisms: amplification of the HGPRT gene, which occurred spontaneously, or point mutation, which predominated after exposure to mutagens.

Animals↗
PubMed1983

Deletion and amplification of the HGPRT locus in Chinese hamster cells.

Somatic cell selective techniques and hybridization analyses with a cloned cDNA probe were used to isolate and identify Chinese hamster cell lines in which the X-linked gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) has been altered. Two of 19 HGPRT-deficient mutants selected were found to have major DNA deletions affecting the HGPRT locus. Cytogenetic studies revealed that the X chromosome of each deletion mutant had undergone a translocation event, whereas those from the remaining 17 mutants were normal. Phenotypic revertants of the thermosensitive HGPRT mutant RJK526 were isolated, and amplification of the mutant allele was shown to be the predominant mechanism of reversion. Comparisons of restriction enzyme fragments of DNA from deletion versus amplification strains identified two regions of the Chinese hamster genome that contained homology to the cDNA probe. One was shown to be much larger than the 1,600-nucleotide mRNA for HGPRT and to be comprised of linked fragments that contained the functional HGPRT gene. The second was neither transcribed nor tightly linked to the functional gene. These initial studies of HGPRT alterations at the level of DNA thus identified molecular mechanisms of phenotypic variation.

Animals↗
PubMed1980

Reversion of a mutation affecting the molecular weight of HGPRT: intragenic suppression and localization of X-linked genes.

RJK39 is a clone of Chinese hamster cells carrying a mutation which inactivates hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and reduces the apparent molecular weight of the enzyme. Using mutagens, we have isolated subclones of RJK39 which will grow in the counterselective HAT medium. Some continue to be HGPRT-deficient and survived the selection because they are resistant to aminopterin. In all but one of the HGPRT-positive revertants, the molecular weight of the enzyme returned to the wild-type value. However, the phenotypes of several of those strains indicate they produce altered forms of HGPRT, and one can conclude that second-site mutations must be able to cause intragenic suppression of the original mutation in RJK39. One of the revertants is pseudotetraploid and functionally heterozygous at the HGPRT locus. Segregation studies with that clone localized the genes for HGPRT, glucose-6-phosphate dehydrogenase, and phosphoglycerate kinase to the short arm of the Chinese hamster X chromosome.

Aminopterin↗
PubMed1977

Mutations affecting the antigenic properties of hypoxanthine-guanine phosphoribosyl transferase in cultured Chinese hamster cells.

Cells of the mutant Chinese hamster strain RJK10 do not contain either hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT) or protein that cross-reacts immunologically with HGPRT. HGPRT+ revertants have been isolated from RJK10 and those strains produce HGPRT with altered antigenic properties. HGPRT from the revertant cells is less reactive with anti-HGPRT serum than enzyme from the wild-type cells, and enzymes from the two sources are immunoprecipitated independently from mixtures of cell extracts. Thus one or more of the antigenic determinants present on Chinese hamster HGPRT are either missing or present in an altered form on HGPRT from revertants of RJK10. This indicates that RJK10 carries a mutation in the structural gene for HGPRT and that secondary mutations in the gene give rise to the revertants that produce the antigenically altered enzymes.

Cell Line↗
PubMed1977

Forward and reverse mutations affecting the kinetics and apparent molecular weight of mammalian HGPRT.

Chinese hamster cells selected for resistance to 8-azaguanine following mutagenesis have hypoxanthine-guanine phosphoribosyltransferase (HGPRT; E.C. 2.4.2.8) with characteristics compatible with different mutations in the structural gene for that enzyme. Using immunopurification and SDS-polyacrylamide electrophoresis, mutants producing antigenically active forms of the enzyme can be analyzed for changes in the molecular weight of HGPRT. Enzyme subunits from mutants RJK3 and RJK39 are reduced in molecular weight by an estimated 4 and 2%, respectively. HGPRT activity is not detectable in RJK39. The enzyme from RJK3 is active but has altered substrate binding properties. Enzymes from two other mutants with altered kinetic properties, RJK44 and RJK47, have normal molecular weights. The genetic alterations of RJK44 and 47 are probably missense mutations, while RJK3 and 39 might contain either deletions or mutations causing premature peptide chain termination. Somatic cell hybridization between RJK39 and a revertant of that strain with HGPRT of normal molecular weight revealed that the revertant probably arose by intragenic mutation rather than extragenic mutation or suppression.

Aminopterin↗
PubMed1977

Photoproducts from DNA pyrimidine bases and polycyclic aromatic hydrocarbons.

The major photoproduct formed between benzo[a]pyrene and thymine is identified as 1-(benzo[a]pyren-6-yl)-thymine by means of spectroscopic analysis and isotopic syntheses. Irradiation of 1-methylcytosine hydrochloride and anthracene gives two isolable photoproducts of which one is assigned the structure 5-(anthracen-9-yl)-1-methylcytosine.

Anthracenes↗
PubMed1975

Mutant chinese hamster cells with a thermosensitive hypoxanthine-guanine phosphoribosyltransferase.

By selecting variants of Chinese hamster cells that were resistant to 6-thioguanine at 39 degrees C, but which would continue to grow in HAT medium at 33 degrees C, we have isolated cell lines with thermosensitive phenotypes. These clones form colonies in HAT medium and incorporate 14-C-hypoxanthine much more efficiently at 33 degrees C than at 39 degrees C. The specific activity of hypoxanthine-guanine phosphoribo-syltransferase is at least 10 times higher in variant cells grown at 33 degrees C than in those grown in 39 degrees C, and the enzymes from the variant clones are inactivated in vitro at 39 degrees C 7-9 times more rapidly than is the enzyme from wild-type cells. The results are consistent with the conclusion that the selected clones have missense mutations in the structural gene for the enzyme.

Adenine↗