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L H Thompson

Publications and source records attributed to L H Thompson.

At least 19 recordsLinked to original sources
PubMed1992

Construction of human XRCC1 minigenes that fully correct the CHO DNA repair mutant EM9.

The human gene that corrects the DNA repair defect of the CHO cell mutant EM9 is designated XRCC1 and is the first human gene to be cloned that has an established role in DNA strand-break repair. In this study, either an XRCC1 cosmid genomic fragment or synthetic oligonucleotides were ligated to an incomplete XRCC1 cDNA to generate two full-length XRCC1 cDNA constructs. The ability of these minigene constructs to restore normal levels of sister chromatid exchange (SCE) to EM9 upon transfection was demonstrated, and the transfectants grew at normal rates in selective medium that is fully toxic to EM9 cells. Constructs in which the XRCC1 open reading frame (ORF) was transcribed from the SV40 early promoter or the genomic XRCC1 native promoter were compared in their efficiency of correction. EM9 transfectants derived from the SV40 promoter displayed fewer SCEs and lower sensitivity to CldUrd than either AA8 wild-type cells or transfectants containing the ORF transcribed from the native promoter.

Animals↗
PubMed1992

Alterations in expression and structure of the DNA repair gene XRCC1.

The repair-associated gene XRCC1 was previously cloned by complementing the hamster mutant EM9, which has a high rate of spontaneous SCE and hypersensitivity to DNA damaging agents. In analyzing XRCC1 gene expression, similar levels of steady-state mRNA were found in normal cells, Bloom's syndrome cells with altered SCE, and in squamous carcinoma cells with differential X-ray sensitivity. An EcoRI restriction fragment-length polymorphism previously identified in XRCC1 did not correlate with the repair phenotypes of these cells. The mRNA of XRCC1 decreased to 20-40% after treatment of cells with a DNA damaging agent. XRCC1 also showed tissue specific expression in rats. The mRNA levels were high in testis (7-8 fold), ovary (3-4 fold) and brain (4-5 fold), when compared with those in intestine, liver and spleen (1-2 fold). These data and the high levels of XRCC1 protein detected in testis indicate that XRCC1 may play an important role in DNA processing during meiogenesis and recombination in germ cells.

Amino Acid Sequence↗
PubMed1992

Epidemiology of bluetongue viruses in the American tropics. Regional Bluetongue Team.

A study of the epidemiology of bluetongue viruses is in progress with the collaboration of 11 Central American and Caribbean countries. To date, over 200 bluetongue virus isolates have been obtained from cattle and sheep in sentinel groups distributed in the participating countries. Bluetongue serotypes identified include 1, 3, 6, and 12, virus types not previously recorded in the Western Hemisphere. Although the clinical impact of bluetongue virus infections in this hyperendemic environment appears to be minimal, the ubiquity of infection causes restrictions on the export of ruminant livestock and germ plasm. The stability of the Caribbean region ecosystem and the long-range implications of the interface with the northern temperate bluetongue virus ecosystem are reviewed.

Animals↗
PubMed1992

Specificity of molecular hybridization techniques for the detection of bluetongue virus serotypes in Culicoides variipennis.

Direct blot hybridization (DBH) and sandwich hybridization (SH) were evaluated for their ability to detect bluetongue virus (BTV) RNA in the biting midge Culicoides variipennis (Coquillett). Probes were derived from the L3 RNA segment of BTV, serotype 17. RNA of the five BTV serotypes occurring in the USA (BTV-2, BTV-10, BTV-11, BTV-13, and BTV-17) was extracted from pools of varying numbers of infected and uninfected biting midges and assayed by direct blot and sandwich hybridization tests. Direct blot hybridization using an RNA transcript probe or cDNA probe was a fast, efficient and sensitive technique, detecting as few as one midge infected with any BTV serotype in a pool of 50 or 100. Sandwich hybridization was able to detect the homologous serotype, BTV-17, in pools containing a single infected midge in a total of 50 or 100. However, detection of the heterologous serotypes, BTV-10, BTV-11, and BTV-13, was limited to pools containing 5 or more infected midges in a total of 50, and BTV-2 was undetectable by SH. Hybridization techniques provide an alternative to the conventional detection methods of inoculation of cell culture or embryonated chicken eggs for detection of BTV.

Animals↗
PubMed1992

A Chinese hamster ovary cell mutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatid exchanges.

We have isolated a Chinese hamster ovary cell mutant hypersensitive to monofunctional alkylating agents. The mutant, designed as EM-C11, showed hypersensitivity to ethyl methanesulfonate (EMS), methyl methanesulfonate and ethylnitrosourea (8-, 7- and 2-fold, respectively, based on D10 values). About 2-fold increased sensitivity towards 4-nitroquinoline-1-oxide and only slightly increased sensitivity to X-rays (1.4-fold) and mitomycin C treatment (1.6-fold) were found in this mutant. EM-C11 was not hypersensitive to UV irradiation nor to adriamycin. The EM-C11 cells showed approximately 10-fold higher level of spontaneous sister chromatid exchange. The level of spontaneous chromosomal aberrations was 2- to 3-fold higher, but the frequency of EMS-induced chromosomal aberrations was approximately 10-fold higher in the mutant cells, in agreement with the increased sensitivity to killing. As measured by alkaline elution, EM-C11 cells showed a defect in the rejoining of single-strand DNA breaks after exposure to X-rays and even more so after the EMS treatment. Genetic analysis revealed that the EM-C11 mutant belongs to the same complementation group as the EM9 mutant described earlier. The XRCC1 gene which complements the defect in EM9 also complements the defect in EM-C11, confirming that these two independently isolated mutants are defective in the same gene.

Alkylating Agents↗
PubMed1992

Observations on "Cost containment and new priorities in the European community" by Brian Abel-Smith.

A commentary on the article by Brian Abel-Smith briefly explores why the United States lags behind Europe in controlling health care costs. Three important factors are, first, that until recently the real cost of health insurance was not apparent to U.S. workers and political leaders; second, Americans prefer to try competition before resorting to other strategies; and third, responsibility for financing health care is divided among a large number of independent entities, while government regulation is split between the state and federal levels. At the same time, governments and private insurers in the United States have developed new health delivery structures and reimbursement mechanisms that the Europeans may find useful. A convergence of the two systems would be to the benefit of both.

Budgets↗
PubMed1992

Expression of the polymorphic human DNA repair gene XRCC1 does not correlate with radiosensitivity in the cells of human head and neck tumor cell lines.

The human X-ray repair cross-complementing gene 1 (XRCC1) is highly polymorphic in the cells of human head and neck squamous cell carcinoma lines and in a variety of other human cell lines. We describe six patterns seen in Southern analysis of EcoRI-digested genomic DNA. Levels of XRCC1 mRNA vary in cells of human head and neck cell lines as seen in northern blots. Expression of this gene in cell culture does not correlate with radiobiological parameters, clinical outcome, or the DNA restriction fragment length polymorphisms.

Blotting, Northern↗
PubMed1991

Introduction of cytochrome P450IA2 metabolic capability into cell lines genetically matched for DNA repair proficiency/deficiency.

We introduced into the CHO cell line the cDNA of the mouse cytochrome P3450 (P450IA2) gene, which oxidizes aromatic amines. A cDNA clone of P3450 was transfected into mutant UV5 cells, which is defective in nucleotide excision repair. Expression of the P3450 cDNA was measured using 9000 x g supernatant (S9) fractions from CHO cells to evaluate Salmonella TA1538 mutagenicity with the mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The P3450-expressing clone UV5P3 was reverted to repair proficiency using ethyl methanesulfonate to obtain the UV-resistant clone 5P3R2, which maintained the same level of P3450 protein activity as UV5P3. These genetically similar cell lines were compared for toxicity and mutation induction at the aprt locus. With 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (the most prevalent mutagen found in fried beef) the differential sensitivity due to repair deficiency/proficiency was approximately 40-fold, and with IQ there were smaller, but significant, differences in sensitivity. These genotoxic effects occurred at doses that were approximately 10 times lower than those that previously gave similar effects in S9-mediated assays. Thus, these cell lines should be valuable for genotoxicity analysis as well as important for assessing DNA repair when evaluating compounds that undergo metabolic activation.

Animals↗
PubMed1991

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.

Animals↗
PubMed1991

Properties and applications of human DNA repair genes.

The importance of understanding DNA repair processes is discussed in terms of the origins of human cancer. Several human repair genes have been mapped to specific human chromosomes using somatic cell hybrids. It is noteworthy that 3 of these genes lie in the same region of chromosome 19: genes ERCC1 and ERCC2, which are involved in nucleotide excision repair, and XRCC1, which is involved in the repair of strand breaks. The genes XRCC1 and ERCC2 were cloned from cosmid libraries prepared from DNA transformants of the CHO mutants EM9 and UV5, respectively. Analysis of the cDNA sequence of ERCC2 showed that the protein encoded by this gene is highly homologous (73%) to the RAD3 repair protein in the yeast Saccharomyces cerevisiae. Thus, the known properties of RAD3 combined with the high homology provide the first insight about the biochemical role of a human repair protein involved in the incision step of nucleotide excision repair. So far XRCC1 is the only cloned mammalian gene involved in repairing damage from ionizing radiation. The UV5 mutant line was also applied to problems in environmental mutagenesis by introducing the mouse cytochrome P(3)450 (P450IA2 subfamily) gene for metabolic activation of aromatic amines. We show in a rapid differential cytotoxicity assay with 2 compounds found in cooked beef (IQ, 2-amino-3-methylimidazo[4,5-f]quinoline and PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) that this gene is efficiently expressed in the transformed UV5P3 cells. Reversion of the repair deficiency in these cells will give a matched pair of cell lines that are metabolically proficient and repair deficient. Such lines will provide a rapid assay for genotoxic heterocyclic amines requiring activation.

Cell Line↗
PubMed1991

Cytogenetic characterization of the ionizing radiation-sensitive Chinese hamster mutant irs1.

The X-ray-sensitive mutant V79 cell line irs1 was characterized with respect to chromosomal aberrations induced by 137Cs, mitomycin C (MMC), and decarbamoyl mitomycin C (DCMMC). To measure chromosome damage induced at different cell cycle stages, irs1 and the parental V79-4 cell lines were pulse-labeled with bromodeoxyuridine (BrdUrd) at the time of exposure and harvested at various intervals corresponding to exposure in G1, S, and G2 phases of the cell cycle. Metaphase spreads were stained with an anti-BrdUrd antibody, followed by a fluorescein-conjugated second antibody. With propidium iodide as a counter stain, cells were scored for aberrations. Compared to the parental V79 cells, irs1 cells had: (1) greatly increased sensitivity to all 3 agents; (2) a high frequency of chromatid exchanges after exposure in each phase of the cell cycle; and (3) more sensitivity to the agent causing crosslinks (MMC) than its monofunctional analog (DCMMC). The finding of chromatid-type damage in cells exposed to ionizing radiation during G1 is atypical of normal cells, but is similar to observations made in several mutant rodent cell lines and in ataxia telangiectasia cells. Our results suggest that the defect in irs1 cells can manifest itself as misrepair or misreplication during all phases of the cell cycle and leads to a high incidence of chromatid exchanges and deletions.

Alkylating Agents↗
PubMed1991

Characterization of revertants of the CHO EM9 mutant arising during DNA transfection.

We have studied reversion in DNA repair deficient EM9 cells, by selection for ethylmethanesulfonate (EMS) resistance. EM9 is a mutant CHO cell line that is hypersensitive to killing by EMS and X-rays and deficient in DNA single-strand break (SSB) repair. EM9 cells were transfected with DNA from a cosmid library, and transfectants resistant to EMS were isolated. Four revertant lines were obtained, which varied in their sensitivity to killing by EMS, ionizing radiation and other genotoxic agents. When the cell lines were analyzed for resistance to killing by chlorodeoxyuridine (CldUrd) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a different relative ranking among the cell lines was observed. The recently cloned human XRCC1 gene is capable of correcting the deficiencies of the EM9 cell line. Using the human XRCC1 cDNA (pXR1-30) as a probe, we determined that the resistant-transfectant cell lines contained only the endogenous hamster XRCC1 gene, implying that a hamster XRCC1 gene was altered during the transfection/selection procedure and was responsible for the EMS resistance. In these cells the levels of XRCC1 mRNA corresponded roughly to the degrees of resistance of the reverted cell lines to killing by EMS or X-rays. The degree of increased resistance to killing by EMS or X-rays also roughly correlated with increased SSB repair. These results suggest that increased cellular levels of the endogenous XRCC1 gene mRNA may largely, though not completely, explain the phenotypes of revertant, EMS-resistant EM9 cell lines.

Animals↗
PubMed1991

Metabolic activation and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) in Chinese hamster ovary cells expressing murine cytochrome P450 IA2.

We have utilized Chinese hamster ovary cell lines which stably express a murine cytochrome P450IA2 (P(3)450) cDNA to characterize more fully the mechanisms of genotoxicity of heterocyclic amines derived from cooked meats. To verify that these cell lines were capable of converting promutagens into active metabolites, we studied the microsomal metabolism and cytogenetic effects of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pridine (PhIP). Microsomal preparations derived from excision repair-deficient Chinese hamster ovary cells expressing the mouse cytochrome P(3)450 cDNA (UV5P3) converted PhIP to the genotoxic N-hydroxy-PhIP metabolite. Cytotoxic activity in UV5P3 was observed at concentrations of PhIP as low as 1 microM. Cytotoxicity of PhIP was an order of magnitude lower in a matched repair-proficient cell line (5P3R2) expressing the P(3)450 cDNA. PhIP produced a concentration-dependent increase in sister chromatid exchange (SCE) in UV5P3. N-Hydroxy-PhIP, at concentrations as low as 0.1 microM, produced an increase in SCE in both UV5P3 and in UV5 cells which lack the P(3)450 cDNA. Incubation of PhIP with UV5P3 cells increased the frequency of micronuclei (MN) in cytokinesis-blocked cells. Chromatid gaps, but not aberrations also were induced by treatment with PhIP. N-Hydroxy-PhIP produced increases in MN and chromatid gaps in both UV5 and UV5P3 cell lines; chromosomal aberrations were induced in UV5P3 cells. These results suggested that UV5P3 cells metabolize sufficient quantities of PhIP to produce cytogenetic damage and further indicated that N-hydroxylation of PhIP was requisite for mammalian genotoxicity.

Animals↗
PubMed1990

ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3.

Human ERCC2 genomic clones give efficient, stable correction of the nucleotide excision repair defect in UV5 Chinese hamster ovary cells. One clone having a breakpoint just 5' of classical promoter elements corrects only transiently, implicating further flanking sequences in stable gene expression. The nucleotide sequences of a cDNA clone and genomic flanking regions were determined. The ERCC2 translated amino acid sequence has 52% identity (73% homology) with the yeast nucleotide excision repair protein RAD3. RAD3 is essential for cell viability and encodes a protein that is a single-stranded DNA dependent ATPase and an ATP dependent helicase. The similarity of ERCC2 and RAD3 suggests a role for ERCC2 in both cell viability and DNA repair and provides the first insight into the biochemical function of a mammalian nucleotide excision repair gene.

Amino Acid Sequence↗
PubMed1990

Myogenin is in an evolutionarily conserved linkage group on human chromosome 1q31-q41 and unlinked to other mapped muscle regulatory factor genes.

Myogenin is a member of a family of muscle-specific regulatory factors which includes MyoD1, Myf-5, and Myf-6 (also called MRF4 and herculin). Extensive regions of sequence homology in genes for these three factors suggest duplication events associated with their evolution. In the present study, the chromosomal location of the myogenin gene in humans (MYOG), mice (Myog), and Chinese hamsters (MYOG) was determined using in situ hybridization to human metaphase chromosomes as well as segregation analysis among interspecific somatic cell hybrid panels and interspecific backcrossed mice. We localize the gene encoding myogenin to human chromosome 1q31-q41 within a linkage group homologous with a region on mouse chromosome 1 and Chinese hamster chromosome 5. The results verify the nonlinkage of MYOG to MYOD1, MYF5, and MYF6 genes and indicate that events associated with the duplication of MYOG with respect to MYOD1, MYF5, or MYF6 loci were not chromosome-wide.

Animals↗
PubMed1990

Cyclobutane-pyrimidine dimer excision in UV-sensitive CHO mutants and the effect of the human ERCC2 repair gene.

Using a radiochromatographic assay, we have examined cis-syn cyclobutane-pyrimidine dimer removal after ultraviolet irradiation in cell lines representative of the first 6 complementation groups of Chinese hamster ovary DNA nucleotide excision repair mutants. AA8, the CHO cell line from which these mutants were derived, consistently showed normal dimer excision for a rodent cell. The mutants uniformly exhibited no significant dimer excision within the limits of determination. Additionally, V-H1, a mutant belonging to complementation group 2 and derived from V79 hamster cells, exhibited no dimer excision. Two UV5 derived transformants that carry the complementing human ERCC2 repair gene showed a capacity for dimer excision comparable to the AA8 wild-type cells.

Animals↗
PubMed1990

Biochemical and genetic analysis of the Chinese hamster mutants irs1 and irs2 and their comparison to cultured ataxia telangiectasia cells.

Two mutants of the Chinese hamster cell line V79-4 (irs1 and irs2) were previously isolated on the basis of their hypersensitivity (2- to 3-fold) to cell inactivation by ionizing radiation. One of these mutants, irs1, displays an unusual phenotype of cross-sensitivity to other varied genotoxic agents including UV light (2- to 3-fold), ethyl methanesulphonate (approximately 10-fold) and mitomycin C (approximately 60-fold). The possibility that these sensitivities might be due to more than one gene mutation in irs1 was investigated. Hybrids formed between irs1 and human lymphocytes were isolated in which the mitomycin C (MMC) sensitivity of irs1 was corrected by complementing human chromosomal material. These MMC-resistant hybrids and their subclones also showed concordant correction of the gamma-ray, UV and EMS sensitivities of irs1, suggesting that a single gene defect is most likely responsible for the phenotype of irs1. In addition it was shown that the MMC-sensitivity of irs1 is complemented by four CHO cell mutants (UV20, UV41, UV-1 and irs1SF), which also display extreme sensitivity to MMC. Mutants irs1, irs1SF and UV-1 define three new complementation groups for MMC sensitivity. The biochemical nature of the ionizing radiation sensitivity of irs1 and irs2 was also investigated. The production and repair of DNA single- and double-strand breaks were studied using the techniques of alkaline and neutral elution, respectively. Irs1 and irs2 both showed repair kinetics for each lesion that are indistinguishable from wild-type. Analysis of the rate of DNA synthesis following gamma-irradiation showed irs1 to have a dose-dependent inhibition similar to that of wild-type.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗