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Biomedical subjects

L H Thompson

Publications and source records attributed to L H Thompson.

At least 109 records · Page 6Linked to original sources
PubMed1988

Repair of (6-4)photoproducts correlates with split-dose recovery in UV-irradiated normal and hypersensitive rodent cells.

Chinese hamster ovary cells and two UV-hypersensitive derivatives were used to determine the importance of DNA excision repair for split-dose recovery. In the wild-type cells 75% of the maximum theoretical recovery was observed when the fractions were delivered at 2-h intervals. Very little recovery was evident in the two hypersensitive cell lines. Using radioimmunoassays specific for (6-4)photoproducts and cyclobutane dimers, the ability of UV-irradiated repair-deficient cells representing 5 complementation groups to repair these 2 photoproducts was determined. Removal of antibody-binding sites specific for (6-4)photoproducts was 80% complete in 6 h and was defective in the UV-sensitive cells. In contrast, only 20-60% of antibody-binding sites specific for cyclobutane dimers were removed 18 h post-irradiation, and the extent of removal was the same in normal and defective cell lines. We conclude that repair of (6-4)photoproducts accounts for split-dose recovery. In addition, we conclude that a consequence of DNA repair in CHO cells is modification rather than removal of cyclobutane dimers.

Animals↗
PubMed1988

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants.

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.

Animals↗
PubMed1988

Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.

The UV-sensitive Chinese hamster ovary (CHO) cell line UV5, which is defective in the incision step of nucleotide excision repair, was used to identify and clone a complementing human gene, ERCC2, and to study the repair process. Genomic DNA from a human-hamster hybrid cell line was sheared and cotransferred with pSV2gpt plasmid DNA into UV5 cells to obtain five primary transformants. Transfer of sheared DNA from one primary transformant resulted in a secondary transformant expressing both gpt and ERCC2. The human repair gene was identified with a probe for Alu-family repetitive sequences. For most primary, secondary, and cosmid transformants, survival after UV exposure showed a return to wild-type levels of resistance. The levels of UV-induced mutation at the aprt locus for secondary and cosmid transformants varied from 50 to 130% of the wild-type level. Measurements of the initial rate of UV-induced strand incision by alkaline elution indicated that, whereas the UV5 rate was 3% of the wild-type level, rates of cosmid-transformed lines were similar to that of the wild type, and the secondary transformant rate was about 165% of the wild-type rate. Analysis of overlapping cosmids determined that ERCC2 is between 15.5 and 20 kilobases and identified a closely linked gpt gene. Cosmids were obtained with functional copies of both ERCC2 and gpt. ERCC2 corrects only the first of the five CHO complementation groups of incision-defective mutants.

Animals↗
PubMed1988

Human creatine kinase genes on chromosomes 15 and 19, and proximity of the gene for the muscle form to the genes for apolipoprotein C2 and excision repair.

The human chromosomal assignments of genes of the creatine kinase (CK) family--loci for brain (CKBB), muscle (CKMM), and mitochondrial (CKMT) forms--were studied by Southern filter hybridization analysis of DNAs isolated from a human x rodent somatic cell hybrid clone panel. Probes for the 3'-noncoding sequences of human CKBB and CKMM hybridized concordantly only to DNAs from somatic cell hybrids containing chromosomes 14 and 19, respectively. Thus the earlier assignment of the gene coding for the CKBB isozyme to chromosome 14 was confirmed by molecular means, as was the provisional assignment of CKMM to the long arm of chromosome 19. A probe containing canine sequences for CKMM cross-hybridized with human sequences on chromosomes 14 and 19, a result consistent with the assignments of CKBB and CKMM. A probe containing human sequences for CKMT enabled the provisional assignment of CKMT to human chromosome 15. Independent hybrids with portions of the long arm of chromosome 19 missing indicated the order of genes on the long arm of chromosome 19 as being cen-GPI-(TGFB, CYP1)-[CKMM, (APOC2-ERCC1)]-(CGB, FTL). The unexpectedly more distal location of APOC2 among the genes on the long arm--and APOC2's close association with CKMM--is discussed with respect to the close linkage relationship of APOC2 to myotonic muscular dystrophy.

Animals↗
PubMed1987

Identification of nucleotide-excision-repair genes on human chromosomes 2 and 13 by functional complementation in hamster-human hybrids.

The CHO UV-sensitive mutants UV24 and UV135 (complementation groups 3 and 5, respectively) are defective in nucleotide excision repair. After fusing each mutant with human lymphocytes, resistant hybrid clones showing genetic complementation were isolated by repeated exposure to UV radiation. Using a combination of isozyme markers, DNA probes, and cytogenetic methods to analyze the primary hybrids and their subclones, correction of the repair defect was shown to be correlated with the presence of a specific human chromosome in each case. Chromosome 2 corrected UV24, and the gene responsible was designated ERCC3. Line UV135 was corrected by human chromosome 13 and the gene designated ERCC5. The UV-sensitive mouse cell line, Q31, was shown not to complement UV135 and thus appears to be mutated in the same genetic locus (homologous to ERCC5) as UV135. Breakage of complementing chromosomes with retention of the genes correcting repair defects allowed the following provisional assignments: regional localization of ERCC5 to 13q14-q34, exclusion of ERCC3 from the region of chromosome 2 distal to p23, and relief of the ambiguity of ACP1 assignment (2p23 or 2p25) to 2p23 proximal to MDH1.

Animals↗
PubMed1987

Genotoxicity of compounds from cooked beef in repair-deficient CHO cells versus Salmonella mutagenicity.

A series of compounds isolated on the basis of their mutagenicity in the Ames/Salmonella reversion assay were previously identified in fried beef and chemically synthesized for further evaluation. In this study three of these compounds were tested for genotoxic effects in the UV5 line of Chinese hamster ovary (CHO) cells, which is deficient in nucleotide excision repair. Both 2-amino-3,4-dimethyl-imidazo]4,5-f]quinoline (MeIQ) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) gave very weak responses for cell killing, hprt mutation induction and sister chromatid exchange. These effects occurred at doses in the range of 100-800 micrograms/ml (approximately solubility limit), and dose-dependent increases were not observed. Induction of chromosomal aberrations did not occur with either compound. Nor did either of these compounds produce differential cytotoxicity in normal CHO cells versus UV5 cells, indicating that potentially repairable DNA damage was not responsible for the observed cell killing. In contrast to these results, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), which constitutes greater than 90% of the mass of bacterial mutagens in beef, was strongly positive for all endpoints at doses in the range 1-3 micrograms/ml. PhIP also gave marked differential cytotoxicity (ratio of 6) and cell survival curves that were strongly dependent on repair capacity. Because PhIP is 50- to 300-fold less mutagenic than MeIQ and MeIQx in Salmonella TA1538, these results point to major differences between the bacterial and mammalian assays in terms of the relative potency of these food-related compounds.

Animals↗
PubMed1987

Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange.

Transformation frequencies were measured in CHO mutant EM9 after transfection with intact or modified plasmid pSV2-gpt. The mutant and wild-type strain behaved similarly under all conditions except when homologous recombination was required to produce an intact plasmid. Therefore, the defect of the mutant which renders it slow in DNA strand break rejoining and high in sister chromatid exchange induction reduces its ability to recombine foreign DNA molecules.

Animals↗
PubMed1987

Recent progress with the DNA repair mutants of Chinese hamster ovary cells.

Repair-deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant, which is noted for its very high sister-chromatid exchange frequencies. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to chlorodeoxyuridine (CldUrd). After EcoRI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25-30 kilobases (kb) was present. Since the DNA molecules used to produce these transformants were sheared to less than 50 kb in size, the correcting gene should be small enough to clone in a cosmid vector. Using drug-resistance markers to select for hybrids after fusion, we have done complementation experiments with ultraviolet light (u.v.)-sensitive mutants and have identified a sixth complementation group, line UV61. Additionally, CHO mutants UV27-1 and MMC-2, isolated in other laboratories, were found to belong to UV group 3, which is represented by line UV24. To study the behaviour of transfected DNA molecules in repair-deficient cells, we treated plasmid pSV2gpt with either u.v. radiation or cis-diamminedichloroplatinum(II) (cis-DDP) and introduced the damaged DNA into normal CHO cells (AA8) and mutants UV4 and UV5. Unrepaired damage to the plasmid was indicated by loss of colony-forming ability of the transfected cells in selective medium containing mycophenolic acid. With u.v. damage, the differential survival of the cell lines was similar to that seen when whole cells are treated with u.v. However, with cis-DDP damage, mutant UV4 did not exhibit the extreme hypersensitivity (50-fold) that occurs when cells are treated. This result suggests that UV4 cells may be able to repair cross-links in transfected DNA.

Animals↗
PubMed1987

The localization of the human myeloperoxidase gene is in close proximity to the translocation breakpoint in acute promyelocytic leukemia.

The human myeloperoxidase (MPO) gene has recently been cloned in our laboratory. Southern blot hybridization of our MPO cDNA to DNA from a somatic cell hybrid clone panel revealed that the MPO cosegregated with human chromosome 17. In situ hybridization mapped the MPO gene to chromosome 17q22-24. Although this location is close to the translocation breakpoint which occurs in acute promyelocytic leukemia (APL), t(15;17)(q22;q21-22), Southern blot hybridization with different restriction-digested genomic DNA samples from four APL patients did not reveal MPO gene rearrangement. However, RNA dot-blot hybridization showed that APL patients with the translocation expressed high levels of MPO mRNA. This observation raises the possibility that the high levels of MPO gene expression in APL could be due to the arrest of leukemic cells at a specific stage of differentiation or a consequence of the translocation.

Chromosome Mapping↗
PubMed1986

DNA repair genes of mammalian cells.

In the Chinese hamster ovary (CHO) cell line, various mutations affecting DNA repair have been obtained. Mutants that belong to 5 genetic complementation groups for ultraviolet (UV) sensitivity and resemble the cells from individuals having the cancer-prone genetic disorder xeroderma pigmentosum (XP) were previously identified. Each mutant is defective in the incision step of nucleotide excision repair and hypersensitive to bulky DNA lesions. These UV mutants can be divided into two subgroups; only Groups 2 and 4 are extremely sensitive to mitomycin C and other DNA cross-linking agents. The clear-cut phenotypes of the CHO mutants have allowed us to construct hybrid cells by fusion with human lymphocytes and thereby identify which human chromosomes carry genes that correct the CHO mutations. The first two mutations analyzed, UV20 (excision-repair deficient; UV Group 2) and EM9, which has a very high frequency of sister chromatid exchange (SCE), are both corrected by chromosome 19. Efforts are underway to isolate complementing repair genes by DNA-mediated gene transfer. The human gene that corrects mutant EM9 and the hamster gene that corrects UV135 (UV Group 5) have been introduced by cotransfer of genomic DNA and the dominant selectable marker gpt (guanine phosphoribosyltransferase) gene. In each case, the DNA repair function was co-selected based on resistance to 5-chlorodeoxyuridine (CldUrd) or repeated UV irradiation, respectively. The presence of a functional human repair gene in the EM9 transformants is shown by the presence of common human DNA sequences on some fragments produced by restriction enzyme cleavage. In UV135, transfer of a repair gene is indicated by a colony distribution containing "jackpots" and by instability of the resistant phenotype.

Animals↗
PubMed1986

Incorporated bromodeoxyuridine enhances the sister-chromatid exchange and chromosomal aberration frequencies in an EMS-sensitive Chinese hamster cell line.

The mutant Chinese hamster cell line, EM9, is characterized by a high baseline sister-chromatid exchange (SCE) frequency, increased sensitivity to cell killing, and a defect in DNA strand-break repair. The molecular basis for this pleotrophic phenotype is not known. We examined, at the chromosomal level, the increased sensitivity of this mutant to incorporated BrdUrd. By varying the amount of BrdUrd in template DNA and measuring the frequency of SCEs and chromosomal aberrations, we demonstrated the enhanced sensitivity of EM9 to BrdUrd present in the template strand of DNA. Our results show that a 6-fold increase in SCEs occurs due to DNA replication over a BrdUrd-substituted template relative to a dThd-substituted template. With regard to aberration production in EM9, there is a significant enhancement of aberrations and a specific bias toward damage for the chromatid with Brdurd in the template strand. While these cells share some phenotypic properties with cells from patients with Bloom's syndrome, the genotypic similarities have not yet been established.

Animals↗
PubMed1986

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19.

The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinese-hamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repair-deficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.

Animals↗
PubMed1986

Evaluation of growth, carcass traits and reproductive organs of young boars in response to zeranol implantation.

Two experiments were conducted to evaluate the effect of implants containing zeranol on growth rate, carcass composition, palatability and reproductive organ development of intact male pigs. In Exp. 1, three treatment groups were evaluated: control barrows, intact control boars and implanted boars (implanted at either 28, 56 or 112 d of age with one 12-mg dose of zeranol). In Exp. 2, four treatment groups were evaluated: control barrows, intact control boars, boars implanted at 28 d with 24 mg of zeranol (single implant) and boars implanted at 28 d and re-implanted at 56 and 112 d of age with 24 mg of zeranol (triple implant). Differences for average daily gain and carcass traits were not consistent between treatment groups with the exception of 10th rib fat, where barrows were fatter than boars. There were no differences among treatments due to zeranol for the reproductive organ characteristics, with the exception that bulbourethal gland and teat weights were heavier for boars in Exp. 2. Penis weights and lengths were lower for barrows in both experiments. Juiciness, tenderness, Warner-Bratzler shear force, pork flavor intensity and sensory scores were not consistently affected by male condition or zeranol treatment. In two of the three comparisons, implanted boars had higher off-flavor intensity scores in Exp. 1, but no treatment difference was observed in Exp. 2. Zeranol implantation did not result in significant changes in growth rate, development of reproductive organs or carcass characteristics of young boars.

Animals↗
PubMed1985

Different genetic alterations underlie dual hypersensitivity of CHO mutant UV-1 to DNA methylating and cross-linking agents.

CHO mutant UV-1, isolated on the basis of hypersensitivity to UV radiation (254 nm), was further characterized with respect to sensitivity to classes of DNA damaging agents in a differential cytotoxicity (DC) assay. Compared to its parental strain, Gly- A, UV-1 was dramatically (10- to 100-fold) hypersensitive to both DNA methylating and cross-linking agents. In addition, UV-1 was moderately (two- to fourfold) hypersensitive to several other classes of mutagens. DNA isolated from UV-1 or Gly- A after exposure to 14C-labeled methylnitrosourea (MNU) contained similar amounts of label, thus ruling out differences in uptake or binding. Three phenotypic revertants of UV-1 were resistant to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and other methylating agents but retained hypersensitivity to cross-linking agents. Moreover, fusion of UV-1 with two different UV-sensitive CHO mutants also having hypersensitivity to cross-link and methylation damage produced hybrids resistant to mitomycin C (MMC) but not to methyl methane sulfonate (MMS). Since the methylation and cross-link sensitivities were uncoupled in both genetic tests, the complex phenotype of UV-1 is likely due to more than one genetic alteration.

Alkylating Agents↗