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Biomedical subjects

L H Thompson

Publications and source records attributed to L H Thompson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Genomic sequence comparison of the human and mouse XRCC1 DNA repair gene regions.

The XRCC1 (X-ray repair cross complementing) gene is involved in the efficient repair of DNA single-strand breaks formed by exposure to ionizing radiation and alkylating agents. The human gene maps to chromosome 19q13.2, and the mouse homologue maps to the syntenic region on chromosome 7. Two cosmids (approximately 38 kb each) containing the human and mouse genes were sequenced to an average 8-fold clonal redundancy. The XRCC1 gene spans a genomic distance of 26 kb in mouse and 31.9 kb in human. Both genes contain 17 exons, are 84% identical within the coding regions, and are 86% identical at the amino acid sequence level. Intron and exon lengths are highly conserved. For the human cosmid, a total of 43 Alu repetitive elements are present, a density of 1.1 Alu/kb, but due to clustering, the local density is as high as 1.8 Alu/kb. In addition, we observed a statistically significant bias for insertion of these elements in the 3'-5' orientation relative to the direction of XRCC1 transcription, predominantly in the second and third introns. This bias may indicate that XRCC1 is more accessible to Alu retroposition events during transcription than genes not expressed during spermatogenesis. The density of B1 and B2 elements in the mouse is 0.4/kb, integrated primarily in the 5'-3' orientation. The human chromosome 19-specific minisatellite PE670 was present in the same orientation in 3 introns in the human gene, and a similar repeat was found at 3 different locations in the mouse cosmid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Genetically modified Chinese hamster ovary (CHO) cells for studying the genotoxicity of heterocyclic amines from cooked foods.

We have developed metabolically competent Chinese hamster ovary (CHO) cells to evaluate the genotoxicity associated with heterocyclic amines, such as those that are present in cooked foods. Into repair-deficient UV5 cells we introduced cDNAs for expressing cytochrome P450IA2 and acetyltransferases. We then genetically reverted these transformed lines to obtain matched metabolically competent repair-deficient/proficient lines. For a high mutagenic response, we find a requirement for acetyltransferase with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) but not with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). This system allows for both quantifying mutagenesis and analyzing the mutational spectra produced by heterocyclic amines.

Animals↗
PubMed1995

Identification of aprt gene mutations induced in repair-deficient and P450-expressing CHO cells by the food-related mutagen/carcinogen, PhIP.

We investigated the specific sequence changes produced by the dietary mutagen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) in UV5P3 cells [a Chinese hamster ovary (CHO) cell line]. Sequence analysis of the PhIP-induced mutations in the adenine phosphoribosyltransferase (aprt) gene, which is heterozygous in the UV5P3 cells, can provide insight into the mutagenic mechanism in these repair-deficient cells expressing P4501A2. Two allele-specific 20 mer oligonucleotide primer pairs were used in the polymerase chain reaction and the allele of interest was amplified. Single-base transversions occurred in 31/32 PhIP-induced mutants; of these, 6 were A.T-->T.A, 18 were C.G-->A.T and 6 were G.C-->T.A. Twenty of the 30 changes altered specific amino acid sequences and the other 10 resulted in a stop codon. On mutant had a change from C.G-->G.C at the 3' splice site of intron 4, thereby creating a new AG splice acceptor site. Another mutant had an insertion of T within a run of repeated sequences and resulted in a frameshift mutation. There were three 'hot-spots', two at the 3' end of exon 2 and one at the beginning of exon 3; 6 (19%) mutants showed a change from A.T-->T.A (exon 2, amino acid residue 57), 11 (34%) mutants from C.G-->A.T (exon 2, amino acid residue 62), and 7 (22%) mutants from C.G-->A.T (exon 3, amino acid residue 66). Consequently, 75% of the mutations were observed at these three sites. In contrast, none of the 20 spontaneous mutants had alterations at these hotspot sites. The mutations induced by PhIP in these repair-deficient CHO cells were unique and specific, and suggest that these sequences, if found in important genes controlling cell replication and survival, may be more susceptible to mutation from these food mutagens than genes not containing these sequences.

Adenine Phosphoribosyltransferase↗
PubMed1995

Effects of temperature on virogenesis of bluetongue virus serotype 11 in Culicoides variipennis sonorensis.

Culicoides variipennis sonorensis females were fed bluetongue virus serotype 11 mixed in sheep blood and were held at constant temperatures of 32, 27, 21 and 15 degrees C. Virogenesis, as measured by enzyme-linked immunosorbent assay (ELISA), proceeded significantly faster at higher temperatures. Based on ELISA absorbance > or = 0.2, some flies first were categorized as infected after 1 day, 2 days and 4 days at 32, 27 and 21 degrees C, respectively. Peak levels of virus antigen were seen after 5-7, 7-13 and 18-22 days for flies held at 32, 27 and 21 degrees C, respectively. There was no significant virus replication in flies held at 15 degrees C for 22 days, but latent virus replicated and was detected easily (44% infection) 4-10 days after these flies were transferred to 27 degrees C. The implications for temperature effects on bluetongue epizootiology are discussed.

Analysis of Variance↗
PubMed1995

Heterocyclic amine mutagenicity/carcinogenicity: influence of repair, metabolism, and structure.

Cooking, heat processing, and pyrolysis of protein-rich foods induce the formation of structurally related heterocyclic aromatic amines that have been found to be mutagenic in bacteria, mammalian cells in culture and mice. All these compounds are potent mutagens and most are active below 1 ng/plate, in Ames/Salmonella tester strain TA1538 in the presence of S9 liver microsomal preparations from rat, mouse, or hamster. They are also potent in strains TA98, TA97, moderately active in TA1537, weakly active in TA100, and virtually inactive in TA1535 and TA102. Thus, they show powerful frameshift activity in reverting specific GC-rich sequences, but do not cause base substitution mutations or revert an AT-rich sequence. They are 100-fold less active in the uvrB+, repair-proficient strain TA1978, and in the case of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), cause insertions and large deletions not seen in TA1538.

Amines↗
PubMed1994

Molecular cloning of the human nucleotide-excision-repair gene ERCC4.

ERCC4 was previously identified in somatic cell hybrids as a human gene that corrects the nucleotide-excision-repair deficiency in mutant hamster cells. The cloning strategy for ERCC4 involved transfection of the repair-deficient hamster cell line UV41 with a human sCos-1 cosmid library derived from chromosome 16. Enhanced UV resistance was seen with one cosmid-library transformant and two secondary transformants of UV41. Cosmid clones carrying a functional ERCC4 gene were isolated from a library of a secondary transformant by selecting in Escherichia coli for expression of a linked neomycin-resistance gene that was present in the sCos-1 vector. The cosmids mapped to 16p13.13-p13.2, the location assigned to ERCC4 by using somatic cell hybrids. Upon transfection into UV41, six cosmid clones gave partial correction ranging from 30% to 64%, although all appeared to contain the complete gene. The capacity for in vitro excision of thymine dimers from a plasmid by transformant cell extracts correlated qualitatively with enhanced UV resistance.

Animals↗
PubMed1994

Isolation and characterization of mouse Xrcc-1, a DNA repair gene affecting ligation.

Human DNA repair gene XRCC1 complements the strand-break rejoining defect in Chinese hamster mutant EM9 and encodes a protein that is apparently required for optimal activity of DNA ligase III. Toward the goal of producing transgenic mice that carry a mutation in the Xrcc-1 locus, the murine homolog of XRCC1 was cloned from both cosmid genomic and cDNA libraries. Upon transfection into EM9 cells, cosmids containing the functional mouse gene efficiently corrected (94-100%) the high sister-chromatid-exchange defect. Mouse Xrcc-1 is 26 kb in length, contains 17 exons, and maps by metaphase in situ hybridization to the 7A3-7B2 region of mouse chromosome 7. Isolated cDNA clones were highly truncated and were extended by anchored polymerase chain reactions. The 1893-bp open reading frame of mouse Xrcc-1 encodes 631 amino acids, compared with 633 for the human homolog. The predicted mouse Xrcc-1 protein of 69.1 kDa and pI of 5.95 is 86% identical and 93% similar to human XRCC1.

Amino Acid Sequence↗
PubMed1994

Virulence-associated antigenic and genetic characteristics of bluetongue virus-17 isolates.

Bluetongue virus (BLU), an orbivirus, is of importance to the sheep and cattle industries. We have obtained 5 United States BLU-17 isolates which have been tested for virulence in sheep and 16 BLU-17 field isolates from the Caribbean and Central America. Using a panel of 15 monoclonal antibodies (MAb) against an avirulent BLU-17, we observed that 6 MAbs had negligible or very low neutralization titers for the virulent isolates in contrast to moderate to high titers for the avirulent isolates. These MAbs also differentiated the field isolates into two groups--inadequate vs effective neutralization. All 6 MAbs immunoprecipitated the outer capsid protein, VP2. Electropherotyping of genomic RNA from all 21 viruses identified an increase in RNA segment 3 mobility for those isolates which were not neutralized by the 6 specific MAbs. RNA segment 3 codes for the inner core protein, VP3. There were no detectable electrophoretic differences for RNA segment 2, which encodes VP2. In summary, the virulent BLU-17 isolates differed from the avirulent isolates in both the antigenicity of the outer capsid protein, VP2, and the electrophoretic mobility of RNA segment 3, and we hypothesize that one or both of these changes may result in BLU virulence.

Animals↗
PubMed1994

Testis and somatic Xrcc-1 DNA repair gene expression.

The human XRCC1 gene has been shown to be involved in DNA strand-break repair using the Chinese hamster ovary cell mutant EM9. The purpose of this study was to characterize the expression of Xrcc-1 to determine if there is tissue-specific expression and to provide a baseline of information for future studies that may involve altering Xrcc-1 expression in mice. Normal young adult male testis and enriched populations of pachytene spermatocytes and round spermatids displayed significantly higher levels of Xrcc-1 expression than other mouse tissues, although Xrcc-1 transcripts were found in low abundance in all tested tissues. Cultured mouse cell lines displayed levels of expression similar to male germ cells, which is a striking contrast to the levels of expression obtained in somatic tissues from the mouse. The relatively high levels of expression identified in male germ cells indicate Xrcc-1 may have an important role in male germ cell physiology.

Animals↗
PubMed1994

The smallest gene of the orbivirus, epizootic hemorrhagic disease, is expressed in virus-infected cells as two proteins and the expression differs from that of the cognate gene of bluetongue virus.

The smallest gene (S10) of the virus of epizootic hemorrhagic disease of deer (EHD, serotype 2) is expressed as two proteins in virus-infected cells. By contrast, the non-structural proteins (NS3 and NS3A) encoded in the smallest gene of bluetongue (BT) viruses are difficult to detect in virus-infected cells. The nucleotide sequence of S10 of EHDV-2 contains two in-frame initiation codons which allow for translation of proteins of mol. wt. 25503 and 23921 analogous to NS3 and NS3A of BT viruses. The S10 genes of BT viruses are highly conserved (82%-99%); the nucleotide sequence similarity of S10 of EHDV-2 and BT viruses is about 64%. Some structural features of NS3 and NS3A are conserved in the two viruses, despite the divergence in the amino acid sequences of the proteins. The hydrophobic domains of the proteins and the putative transmembrane sequences are conserved, as are potential glycosylation sites in the proteins. A cluster of proline residues, which is conserved at residues 36-50 in all of the published sequences of NS3 of BT viruses, is conserved exactly in the alignment of the sequence of NS3 of EHDV-2 with that of the BT viruses. An explanation for the differences in expression of NS3/NS3A in EHD and BT viruses was not evident in comparing the nucleotide sequences of S10 of the viruses.

Amino Acid Sequence↗
PubMed1994

Complementation group assignments of moderately UV-sensitive CHO mutants isolated by large-scale screening (FAECB).

The complementation group (CG) assignment is presented for 74 moderately sensitive (approximately 2-4x sensitivity of parental line based on ratio of D10s) UV-sensitive mutants of Chinese hamster ovary (CHO) cells from the Facility for Automated Experiments in Cell Biology (FAECB) collection. The distribution of mutants within the first five rodent UV CGs was similar to that of previously reported highly sensitive (> 4x wild-type UV sensitivity) mutants from this collection. This analysis nearly completes the identification of this large collection of over 200 mutant lines isolated after screening an estimated 3 million total colonies of mutagenized CHO cells from approximately 20 mutant hunts with up to about 400,000 colonies screened. Only eight lines with less than about 2x parental line UV sensitivity remain unassigned. One CG of UV mutants (CG6), which now has five identified representatives in the collection, has only been found among moderately UV-sensitive CHO cells. Mutant UV40, a mitomycin C (MMC)- and X-ray-sensitive line with moderate UV cross-sensitivity, is not in CGs 1-6 and apparently is not a nucleotide excision repair mutant. Also identified were new alleles of CG1 and CG4 mutants with profoundly deficient unscheduled DNA synthesis and moderate UV sensitivity but low sensitivity to MMC. The first CG5 mutant derived from MMC-sensitive MC5 cells has been identified as the second CG5 mutant in the collection. No representatives of rodent CGs 7-11 were found, suggesting that AA8 cells have a chromosomal makeup that precludes easy isolation of mutants in these CGs.

Animals↗
PubMed1994

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

Amino Acid Sequence↗
PubMed1994

The advantages and disadvantages of different social welfare strategies.

The following was delivered by the author to the High Level American Meeting of Experts on The Challenges of Social Reform and New Administrative and Financial Management Techniques. The meeting, which took place September 5-7, 1994, in Mar del Plata, Argentina, was sponsored by the International Social Security Association at the invitation of the Argentine Secretariat for Social Security in collaboration with the ISSA Member Organizations of that country.

Economic Competition↗
PubMed1994

Experimentally induced infection with bluetongue virus serotype 11 in cows.

The consequences of inoculation of bluetongue virus (BTV) serotype 11 into 16 susceptible cows either at the time of breeding or at specified stages of pregnancy were studied. The cows were free of BTV or epizootic hemorrhagic disease virus, and none had antibodies to BTV before virus inoculation. A group of 4 cows was mated naturally to a bull reported to shed BTV-11 (CO75B300 strain) in the semen. The bull was suspected of infecting cows at mating with BTV-11, which subsequently transplacentally infected the developing fetuses and induced persistently infected and congenitally malformed progeny. Two groups of 4 pregnant cows were inoculated with an insect-derived strain of BTV-11 (CO75B300), one group by direct deposit into the uterus at estrus, the other, by intradermal and SC administrations. A 90-day fetus was inoculated in utero with virus from the same pool. Four pregnant cows were inoculated with sheep blood-passaged virus of the same BTV-11 strain (CO75B300) by intradermal and SC routes. Three cows were inoculated with BTV-free suspending fluids and ovine erythrocytes by the intrauterine and intradermal-SC routes and were used as in-contact controls. Infection with insect-derived BTV-11 was confirmed in 3 cows of 1 group by virus isolation and by detection of serum antibodies. The 4 cows inoculated with sheep blood suspension of BTV-11 developed viremia and produced antibodies to the virus. None of the cattle had clinical signs of bluetongue, other than 2 cows that had a slight rectal temperature increase on postinoculation day 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Bluetongue virus isolations from vectors and ruminants in Central America and the Caribbean. Interamerican Bluetongue Team.

A regional prospective study of the epidemiology of bluetongue virus (BTV) serotypes covering 11 countries in Central America and the Caribbean took place between 1987 and 1992. Active surveillance revealed BTV infection to be endemic in the absence of confirmed indigenous cases of bluetongue. During the 6-year span of the study, over 300 BTV isolations were obtained from cattle and sheep. Results of the earlier years of the study were summarized, and surveillance activities in the concluding months of the study from November 1990 to February 1992 were evaluated. Forty-five BTV isolations were made during this time, 44 from sentinel cattle and 1 from a ram with clinical signs compatible with contagious ecthyma. Virus isolation from potential vectors also was attempted, yielding a further 9 BTV isolates from parous Culicoides insignis and C pusillus, 2 BTV isolates from blood-engorged C filarifer, and 1 epizootic hemorrhagic disease virus type-2 isolate from parous C pusillus. Our extensive network of sentinel herds in the region detected BTV-1 as the predominant serotype in Central America in 1991, after an apparent absence of 1 year in the sentinel animals. Other serotypes in Central America at that time included BTV-3 and BTV-6. In Puerto Rico and the Dominican Republic, BTV-4 became the predominant serotype, without detection of BTV-8 and BTV-17, which were common in recent years of the study. The serotypes found in the Caribbean Basin continued to have marked differences from those in North America. The importance of viewing bluetongue as an infection, the distribution of which is determined principally by ecologic factors, is emphasized.

Animals↗