87 results on '"Fuscoe, J C"'
Search Results
2. High-Speed Chromosome Sorting
- Author
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Gray, J. W., Dean, P. N., Fuscoe, J. C., Peters, D. C., Trask, B. J., Van Den Engh, G. J., and Van Dilla, M. A.
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- 1987
3. DNA Repair Genes of Mammalian Cells
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Thompson, L. H., Brookman, K. W., Salazar, E. P., Fuscoe, J. C., Weber, C. A., Hollaender, Alexander, editor, Shankel, Delbert M., editor, Hartman, Philip E., editor, Kada, Tsuneo, editor, Wilson, Claire M., editor, and Kuny, Gregory, editor
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- 1986
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4. Genetics of formamidase-5 (brain formamidase) in the mouse: Localization of the structural gene on chromosome 14
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Cumming, R. B., Walton, Marva F., Fuscoe, J. C., Taylor, B. A., Womack, J. E., and Gaertner, F. H.
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- 1979
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5. Relationship Between Cytotoxicity and Site-Specific DNA Recombination After In Vitro Exposure of Leukemia Cells to Etoposide
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Chen, C.-L., primary, Fuscoe, J. C., additional, Liu, Q., additional, Pui, C.-H., additional, Mahmoud, H. H., additional, and Relling, M. V., additional
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- 1996
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6. Quantification and molecular characterization of hprt mutants of human T-lymphocytes.
- Author
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Moore, M M, primary, Harrington-Brock, K, additional, Zimmerman, L J, additional, Burnette, L P, additional, Smith, T W, additional, Everson, R B, additional, O'Neill, J P, additional, and Fuscoe, J C, additional
- Published
- 1993
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7. In vivo mutations in human blood cells: biomarkers for molecular epidemiology.
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Albertini, R J, primary, Nicklas, J A, additional, Fuscoe, J C, additional, Skopek, T R, additional, Branda, R F, additional, and O'Neill, J P, additional
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- 1993
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8. Detection of early gene expression changes by differential display in the livers of mice exposed to dichloroacetic acid.
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Thai, S F, Allen, J W, DeAngelo, A B, George, M H, and Fuscoe, J C
- Abstract
Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in mice when administered in drinking water. The mechanism of DCA carcinogenicity is not clear and we speculate that changes in gene expression may be important. In order to analyze early changes in gene expression induced by DCA treatment we used the differential display method. Mice were treated with 2 g/l DCA in drinking water for 4 weeks. Total RNAs were obtained from livers of both control and treated mice for analysis. Of approximately 48 000 bands on the differential display gels representing an estimated 96% of RNA species, 381 showed differences in intensity. After cloning and confirmation by both reverse-northern and northern analyses, six differentially expressed genes were found. The expression of five of these genes was suppressed in the DCA-treated mice while one was induced. After sequencing, four genes were identified and two were matched to expressed sequence tags through the BLAST program. These genes are alpha-1 protease inhibitor, cytochrome b5, stearoyl-CoA desaturase and carboxylesterase. Stearoyl-CoA desaturase was induced approximately 3-fold in the livers of DCA-treated mice and the other three genes were suppressed approximately 3-fold. Stearoyl-CoA desaturase, cytochrome b5 and carboxylesterase are endoplasmic reticulum membrane-bound enzymes involved in fatty acid metabolism. The expression pattern of four of these genes was similar in DCA-induced hepatocellular carcinomas and the 4 week DCA-treated mouse livers. The expression of stearoyl-CoA desaturase and one of the unidentified genes returned to control levels in the carcinomas. Understanding the roles and interactions between these genes may shed light on the mechanism of DCA carcinogenesis.
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- 2001
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9. Human Chromosome–Specific DNA Libraries: Construction and Availability
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Van Dilla, M. A., Deaven, L. L., Albright, K. L., Allen, N. A., Aubuchon, M. R., Bartholdi, M. F., Brown, N. C., Campbell, E. W., Carrano, A. V., Clark, L. M., Cram, L. S., Crawford, B. D., Fuscoe, J. C., Gray, J. W., Hildebrand, C. E., Jackson, P. J., Jett, J. H., Longmire, J. L., Lozes, C. R., Luedemann, M. L., Martin, J. C., McNinch, J. S., Meincke, L. J., Mendelsohn, M. L., Meyne, J., Moyzis, R. K., Munk, A. C., Perlman, J., Peters, D. C., Silva, A. J., and Trask, B. J.
- Abstract
The goal of the National Laboratory Gene Library Project at the Los Alamos and Lawrence Livermore National Laboratories is the production of chromosome–specific human gene libraries and their distribution to the scientific community for studies of the molecular biology of genes and chromosomes, and for the study and diagnosis of genetic disease. The specific aim of Phase I of the project is the production of complete digest (4 kb average insert size) libraries from each of the 24 human chromosomal types purified by flow sorting. The bacteriophage vector is Charon 21A, which has both Eco R1 and Hind III insertion sites accommodating human DNA fragments up to 9.1 kb in size. Each laboratory has undertaken production of a complete set of chromosome–specific libraries, Los Alamos with Eco R1 and Livermore with Hind III; most of this task has now been accomplished. Close to 1200 library aliquots have been sent to about 300 laboratories world–wide through February 1986, at which time repository and distribution functions were transferred to the American Type Culture Collection, Rockville, Md. Following Phase I, we will begin the construction of libraries with large inserts in a more ad vanced, recently developed bacteriophage vector (about 20 kb inserts) or in a cosmid vector (about 40 kb inserts), and with characteristics better suited to basic studies of gene structure and function.
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- 1986
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10. A human lymphoid leukemia cell line with a V(D)J recombinase-mediated deletion of hprt
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Chen, C.-L., Woo, M. H., Neale, G. A., Goorha, R. M., Fuscoe, J. C., Behm, F. G., Mathew, S., and Relling, M. V.
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- 1998
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11. Human in vivo somatic mutation measured at two loci: individuals with stably elevated background erythrocyte glycophorin A (gpa) variant frequencies exhibit normal T-lymphocyte hprt mutant frequencies
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Bigbee, W. L., Fuscoe, J. C., Grant, S. G., Jones, I. M., Gorvad, A. E., Harrington-Brock, K., Strout, C. L., Thomas, C. B., and Moore, M. M.
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- 1998
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12. Molecular Analysis of X-ray-induced Mutants at the HPRT Locus in V79 Chinese Hamster Cells
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Fuscoe, J. C., Ockey, C. H., and Fox, Margaret
- Abstract
Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.
- Published
- 1986
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13. Recombination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange
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Hoy, C A, Fuscoe, J C, and Thompson, L H
- Abstract
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.
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- 1987
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14. DNA-mediated transfer of a human DNA repair gene that controls sister chromatid exchange
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Thompson, L H, Brookman, K W, Minkler, J L, Fuscoe, J C, Henning, K A, and Carrano, A V
- Abstract
The Chinese hamster cell line mutant EM9, which has a reduced ability to repair DNA strand breaks, is noted for its highly elevated frequency of sister chromatid exchange, a property shared with cells from individuals with Bloom's syndrome. The defect in EM9 cells was corrected by fusion hybridization with normal human fibroblasts and by transfection with DNA from hybrid cells. The transformants showed normalization of sister chromatid exchange frequency but incomplete correction of the repair defect in terms of chromosomal aberrations produced by 5-bromo-2'-deoxyuridine.
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- 1985
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15. Deletion and amplification of the HGPRT locus in Chinese hamster cells
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Fuscoe, J C, Fenwick, R G, Ledbetter, D H, and Caskey, C T
- Abstract
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.
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- 1983
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16. Quantification of t(14;18) in the lymphocytes of healthy adult humans as a possible biomarker for environmental exposures to carcinogens.
- Author
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Fuscoe, J C, Setzer, R W, Collard, D D, and Moore, M M
- Abstract
A t(14;18) chromosomal translocation is found in approximately 85% of follicular lymphomas by both cytogenetic and molecular analyses. This rearrangement deregulates expression of the bcl-2 proto-oncogene by translocation into the immuno-globulin heavy chain locus and is probably mediated by illegitimate V(D)J recombination. We have developed a quantitative nested PCR method for detecting this event in lymphocytes of healthy individuals. Genomic DNA is purified from peripheral blood lymphocytes, and 2.5 microg (representing 4 X 10(5) cells) are amplified with translocation-specific primers under conditions in which a single copy, if present, will give a detectable PCR product. Multiple replicates are analyzed for each individual, and Poisson statistics are then used to estimate the translocation mutant frequency. We have examined lymphocyte DNA from 34 healthy individuals by this assay and found the frequency of cells with t(14;18) to range from <0.8-96X10(-7). The molecular nature of the translocations has been investigated by determining the DNA sequence at the translocation junctions. In several individuals, multiple isolates of the same translocation event were recovered, indicating that the cell with the original translocation had undergone clonal expansion. In addition, multiple independent translocations were shown to occur within an individual. Since this translocation appears to be one step in the progression of a normal cell to a cancer cell, this assay may have utility as an effects biomarker for environmental carcinogen exposure.
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- 1996
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17. Microarray analysis of virulence gene profiles in Salmonella serovars from food/food animal environment
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Wen Zou, Al-Khaldi, S. F., Branham, W. S., Han, T., Fuscoe, J. C., Han, J., Foley, S. L., Xu, J., Fang, H., Cerniglia, C. E., and Nayak, R.
18. Human Chromosome–Specific DNA Libraries: Construction and Availability
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Van Dilla, M. A., primary, Deaven, L. L., additional, Albright, K. L., additional, Allen, N. A., additional, Aubuchon, M. R., additional, Bartholdi, M. F., additional, Brown, N. C., additional, Campbell, E. W., additional, Carrano, A. V., additional, Clark, L. M., additional, Cram, L. S., additional, Crawford, B. D., additional, Fuscoe, J. C., additional, Gray, J. W., additional, Hildebrand, C. E., additional, Jackson, P. J., additional, Jett, J. H., additional, Longmire, J. L., additional, Lozes, C. R., additional, Luedemann, M. L., additional, Martin, J. C., additional, McNinch, J. S., additional, Meincke, L. J., additional, Mendelsohn, M. L., additional, Meyne, J., additional, Moyzis, R. K., additional, Munk, A. C., additional, Perlman, J., additional, Peters, D. C., additional, Silva, A. J., additional, and Trask, B. J., additional
- Published
- 1986
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19. Analysis of X-ray-induced HPRT mutations in CHO cells: Insertion and deletions
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Fuscoe, J. C., Zimmerman, L. J., Fekete, A., and Setzer, R. W.
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- 1992
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20. Erythrocyte and spermatid micronucleus analyses in mice chronically exposed to potassium bromate in drinking water.
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Allen JW, Collins BW, Lori A, Afshari AJ, George MH, DeAngelo AB, and Fuscoe JC
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- Animals, Bromates administration & dosage, Carcinogens toxicity, Dose-Response Relationship, Drug, Male, Mice, Mice, Inbred Strains, Micronucleus Tests, Water Supply, Bromates toxicity, Erythrocytes drug effects, Mutagens toxicity, Spermatids drug effects
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- 2000
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21. Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults.
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Scheerer JB, Xi L, Knapp GW, Setzer RW, Bigbee WL, and Fuscoe JC
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- Adult, Base Sequence, Chromosome Breakage, Chromosomes, Human, Pair 14, Chromosomes, Human, Pair 18, DNA Nucleotidyltransferases drug effects, Exons, Female, Fetal Blood physiology, Humans, Hypoxanthine Phosphoribosyltransferase drug effects, Hypoxanthine Phosphoribosyltransferase metabolism, Infant, Newborn, Lymphocytes drug effects, Molecular Sequence Data, Phytohemagglutinins pharmacology, Polymerase Chain Reaction methods, Sequence Deletion, Translocation, Genetic, VDJ Recombinases, DNA Nucleotidyltransferases metabolism, Hypoxanthine Phosphoribosyltransferase genetics, Lymphocytes physiology, Mutation
- Abstract
We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2 + 3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (< 0.049 x 10(-7)). The frequency of HPRT exons 2 + 3 deletions was 0.10 x 10(-7) per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%-70%) and previous results using the T-cell cloning assay (approximately 2-3 x 10(-7) per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7 x 10(8) leukocytes analyzed), and which occurred at frequencies from < 1 x 10(-7) (undetected) to 1.6 x 10(-4) among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2 + 3 deletions in the leukocytes of the newborns, from 0.07 x 10(-7) to 0.23 x 10(-7). We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2 + 3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.
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- 1999
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22. Effects of arsenic exposure on the frequency of HPRT-mutant lymphocytes in a population of copper roasters in Antofagasta, Chile: a pilot study.
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Harrington-Brock K, Cabrera M, Collard DD, Doerr CL, McConnell R, Moore MM, Sandoval H, and Fuscoe JC
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- Aged, Biomarkers, Chile, Chromosome Aberrations, DNA Mutational Analysis, Dose-Response Relationship, Drug, Genetics, Population, Humans, Lymphocytes drug effects, Lymphocytes physiology, Male, Metallurgy, Middle Aged, Pilot Projects, Arsenic toxicity, Hypoxanthine Phosphoribosyltransferase drug effects, Hypoxanthine Phosphoribosyltransferase genetics, Mutation, Occupational Exposure
- Abstract
A pilot biomarker study was conducted to investigate the feasibility of using the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in peripheral blood lymphocytes as a biomarker for detecting genetic effects of arsenic exposure. Blood and urine samples were obtained from workers highly exposed to arsenic in a copper roasting plant in Antofagasta, Chile. Individuals were classified according to their job titles into three potential exposure groups: high, medium, and low. To confirm exposure, arsenic concentration was determined in urine samples. The HPRT mutant frequencies were measured in lymphocytes from 15 individuals ranging in age from 24 to 66 years. The mean mutant frequencies for the three exposure groups were: low (9 x 10(-6)), medium (11 x 10(-6)), and high (24 x 10(-6)). An increased mutant frequency was observed in the highly exposed group, but the response was so slight that it is not likely that this assay will be capable of providing dose-response information across a range of lower, more typical environmental arsenic levels.
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- 1999
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23. The frequency of illegitimate TCRbeta/gamma gene recombination in human lymphocytes: influence of age, environmental exposure and cytostatic treatment, and correlation with frequencies of t(14;18) and hprt mutation.
- Author
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Meydan D, Nilsson T, Törnblom M, Hagmar L, Hellgren D, Fuscoe JC, and Lambert B
- Subjects
- Adult, Age Factors, Aged, Aged, 80 and over, Etoposide therapeutic use, Humans, Male, Middle Aged, Testicular Neoplasms drug therapy, Testicular Neoplasms genetics, Chromosomes, Human, Pair 14, Chromosomes, Human, Pair 18, Gene Rearrangement, beta-Chain T-Cell Antigen Receptor, Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor, Hypoxanthine Phosphoribosyltransferase genetics, Lymphocytes metabolism, Mutation, Translocation, Genetic
- Abstract
Chromosome translocations in lymphoid malignancies often involve V(D)J recombinase mediated events giving rise to aberrant T-cell receptor (TCR) and immunoglobulin genes, which have been suggested to be useful as markers of genomic instability, genotoxic exposure and cancer risk. Illegitimate rearrangements involving the TCRbeta/gamma loci on chromosome 7 create TCRbeta/gamma hybrid genes which occur at low frequency in peripheral blood lymphocytes (PBLs) of normal healthy individuals. To evaluate the utility of this marker, we studied the possible effects of age and genotoxic exposures on the TCRbeta/gamma gene variant frequency (VF), and compared the frequencies of hypoxanthine guanine phosphoribosyl transferase (hprt) mutation, hprt exon 2/3 deletion, t(14;18) and TCRbeta/gamma gene rearrangements in cells from the same donors. The TCRbeta/gamma VF ranged five-fold among 16 middle aged blood donors with a mean of 0.74+/-0.29/10(5) PBLs, which is consistent with our previous estimate in healthy subjects. The TCRbeta/gamma VF was found to increase from birth until early adult life, and then to decrease with increasing age. Four testis cancer patients, who 6 years earlier had been treated with etoposide and other cytostatic drugs, showed TCRbeta/gamma VF similar to that in healthy controls. No increase of the TCRbeta/gamma VF was found among non-smoking PAH-exposed aluminum smelter workers compared to non-smoking controls. Smoking smelter workers showed decreased TCRbeta/gamma VF compared to non-smoking workers and controls, but in a follow-up study 2 years later the difference was no longer statistically significant, although the smoking smelter workers still showed a lower TCRbeta/gamma VF than the controls. No correlation was obtained between the TCRbeta/gamma VF and the t(14;18) or hprt mutant frequency (MF) in a group of healthy individuals. However, there was a statistically significant correlation between the TCRbeta/gamma VF and the hprt exon 2/3 deletion frequency in PBL DNA from the same donors. These results show that the TCRbeta/gamma VF in healthy individuals changes with age and correlates with the frequency of hprt exon 2/3 deletion, another marker of aberrant V(D)J recombination in T-cells. However, no effect of smoking or present or previous exposure to genotoxic agents on TCRbeta/gamma VF was observed in this study. Thus, further studies are needed to prove the utility of TCRbeta/gamma gene rearrangement as a marker of genotoxic exposure.
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- 1999
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24. The frequency of illegitimate V(D)J recombinase-mediated mutations in children treated with etoposide-containing antileukemic therapy.
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Fuscoe JC, Knapp GW, Hanley NM, Setzer RW, Sandlund JT, Pui CH, and Relling MV
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- Base Sequence, Child, Child, Preschool, Chromosome Deletion, Chromosomes, Human, Pair 14, Chromosomes, Human, Pair 18, Humans, Lymphocytes ultrastructure, Polymerase Chain Reaction, Precursor Cell Lymphoblastic Leukemia-Lymphoma drug therapy, Translocation, Genetic, VDJ Recombinases, Chromosome Aberrations, DNA Nucleotidyltransferases metabolism, Etoposide adverse effects
- Abstract
Etoposide is among the most widely used anti-cancer drugs. Its use, however, has been associated with increased risk of secondary acute myeloid leukemia (AML) which is characterized by chromosomal translocations suggesting involvement of recombination-associated motifs at the breakpoints. A PCR-based assay was developed to quantitate the frequency of two illegitimate V(D)J recombinase-mediated genomic rearrangements-a 20-kb deletion in the hprt gene and the bcl2/IgH translocation (t(14;18)) found in non-Hodgkin's lymphoma. We examined both lymphocyte and non-lymphocyte blood cell DNA of children with acute lymphoblastic leukemia (ALL) for changes in the frequencies of these biomarkers during etoposide therapy to determine the level of illegitimate V(D)J recombination changes during therapy. A low level of t(14;18) was found in the lymphocytes before etoposide treatment, which was significantly reduced during etoposide therapy. In before-etoposide samples, no t(14;18) were found among 7.72x107 non-lymphocytes; during treatment none were found among 1.87x108 non-lymphocytes. Deletions were not found before etoposide treatment in either the lymphocytes (6.67x107) or non-lymphocytes (5.43x107) and were non-significantly elevated during etoposide therapy (1 in 1.4x108 lymphocytes and 1 in 1.39x108 non-lymphocytes). It is interesting to note the one patient with an hprt deletion mutation in non-lymphocytes; V(D)J recombination is not normally found in this cell type, but is the cell type from which AML derives. Several patients had clones of t(14;18)-bearing cells as determined by DNA sequence analysis. These results suggest that this etoposide-based chemotherapy was ineffective in producing genomic rearrangements mediated by illegitimate V(D)J recombination in these patients., (Copyright 1998 Elsevier Science B.V.)
- Published
- 1998
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25. Quantification of hprt gene deletions mediated by illegitimate V(D)J recombination in peripheral blood cells of humans.
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Fuscoe JC, Vira LK, Collard DD, and Moore MM
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- Adolescent, Adult, Base Sequence, DNA Nucleotidyltransferases metabolism, Female, Humans, Male, Middle Aged, Molecular Sequence Data, Reference Values, Sequence Analysis, DNA, VDJ Recombinases, DNA Nucleotidyltransferases genetics, Hypoxanthine Phosphoribosyltransferase blood, Hypoxanthine Phosphoribosyltransferase genetics, Polymerase Chain Reaction methods, Sequence Deletion
- Abstract
V(D)J recombinase is normally involved in the highly regulated rearrangement of immunoglobulin and T-cell-receptor gene segments (in B and T cells, respectively) to form functional antibody genes and T-cell-receptor genes. Occasionally, this tightly controlled process acts on inappropriate places in the genome and results in deletions and translocations. Some of these illegitimate V(D)J recombinase-mediated events have been implicated in the genetic changes associated with several forms of leukemia and lymphoid malignancy. We have developed a sensitive, specific polymerase chain reaction (PCR)-based assay to quantify such events in the peripheral blood cells of humans. This assay detects a V(D)J recombinase-mediated deletion in the hprt gene, which codes for a housekeeping enzyme and is not implicated in cancer development. Alterations in this gene serve as a surrogate indicator for these illegitimate events, which may be occurring throughout the genome. The assay involves a hemi-nested PCR with two sets of primers. Multiple replicates of genomic DNA (each representing 4 x 10(5) cells) are amplified with specific primers under conditions in which a single copy will give a detectable PCR product. Poisson statistics are then used to estimate the deletion mutant frequency. The frequency of cells with the hprt deletion among 20 healthy young adults ranged from <1.3 x 10(-7) to 4.1 x 10(-7) and was compared with the frequency of t(14;18) previously determined in these same individuals. No correlation was found between the frequencies of these two measures of genomic rearrangement. The DNA sequences at the deletion junctions were determined and provided evidence for multiple independent mutations in some individuals. This assay may serve as a biomarker for the level of illegitimate V(D)J recombination occurring in peripheral blood cells of humans.
- Published
- 1997
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26. Etoposide causes illegitimate V(D)J recombination in human lymphoid leukemic cells.
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Chen CL, Fuscoe JC, Liu Q, and Relling MV
- Subjects
- Base Sequence, DNA Primers chemistry, Exons, Humans, Leukemia, Lymphoid enzymology, Molecular Sequence Data, Tumor Cells, Cultured, VDJ Recombinases, DNA Nucleotidyltransferases metabolism, Etoposide pharmacology, Hypoxanthine Phosphoribosyltransferase genetics, Leukemia, Lymphoid genetics, Recombination, Genetic drug effects
- Abstract
Etoposide is one of the most widely used antineoplastics. Unfortunately, the same treatment schedules associated with impressive efficacy are associated with an increased risk of secondary acute myeloid leukemia (AML), which has prompted its withdrawal from some treatment regimens, thereby potentially compromising efficacy against the original tumor. Because etoposide-associated AML is characterized by site-specific illegitimate DNA recombination, we studied whether etoposide could directly cause site-specific deletions of exons 2 and 3 in the hprt gene. Human lymphoid CCRF-CEM cells were treated with etoposide for 4 hours, and DNA was isolated after subculturing. The deletion of exons 2 and 3 from hprt was assayed by a quantitative polymerase chain reaction (PCR) method. In the absence of etoposide treatment, the frequency of deletions of exons 2 and 3 was very low (5.05 x 10(-8)). After exposure to 10 mumol/ L etoposide, the frequency of the exon 2 + 3 deletion was increased immediately after and at 24 hours after etoposide treatment (65 to 89 x 10(-8)) and increased to higher levels (128 to 173 x 10(-8)) after 2 and 6 days of subculture (P < .001 overall). The frequency of the exon 2 + 3 deletion assessed at 6 days of subculture after 4 hours of 0, 0.25, 1, 2.5, 5, and 10 mumol/L etoposide treatment increased with etoposide concentration, ie, 5.05 x 10(-8), 89.2 x 10(-8), 108 x 10(-8), 142 x 10(-8), 163 x 10(-8), and 173 x 10(-8), respectively (P < .0001). Sequencing of a subset of amplified products confirmed the presence of DNA sequences at the breakpoints consistent with V(D)J recombination. By contrast, exon 2 + 3 deletions after etoposide treatment in the myeloid cell lines KG-1A and K562 showed no evidence of V(D)J recombinase in their genesis. We conclude that etoposide can induce the illegitimate site-specific action of V(D)J recombinase on an unnatural DNA substrate after a single treatment in human lymphoid cells.
- Published
- 1996
27. In vivo genotoxicity of dichloroacetic acid: evaluation with the mouse peripheral blood micronucleus assay and the single cell gel assay.
- Author
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Fuscoe JC, Afshari AJ, George MH, DeAngelo AB, Tice RR, Salman T, and Allen JW
- Subjects
- Animals, Anticarcinogenic Agents pharmacology, Antioxidants pharmacology, Bone Marrow drug effects, Electrophoresis, Agar Gel, Erythrocytes drug effects, Kinetochores drug effects, Leukocytes drug effects, Male, Mice, Micronucleus Tests, Vitamin E pharmacology, Water Supply, DNA Damage, Dichloroacetic Acid toxicity, Water Pollutants, Chemical toxicity
- Abstract
Chlorination is a widely used method for disinfection of drinking water supplies. Reaction of chlorine with naturally present organic compounds can result in toxic by-products. One major disinfection by-product from the chlorination of drinking water is dichloroacetic acid (DCA). This chemical has been shown to be carcinogenic in rodents, yet little genotoxicity data are available to assess the possible role of DNA and/or chromosomal damage in this process. We have used the peripheral blood erythrocyte micronucleus (MN) assay and the alkaline single cell gel electrophoresis (SCG) technique to investigate the in vivo genotoxicity of DCA in bone marrow and blood leukocytes, respectively. The MN assay detects chromosome breakage and/or malsegregation, while the SCG assay detects DNA damage (e.g., single strand breaks, alkali-labile sites, crosslinking). Mice were exposed to this compound in drinking water, available ad libitum, for up to 31 weeks. Our results show a small but statistically significant dose-related increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) after subchronic exposure to DCA for 9 days. In addition, at the highest dose of DCA tested (3.5 g/l), a small but significant increase in the frequency of micronucleated normochromatic erythrocytes (NCE) was detected following exposure for > or = 10 weeks. Coadministration of the antioxidant vitamin E did not affect the ability of DCA to induce this damage, indicating that the small induction of MN by DCA was probably not due to oxidative damage. Based on the lack of any difference observed in the proportion of kinetochore-positive micronuclei between the treated and control animals, we interpret MN as arising from clastogenic events. The SCG technique suggested the presence of DNA crosslinking in blood leukocytes in mice exposed to 3.5 g/l DCA for 28 days. These data provide evidence that DCA may be an extremely weak inducer of chromosome damage when provided to mice in drinking water under conditions which lead to increased levels of tumors.
- Published
- 1996
- Full Text
- View/download PDF
28. Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines.
- Author
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Nelson SL, Jones IM, Fuscoe JC, Burkhart-Schultz K, and Grosovsky AJ
- Subjects
- B-Lymphocytes radiation effects, Cell Division drug effects, Cell Line, Cell Survival radiation effects, Chromosome Mapping, DNA Primers, Genetic Markers, Humans, Polymerase Chain Reaction, T-Lymphocytes radiation effects, X-Rays, B-Lymphocytes enzymology, Gene Deletion, Genes radiation effects, Hypoxanthine Phosphoribosyltransferase genetics, T-Lymphocytes enzymology, X Chromosome
- Abstract
We have examined the extent of HPRT- total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT- deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (> 1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3' total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT- total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT- large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions.
- Published
- 1995
29. Deletion mutations in the hprt gene of T-lymphocytes as a biomarker for genomic rearrangements important in human cancers.
- Author
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Fuscoe JC, Zimmerman LJ, Harrington-Brock K, and Moore MM
- Subjects
- Adult, Base Sequence, Biomarkers, Humans, Male, Middle Aged, Molecular Sequence Data, Mutation, Gene Deletion, Gene Rearrangement, T-Lymphocyte, Hypoxanthine Phosphoribosyltransferase genetics, Neoplasms genetics, T-Lymphocytes enzymology
- Abstract
The DNA sequence of 11 in vivo-arising intragenic deletion junctions occurring in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of human T-lymphocytes was determined. These deletions ranged in size from 16 bp to 4057 bp. Extensive homology was not found at the deletion breaksites, indicating that non-homologous recombination was responsible for these deletions. Short regions of homology (1-3 nucleotides) at the deletion termini, which may direct the recombination event, were found in seven of the mutations. Only one mutation had an unaccounted for nucleotide at the junction. V(D)J recombinase recognition sequences, previously identified at other hprt deletion breaksites, were not present. Such features are also found at the deletion and translocation junctions of rearranged oncogenes and suppressor oncogenes. The ability to isolate and molecularly analyze deletion mutations occurring in vivo in peripheral human T-lymphocytes allows the assay of DNA breakage/rejoining events. Such a system may serve as a biomarker of exposure to environmental and occupational agents which may be important in the etiology of cancer.
- Published
- 1994
- Full Text
- View/download PDF
30. Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes.
- Author
-
Fuscoe JC, Zimmerman LJ, Harrington-Brock K, and Moore MM
- Subjects
- Adult, Aged, Environmental Monitoring, Humans, Male, Middle Aged, Hypoxanthine Phosphoribosyltransferase genetics, Mutagenicity Tests methods, Polymerase Chain Reaction methods, Sequence Deletion, T-Lymphocytes enzymology
- Abstract
A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of deletions of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes. The hprt clonal assay was used to isolate in vivo-arising hprt-deficient T-cells from six healthy males. Mutant frequencies ranged from 9-27 x 10(-6). Simple crude cellular extracts from 223 mutants were analyzed for hprt gene deletion. Sixteen (7.2%) were found to be due to total gene deletion and 22 (9.9%) were due to partial gene deletion. The relatively high frequency of total gene deletions was caused by replicate isolates of a single mutational event as shown by single-strand conformation polymorphism (SSCP) analysis of rearranged T-cell receptor (TCR)-gamma genes. Eighteen of the 22 partial hprt gene deletion mutants were determined to be of independent origin based on a unique hprt mutation or SSCP-TCR -gamma pattern. One-half (9/18) of the partial deletion mutants involved all or part of exon 4 alone, suggesting that this region of the hprt gene is prone to deletion. The small deletions effecting exon 1 (1 mutant), exon 2 (2 mutants), and exon 4 (6 mutants) would not have been detected by conventional Southern blot analysis and may represent a new, previously unrecognized class of mutations. The ready isolation of such intragenic deletions will allow the characterization of breakpoint junctions and may provide insights into the important processes of DNA breakage and rejoining.
- Published
- 1994
- Full Text
- View/download PDF
31. Molecular description of a hypoxanthine phosphoribosyltransferase gene deletion in Lesch-Nyhan syndrome.
- Author
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Fuscoe JC and Nelsen AJ
- Subjects
- Base Sequence, Blotting, Southern, Cell Line, DNA genetics, DNA Primers, Exons, Humans, Lesch-Nyhan Syndrome enzymology, Male, Molecular Sequence Data, Polymerase Chain Reaction, Repetitive Sequences, Nucleic Acid, Gene Deletion, Hypoxanthine Phosphoribosyltransferase genetics, Lesch-Nyhan Syndrome genetics
- Published
- 1994
- Full Text
- View/download PDF
32. Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis.
- Author
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Fuscoe JC, Nelsen AJ, and Pilia G
- Subjects
- Adult, Base Sequence, Cell Line, DNA Primers, History, 16th Century, Humans, Male, Molecular Sequence Data, Sequence Analysis, DNA, Gene Deletion, Hypoxanthine Phosphoribosyltransferase genetics, Polymerase Chain Reaction methods
- Abstract
A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking the hprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and including hprt. We used "bubble" PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream of hprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream of hprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay, hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.
- Published
- 1994
- Full Text
- View/download PDF
33. Centromere analysis of micronuclei induced by 2-aminoanthraquinone in cultured mouse splenocytes using both a gamma-satellite DNA probe and anti-kinetochore antibody.
- Author
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Afshari AJ, McGregor PW, Allen JW, and Fuscoe JC
- Subjects
- Animals, Antibodies, Cells, Cultured, DNA Probes, DNA, Satellite, In Situ Hybridization, Fluorescence, Male, Mice, Mice, Inbred C57BL, Spleen ultrastructure, Anthraquinones toxicity, Centromere immunology, Micronuclei, Chromosome-Defective, Mutagens toxicity, Spleen drug effects
- Abstract
We have tested 2-aminoanthraquinone (2-AAQ) as a potential aneugen in a cytokinesis-blocked mouse splenocyte micronucleus (MN) assay. Binucleated cells (BNC) were evaluated for MN, and the MN were further probed with two indicators of centromere presence: an anti-kinetochore autoantibody and a DNA probe for the mouse gamma-satellite locus. A dose-dependent increase in the frequency of BNC with MN was observed. At the highest 2-AAQ concentration (10 micrograms/ml), the frequency of BNC containing MN was increased greater than 10-fold over background. Both centromere-positive and centromere-negative MN were significantly increased. At least 62% of MN at all 2-AAQ doses were positive for the gamma-satellite DNA probe, while 30-53% were labeled with the antikinetochore antibody. In contrast with the 2-AAQ results, after treatment with the aneugen demecolcine (positive control), greater than 80% of MN labelled positive with both probes. This discordance in the results with the two probes after 2-AAQ exposure suggests that the mode of action of this chemical may be as an aneugen by disruption of the kinetochore proteins, as a clastogen with a preferential cleavage site at or near the gamma-satellite locus, or both. Our results also suggest that the use of either of these probes individually may not be an adequate measure of centromere presence. Nevertheless, positive results for both markers provides strong evidence that 2-AAQ is aneugenic.
- Published
- 1994
- Full Text
- View/download PDF
34. PCR cloning of a repeated DNA fragment from Chinese hamster ovary (CHO) cell X chromosomes and mapping by fluorescence in situ hybridization.
- Author
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Weier HU, Miller BM, Yu LC, and Fuscoe JC
- Subjects
- Animals, Base Sequence, CHO Cells, Chromosome Mapping, Cloning, Molecular, Cricetinae, DNA, Humans, Hybrid Cells, In Situ Hybridization, Fluorescence, Molecular Sequence Data, Polymerase Chain Reaction, Repetitive Sequences, Nucleic Acid, X Chromosome
- Abstract
Hamster chromosome-specific DNA sequences were amplified by primer directed DNA amplification using mixed base oligonucleotides in an arbitrarily primed polymerase chain reaction (AP-PCR) protocol. The template DNA was comprised of approximately 3000 chinese hamster ovary cell (CHO) chromosomes enriched by flow sorting from a human x hamster hybrid cell line. Labeling of the PCR product pool and fluorescence in situ hybridization (FISH) demonstrated preferential binding to the distal long arm of the CHO X chromosome. The PCR products were cloned, labeled by PCR and hybridized to metaphase spreads. Clones containing highly reiterated DNA were identified by FISH and sequenced. Here, we present the sequence and chromosomal location of one of the repeat clones that maps close to the secondary constriction on the long arm of the CHO X chromosome, pCAT2066-24.
- Published
- 1993
- Full Text
- View/download PDF
35. Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes.
- Author
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Fuscoe JC, Zimmerman LJ, Harrington-Brock K, and Moore MM
- Subjects
- Adult, Base Sequence, Cloning, Molecular, Genes drug effects, Humans, Male, Molecular Sequence Data, Mutagenesis, Site-Directed, Polymerase Chain Reaction, Thioguanine toxicity, Gene Deletion, Hypoxanthine Phosphoribosyltransferase genetics, T-Lymphocytes drug effects, X Chromosome drug effects
- Abstract
A cloning assay was used to recover hprt- T-lymphocytes from adult human males. Analysis of crude cellular extracts by polymerase chain reactions (PCRs) demonstrated that 7% (16/218) of the hprt mutations were due to total deletion of the hprt gene. 14 of the 16 mutants were examined by PCR for the presence of flanking DNA to determine the extent of the deletions. The deletion mutation in 13 mutants was at least 350 kb with 5 of these deletions being at least 700 kb. The largest deletions were greater than 15 times the size of the hprt gene. Therefore, large deletions are tolerated at the hprt locus of human T-lymphocytes.
- Published
- 1992
- Full Text
- View/download PDF
36. V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans.
- Author
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Fuscoe JC, Zimmerman LJ, Harrington-Brock K, Burnette L, Moore MM, Nicklas JA, O'Neill JP, and Albertini RJ
- Subjects
- Adult, Base Sequence, Clone Cells, DNA genetics, Exons, Humans, Introns, Male, Molecular Sequence Data, Oligodeoxyribonucleotides, Polymerase Chain Reaction methods, VDJ Recombinases, Chromosome Deletion, DNA Nucleotidyltransferases metabolism, Genes, Hypoxanthine Phosphoribosyltransferase genetics, T-Lymphocytes enzymology
- Abstract
The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. We have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mutations in T-lymphocytes of human newborns as measured with umbilical cord blood samples (Fuscoe et al., 1991). This mechanism results in deletion of hprt exons 2 + 3. In this report, we examined a collection of 314 HPRT-deficient clones derived from adult humans for evidence that the mutations were caused by this mechanism by analyzing exons 2 + 3 deletion mutations. DNA sequence analysis of deletion breakpoint junctions showed that 8 of the mutations were the result of V(D)J recombinase activity. The frequency of the recombinase-mediated mutations was similar in the adults and newborns (2-4 x 10(-7). However, since the hprt mutant frequency is about 10-fold higher in the adult than in the newborn, the recombinase-mediated mutations account for only a few percent of the adult mutations. These mutations are likely to have occurred during early development and persist into adulthood. Unregulated expression of V(D)J recombinase activity may be an important mechanism for genomic rearrangements in the genesis of cancer.
- Published
- 1992
- Full Text
- View/download PDF
37. V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes.
- Author
-
Fuscoe JC, Zimmerman LJ, Lippert MJ, Nicklas JA, O'Neill JP, and Albertini RJ
- Subjects
- Base Sequence, Cells, Cultured, Clone Cells, Exons, Humans, Infant, Newborn, Introns, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Oligodeoxyribonucleotides, Polymerase Chain Reaction, VDJ Recombinases, Chromosome Deletion, DNA Nucleotidyltransferases metabolism, Gene Expression Regulation, Enzymologic, Hypoxanthine Phosphoribosyltransferase genetics, T-Lymphocytes enzymology
- Abstract
Studies from several laboratories worldwide have developed a large database for in vivo hypoxanthine-guanine phosphoribosyltransferase gene mutations in human T-lymphocytes. Sufficient differences have been found thus far between the spectrum for spontaneous mutations in adults and that observed in the fetus to suggest fundamental differences in in vivo mutagenic mechanisms at these two life stages. In adults, only approximately 15% of hypoxanthine-guanine phosphoribosyltransferase mutations have structural alterations on Southern blots, while in the fetus 75% of mutations show alterations of which one-half are deletions of exons 2 and 3. We have now sequenced the breakpoint sites for these specific deletions in 18 mutant lymphocyte clones isolated from 13 normal newborns. Three classes of deletions were found. Each class had the same intron 1 breakpoint but a different intron 3 breakpoint. These mutations have all the signatures of a V(D)J recombinase-mediated event (a 5' consensus heptamer, 3' consensus heptamer and nonamer, nibbling, non-germline-encoded nucleotides, P-nucleotides). At the 3' breakpoint of the most common class (comprising 83% of the mutants) a perfect heptamer can be created by postulating a hairpin loop which could attain a Z-DNA configuration. This feature may indicate recombinase preference for certain DNA structures. These results implicate the V(D)J recombinase in illegitimate events causing mutation in this housekeeping gene during T-cell development. Inactivation of genes involved in the control of growth and differentiation (e.g., tumor suppressor genes) by this mechanism may have important implications for cancer development.
- Published
- 1991
38. The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen.
- Author
-
Rossiter BJ, Fuscoe JC, Muzny DM, Fox M, and Caskey CT
- Subjects
- Animals, Base Sequence, Blotting, Southern, Cell Line, Chromosome Deletion, Cloning, Molecular, Cricetinae, Cricetulus, Exons, Humans, Mice, Molecular Sequence Data, Sequence Alignment, Sequence Homology, Nucleic Acid, DNA, Hypoxanthine Phosphoribosyltransferase genetics, Polymerase Chain Reaction methods, Restriction Mapping
- Abstract
The fine structure of the Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene has been determined; the gene has nine exons and is dispersed over 36 kb DNA. Exons 2-9 are contained within overlapping lambda bacteriophage clones and exon 1 was obtained by an inverse polymerase chain reaction (PCR). All the exons have been sequenced, together with their immediate flanking regions, and these sequences compared to those of the mouse and human HPRT genes. Sequences immediately flanking all exons but the first show considerable homology between the different species but the region around exon 1 is less conserved, apart from the preserved location of putative functional elements. Oligonucleotide primers derived from sequences flanking the HPRT gene exons were used to amplify simultaneously seven exon-containing fragments in a multiplex PCR. This simple procedure was used to identify total and partial gene deletions among Chinese hamster HPRT-deficient mutants. The multiplex PCR is quicker to perform than Southern analysis, traditionally used to study such mutants, and also provides specific exon-containing fragments for further analysis. The Chinese hamster HPRT gene is often used as a target for mutation studies in vitro because of the ease of selection of forward and reverse mutants; the information presented here will enhance the means of investigating molecular defects within this gene.
- Published
- 1991
- Full Text
- View/download PDF
39. Direct DNA sequencing of PCR products.
- Author
-
Zimmerman LJ and Fuscoe JC
- Subjects
- Chromosome Deletion, Humans, Base Sequence, DNA Mutational Analysis, Hypoxanthine Phosphoribosyltransferase genetics, Polymerase Chain Reaction methods
- Abstract
Experiments are described which elucidate some of the technical problems associated with the direct sequencing of polymerase chain reaction (PCR) amplified DNA. Sequencing primer purity, labeling methodology, and template preparation were explored. Conditions are presented for the routine sequencing of single- and double-stranded PCR products.
- Published
- 1991
- Full Text
- View/download PDF
40. Workshop overview: new molecular techniques in genome analysis.
- Author
-
DeMarini DM and Fuscoe JC
- Subjects
- Chromosome Aberrations, Electrophoresis, Humans, Nucleic Acid Denaturation, Nucleic Acid Hybridization, Polymerase Chain Reaction methods, Salmonella typhimurium genetics, DNA Mutational Analysis, Mutagenicity Tests methods
- Abstract
An overview of the application of various molecular techniques to the analysis of genomic DNA is presented. For the analysis of small-scale changes, the polymerase chain reaction (PCR), colony probe hybridization, mismatch hybridization, and denaturing gradient gel electrophoresis (DGGE) are providing information on mutations within prokaryotic and eukaryotic genes. For large-scale changes, fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), Southern blotting, multiplex PCR, hybridization of linked probes, and restriction enzyme mapping are permitting analysis of genomic alterations that are larger than point mutations but below the resolution of standard cytogenetic analysis. Many of these techniques, either alone or in combination, produce DNA that can be subjected to DNA sequence analysis, which provides the most detailed information regarding genomic changes.
- Published
- 1991
- Full Text
- View/download PDF
41. Human chromosome-specific DNA libraries: use of an oligodeoxynucleotide probe to detect non-recombinants.
- Author
-
Fuscoe JC
- Subjects
- Base Sequence, DNA, Recombinant, Humans, Nucleic Acid Hybridization, Oligodeoxyribonucleotides, Chromosomes, Human, DNA genetics, Information Systems
- Abstract
The frequencies of non-recombinants are directly determined in 21 human DNA libraries prepared from flow-purified chromosomes. A radiolabeled 18-base oligodeoxynucleotide, whose sequence spans the cloning site, stably hybridizes to non-recombinants. When the cloning site is interrupted by an insert, less stable hybridization occurs. Non-recombinant frequencies range from 1-34% with the frequency of false positives being less than 1%. Libraries are being prepared for each human chromosome as part of the National Laboratory Gene Library Project and the specifications of seven new libraries are presented.
- Published
- 1987
- Full Text
- View/download PDF
42. Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium.
- Author
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Fuscoe JC, Wu R, Shen NH, Healy SK, and Felton JS
- Subjects
- Aflatoxin B1, Aflatoxins pharmacology, Base Composition, Base Sequence, Benzo(a)pyrene pharmacology, Carcinogens, Cloning, Molecular, DNA, Bacterial drug effects, Imidazoles pharmacology, Molecular Sequence Data, Quinolines pharmacology, DNA, Bacterial genetics, Food, Histidine genetics, Mutagens pharmacology, Operon, Salmonella typhimurium genetics
- Abstract
This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.
- Published
- 1988
- Full Text
- View/download PDF
43. Amplification versus mutation as a mechanism for reversion of an HGPRT mutation.
- Author
-
Fenwick RG Jr, Fuscoe JC, and Caskey CT
- Subjects
- Animals, Cell Line, Cricetinae, Cricetulus, Lung, RNA, Messenger genetics, Cloning, Molecular, DNA metabolism, Gene Amplification, Genes, Hypoxanthine Phosphoribosyltransferase genetics, Mutation
- Abstract
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.
- Published
- 1984
- Full Text
- View/download PDF
44. Construction of fifteen human chromosome-specific DNA libraries from flow-purified chromosomes.
- Author
-
Fuscoe JC, Clark LM, and Van Dilla MA
- Subjects
- Cell Line, DNA isolation & purification, Flow Cytometry, Humans, Infant, Newborn, Male, Metaphase, Nucleic Acid Hybridization, Skin cytology, Chromosomes, Human ultrastructure, Cloning, Molecular, DNA genetics
- Abstract
We report the construction of 15 human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow sorting and the DNA was extracted and cleaved with HindIII before cloning into the lambda vector Charon 21A. A sensitive miniblot hybridization method was used to monitor the physical and biochemical steps in the cloning procedure. Using this method, we have developed a highly efficient protocol for producing large numbers of recombinant phage from 0.2-1.0 X 10(6) sorted chromosomes. DNA from the following chromosomes was cloned: #4, 6, 8, 9, 11, 13, 14 + 15, 16, 17, 18, 19, 20, 21, 22 and Y. These libraries are available to the scientific research community and will be valuable in the genetic analysis of the human genome.
- Published
- 1986
- Full Text
- View/download PDF
45. Mutagenicity and cytotoxicity of nineteen heterocyclic mustards (ICR compounds) in cultured mammalian cells.
- Author
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Fuscoe JC, O'Neill JP, Peck RM, and Hsie AW
- Subjects
- Animals, Cell Line, Cricetinae, Drug Evaluation, Preclinical, Female, Ovary, Structure-Activity Relationship, Cell Survival drug effects, Mutagens, Nitrogen Mustard Compounds pharmacology
- Abstract
The mutagenicity and cytotoxicity of 19 ICR compounds, including 6 reported previously, have been determined in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyltransferase system. As with other physical and chemical agents, ICR 170 and 191 exhibit a phenotypic expression time of 7 to 9 days, independent of concentrations tested. Thirteen of these compounds are mutagenic. At equimolar concentrations, the compounds with the tertiary amine-type side chain (ICR 217, 340, 355, 368, 170, and 292) are more mutagenic than the compounds with the secondary amine-type side chain (ICR 449, 371, 191, and 372). All secondary amine types show a "plateau" in their concentration-dependent mutagenesis curves at 3 to 4 microM. Shortening of the side chain by one carbon (ICR 171) results in a reduced mutagenicity. Substitution of a sulfur atom for a nitrogen in the side chain (ICR 342) increases both mutagenicity and cytotoxicity. The presence of two 2-chloroethyl groups on the side chain (ICR 220) also results in greatly increased cytotoxicity and mutagenicity. When the 2-chloroethyl group of ICR 340, 372, 292, 191, or 170 is replaced by a 2-hydroxyethyl group (ICR 340-OH, 372-OH, 292-OH, 191-OH, or 170-OH), a mutagenically inactive compound results which remains toxic. Replacement of the amine linkage with an ether linkage (ICR 283) also yields a mutagenically inactive compound.
- Published
- 1979
46. An efficient method for selecting unique-sequence clones from DNA libraries and its application to fluorescent staining of human chromosome 21 using in situ hybridization.
- Author
-
Fuscoe JC, Collins CC, Pinkel D, and Gray JW
- Subjects
- Bacteriophage lambda genetics, Blotting, Southern, Cloning, Molecular, DNA, Viral isolation & purification, Electrophoresis, Agar Gel, Fluorescence, Gene Amplification, Genetic Techniques, Genetic Vectors, Humans, Plasmids, Repetitive Sequences, Nucleic Acid, Chromosomes, Human, Pair 21, DNA Probes, DNA, Recombinant isolation & purification, Nucleic Acid Hybridization
- Abstract
This paper describes an efficient procedure for selecting large numbers of unique-sequence or very low repeat-sequence probes from recombinant phage libraries. Probes were selected from the Charon 21A library LL21NS02 (made from DNA from human chromosome 21) in a multistep process in which (1) inserts from LL21NS02 were subcloned into Bluescribe plasmids, (2) plasmids were grown at high density in colonies on nitrocellulose, and (3) plasmids were selected as containing unique-sequence inserts if DNA from the colonies failed to hybridize, at low stringency, to radiolabeled total human DNA. In this manner, 1530 colonies were picked to form the library pBS-U21/1530. About 80% of the recombinants constituting pBS-U21/1530 were shown by Southern analysis to carry inserts that are present in only one copy in haploid genomic human DNA. Approximately 70% of the sequences mapped to human chromosome 21. Fluorescence in situ hybridization with DNA from pBS-U21/1530 allowed specific, intense staining of the number 21 chromosomes in metaphase spreads made from human lymphocytes.
- Published
- 1989
- Full Text
- View/download PDF
47. Human chromosome-specific DNA libraries: construction and purity analysis.
- Author
-
Fuscoe JC, McNinch JS, Collins CC, and Van Dilla MA
- Subjects
- Animals, Cell Line, Chromosomes, Human, Pair 21, Cloning, Molecular, Cricetinae, Deoxyribonuclease HindIII, Escherichia coli genetics, Humans, Hybrid Cells, Nucleic Acid Hybridization, Recombination, Genetic, Species Specificity, DNA genetics, DNA isolation & purification, Gene Library
- Abstract
We report the construction of eight human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow-sorting, and the extracted DNA was cleaved with HindIII before cloning into lamba Charon 21A. There is now a complete digest HindIII library containing greater than five chromosome equivalents for each human chromosome. These are available to the scientific community through the American Type Culture Collection in Rockville, MD. The amount of hamster DNA in libraries in which the chromosome was sorted from human x hamster hybrid cells was estimated by species-specific hybridization. It ranged from 5% to 39%. The sorted chromosomes were examined by fluorescence in situ hybridization with species-specific DNA, and the main source of the hamster DNA contamination was found to be intact hamster chromosomes. In addition, we examined a chromosome 21 library, LL21NS02, for clones that fail to grow on the rec+ host LE392. Less than 0.6% of the recombinant phage exhibited the rec+-inhibited phenotype.
- Published
- 1989
- Full Text
- View/download PDF
48. Construction and availability of human chromosome-specific gene libraries.
- Author
-
Fuscoe JC, Van Dilla MA, and Deaven LL
- Subjects
- Cloning, Molecular, Humans, Chromosomes, Human, DNA, Recombinant, Genes, Genetic Engineering
- Published
- 1986
49. Quantitative analyses of radiation- and chemical-induced lethality and mutagenesis in Chinese hamster ovary cells.
- Author
-
Hsie AW, O'Neill JP, Couch DB, SanSebastian JR, Brimer PA, Machanoff R, Fuscoe JC, Riddle JC, Li AP, Forbes NL, and Hsie MH
- Subjects
- Animals, Cell Survival drug effects, Cell Survival radiation effects, Cells, Cultured, Cricetinae, Dose-Response Relationship, Drug, Mesylates toxicity, Mutation radiation effects, Nitrogen Mustard Compounds toxicity, Nitrosamines toxicity, Ploidies, Structure-Activity Relationship, Sunlight, Technology, Radiologic, Ultraviolet Rays, X-Rays, Alkylating Agents toxicity, Carcinogens, Mutagens, Toxicology methods
- Published
- 1978
50. Mutagenicity of heterocyclic nitrogen mustards (ICR compounds) in cultured mammalian cells.
- Author
-
O'Neill JP, Fuscoe JC, and Hsie AW
- Subjects
- Cell Line, Chemical Phenomena, Chemistry, Drug Evaluation, Preclinical, Drug Resistance, Structure-Activity Relationship, Mutagens, Nitrogen Mustard Compounds pharmacology
- Abstract
The mutagenicity of six heterocylic nitrogen mustards (ICR compounds) has been determined in a cultured mammalian cell system by use of resistance to the purine analog 6-thioguanine to select for mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells. The six compounds tested are ICR 191, 170, 292, 372, 191-OH, and 170-OH. The first four contain a single 2-chloroethyl group (nitrogen half-mustard) on the side chain and are mutagenic, with the tertiary amine types (170 and 292) 3 to 5 times more mutagenic than the secondary amine types (191 and 372). The remaining two compounds (191-OH and 170-OH) are not mutagenic, indicating that the 2-chloroethyl group is needed for mutation induction.
- Published
- 1978
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