Abstract
The HPRT assay uses incorporation of toxic nucleotide analogs to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosporibosyltransferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.
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Brennand, J., Chinault, A. C, Konecki, D. S., Melton, D. W., and Caskey, C. T. (1982) Cloned cDNA sequences of the hypoxanthine/guanine phosphoribosyltransferase gene from a mouse neuroblastoma cell line found to have amplified genomic sequences. Proc. Natl. Acad. Sci. USA 79, 1950–1954.
Patel, P. I., Yang, T. P., Stout, J. T., Konecki, D. S., Chinault, A. C, and Caskey, C. T. (1986) Mutational diversity at the human HPRT locus. Prog. Clin. Biol. Res. 209A, 457–463.
Stout, J. T. and Caskey, C. T. (1988) The Lesch-Nyhan syndrome: clinical, molecular and genetic aspects. Trends Genet. 4, 175–178.
Jinnah, H. A., De Gregorio, L., Harris, J. C, Nyhan, W. L, and O’Neill, J. P. (2000) The spectrum of inherited mutations causing HPRT deficiency: 75 new cases and a review of 196 previously reported cases. Mutat. Res. 463, 309–326.
Szybalski, W. (1959) Genetics of human cell lines. II. Methods or determination of mutation rates to drug resistance. Exp. Cell Res. 18, 588–591.
Szybalski, W., Szybalski, E. H., and Ragni, G. (1962) Genetic studies with human cell lines. Natl. Cancer Inst. Monogr. 7, 75–78.
Chu, E. H. Y. and Malling, H. V. (1968) Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro. Proc. Natl. Acad. Sci. USA 61, 1306–1312.
Albertini, R. J., Castle, K. L., and Borcherding, W. R. (1982) T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood. Proc. Natl. Acad. Sci. USA 79, 6617–6621.
Morley, A. A., Cox, S., Wigmore, D., Seshadri, R., and Dempsey, J. L. (1982) Enumeration of thioguanine-resistant lymphocytes using autoradiography. Mutat. Res. 95, 363–375.
Jones, I. M., Burkhart-Schultz, K., and Carrano, A. V. (1985) A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes. Mutat. Res. 147, 97–105.
Zimmer, D. M., Aaron, C. S., O’Neill, J. P., and Albertini, R. J. (1991) Enumeration of 6-thioguanine-resistant T-lymphocytes in the peripheral blood of nonhuman primates (Cynomolgus monkeys). Environ. Mol. Mutagen. 18, 161–167.
Aidoo, A., Morris, S. M., and Casciano, D. A. (1997) Development and utilization of the rat lymphocyte hprt mutation assay. Mutat. Res. 387, 69–88.
Grant, S. G. and Jensen, R. H. (1993) Use of hematopoietic cells and markers for the detection and quantitation of human in vivo somatic mutation, in Immunobiology of Transfusion Medicine (Garratty, G., ed.), Marcel Dekker, New York, pp. 299–323.
Clements, G. B. (1975) Selection of biochemically variant, in some cases mutant, mammalian cells in culture. Adv. Cancer Res. 21, 273–390.
Epstein, J., Ghangas, G. S., Leyva, A., Milman, G, and Littlefield, J. W. (1979) Analysis of HGPRT− CRM+ human lymphoblast mutants. Somatic Cell Genet. 5, 809–820.
Muir, P., Osborne, Y., Morley, A. A., and Turner, D. R. (1988) Karyotypic abnormality of the X chromosome is rare in mutant HPRT− lymphocyte clones. Mutat. Res. 197, 157–160.
Thacker, J. (1981) The chromosomes of a V79 Chinese hamster line and a mutant subline lacking HPRT activity. Cytogenet. Cell Genet. 29, 16–25.
Fuscoe, J. C, Zimmerman, L. J., Fekete, A., Setzer, R.W., and Rossiter, B. J. (1992) Analysis of X-ray-induced HPRT mutations in CHO cells: insertion and deletions. Mutat. Res. 269, 171–183.
Kodama, Y., Hakoda, M., Shimba, H., Awa, A. A., and Akiyama, M. (1989) A chromosome study of 6-thioguanine-resistant mutants in T lymphocytes of Hiroshima atomic bomb survivors. Mutat. Res. 227, 31–38.
Turner, D. R., Morley, A. A., Haliandros, M., Kutlaca, R., and Sanderson, B. J. (1985) In vivo somatic mutations in human lymphocytes frequently result from major gene alterations. Nature 315, 343–345.
Albertini, R. J., O’Neill, J. P., Nicklas, J. A., Heintz, N. H., and Kelleher, P. C. (1985) Alterations of the hprt gene in human in vivo-derived 6-thioguanine-resistant T lymphocytes. Nature 316, 369–371.
Nicklas, J. A., Hunter, T. C, Sullivan, L. M., Berman, J. K., O’Neill, J. P., and Albertini, R. J. (1987) Molecular analyses of in vivo hprt mutations in human T-lymphocytes. I. Studies of low frequency,’ spontaneous’ mutants by Southern blots. Mutagenesis 2, 341–347.
Bradley, W. E. C, Gareau, J. L., Seifert, A. M., and Messing, K. (1987) Molecular characterization of 15 rearrangements among 90 human in vivo somatic mutants shows that deletions predominate. Mol. Cell. Biol. 7, 956–960.
Skulimowski, A. W., Turner, D. R., Morley, A. A., Sanderson, B. J. S., and Haliandros, M. (1986) Molecular basis of X-ray-induced mutation at the HPRT locus in human lymphocytes. Mutat. Res. 162, 105–112.
O’Neill, J. P., Hunter, T. C, Sullivan, L. M., Nicklas, J. A., and Albertini, R. J. (1990) Southern-blot analyses of human T-lymphocyte mutants induced in vitro by γ-irradiation. Mutat. Res. 240, 143–149.
Hakoda, M., Hirai, Y., Kyoizumi, S., and Akiyama, M. (1989) Molecular analyses of in vivo hprt mutant T cells from atomic bomb survivors. Environ. Mol. Mutagen. 13, 25–33.
Nicklas, J. A., O’Neill, J. P., Hunter, T. C, et al. (1991) In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes. Mutat. Res. 250, 383–396.
Nicklas, J. A., Lippert, M. J., Hunter, T. C, O’Neill, J. P., and Albertini, R. J. (1991) Analysis of human HPRT deletion mutations with X-linked probes and pulsed field gel electrophoresis. Environ. Mol. Mutagen. 18, 270–273.
Lippert, M. J., Nicklas, J. A., Hunter, T. C, and Albertini, R. J. (1995) Pulsed field analysis of hprt T-cell large deletions: telomeric region breakpoint spectrum. Mutat. Res. 326, 51–64.
Fuscoe, J. C, Zimmerman, L. J., Harrington-Brock, K., and Moore, M. M. (1992) Large deletions are tolerated at the hprt locus of in vivo derived human T-lymphocytes. Mutat. Res. 283, 255–262.
Fuscoe, J. C, Nelsen, A. J., and Pilia, G. (1994) Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis. Somat. Cell Mol. Genet. 20, 39–46.
Nelson, S. L., Jones, I. M., Fuscoe, J. C, Burkhart-Schultz, K., and Grosovsky, A. J. (1995) Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines. Radiat. Res. 141, 2–10.
Recio, L., Cochrane, J., Simpson, D., et al. (1990) DNA sequence analysis of in vivo hprt mutation in human T lymphocytes. Mutagenesis 5, 505–510.
Gibbs R. A., Nguyen, P. N., Edwards, A., Civitello, A. B., and Caskey, C. T. (1990) Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch-Nyhan families. Genomics 7, 235–244.
Rossi, A. M., Tates, A. D., van Zeeland, A. A., and Vrieling, H. (1992) Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo. Environ. Mol. Mutagen. 19, 7–13.
Steingrimsdottir, H., Rowley, G, Dorado, G, Cole, J., and Lehmann, A. R. (1992) Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene. Nucleic Acids Res. 20, 1201–1208.
Caggana, M., Benjamin, M. B., Little, J. B., Liber, H. L., and Kelsey, K. T. (1991) Single-strand conformation polymorphisms can be used to detect T cell receptor gene rearrangements: an application to the in vivo hprt mutation assay. Mutagenesis 6, 375–379.
Fuscoe, J. C, Zimmerman, L. J., Harrington-Brock, K., and Moore, M. M. (1994) Multiplex PCR analysis of in vivo-arising deletion mutations in the hprt gene of human T-lymphocytes. Environ. Mol. Mutagen. 23, 89–95.
Keohavong, P. and Thilly, W. G. (1992) Determination of the point mutational spectra of benzo[a]pyrene-diol epoxide in human cells. Environ. Health Perspect. 98, 215–219.
Cariello, N. F. and Skopek, T. R. (1993) Mutational analysis using denaturing gel electrophoresis and PCR. Mutat. Res. 288, 103–112.
Keohavong, P. and Thilly, W. G. (1992) Mutational spectrometry: a general approach for hot spot point mutations in selectable genes. Proc. Natl. Acad. Sci. USA 89, 4623–4627.
Molholt, B. and Finette, B. A. (2000) Distinguishing potential sources of genotoxic exposure via HPRT mutations. Radiat. Biol. Radioecol. 40, 529–534.
Hsu, I. C, Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., and Harris, C. C. (1991) Mutational hotspot in the pS3 gene in human hepatocellular carcinomas. Nature 350, 427–428.
Wang, S. S., O’Neill, J. P., Qian, G. S., et al. (199) Elevated HPRT mutation frequencies in aflatoxin-exposed residents of daxin, Qidong county, People’s Republic of China. Carcinogenesis 20, 2181–2184.
Cariello, N. F., Cui, L., and Skopek, T. R. (1994) In vitro mutational spectrum of aflatoxin B1 in the human hypoxanthine guanine phosphoribosyltransferase gene. Cancer Res. 54, 4436–4441.
Cariello, N. F., Craft, T. R., Vrieling, H., van Zeeland, A. A., Adams, T., and Skopek, T. R. (1992) Human HPRT mutant database: software for data entry and retrieval. Environ. Mol. Mutagen. 20, 81–83.
Cariello, N. F., Douglas, G. R., Gorelick, N. J., Hart, D. W., Wilson, J. D., and Soussi, T. (1998) Databases and software for the analysis of mutations in the human p53 gene, human hprt gene and both the lacI and lacZ gene in transgenic rodents. Nucleic Acids Res. 26, 198–199.
Fuscoe, J. C, Zimmerman, L. J., Lippert, M. J., Nicklas, J. A., O’Neill, J. P., and Albertini, R. J. (1991) V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes. Cancer Res. 51, 6001–6005.
Fuscoe, J. C, Zimmerman, L. J., Harrington-Brock, K., et al. (1992) V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans. Mutat. Res. 283, 13–20.
Davila, M., Foster, S., Kelsoe, G., and Yang, K. (2001) A role for secondary V(D)J recombination in oncogenic chromosomal translocations? Adv. Cancer Res. 81, 61–92.
Fuscoe, J. C, Vira, L. K., Collard, D. D., and Moore, M. M. (1997) Quantification of hprt gene deletions mediated by illegitimate V(D)J recombination in peripheral blood cells of humans. Environ. Mol. Mutagen. 29, 28–35.
O’Neill, J. P., Nicklas, J. A., Hunter, T. C, et al. (1994) The effect of T-lymphocyte ‘clonality’ on the calculated hprt mutation frequency occurring in vivo in humans. Mutat. Res. 313, 215–225.
Nicklas, J. A., O’Neill, J. P., Sullivan, L. M., et al. (1988) Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo. Environ. Mol. Mutagen. 12, 271–284.
Nicklas, J. A., O’Neill, J. P., and Albertini, R. J. (1986) Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes. Mutat. Res. 173, 67–72.
de Boer, J. G., Curry, J. D., and Glickman, B. W. (1993) A fast method to determine the clonal relationship among human T-cell lymphocytes. Mutat. Res. 288, 173–180.
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Keohavong, P., Xi, L., Grant, S.G. (2005). Molecular Analysis of Mutations in the Human HPRT Gene. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:161
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DOI: https://doi.org/10.1385/1-59259-840-4:161
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