HPRT mutants

Ward, J.B., Jr, Ammenheuscr, M.M., Bechtold, W.E., Whorton, E.B., Jr Legator, M.S. (1994) hprt Mutant lymphocyte frequencies in workers at a 1,3-butadiene production plant. Environ. Health Perspect., 102 (Suppl. 9), 79-85... 

Detection of hprt mutant in peripheral blood T lymphocytes or splenocytes, using 6-thi( tianjne (6TG)-resistant [dienotype. 

Casciano, D. A., Aidoo, A., Chen, T, Mitlelstaedt, R. A., Manjanatha, M. G., and Heflich, R. H. (1999). Hprt mutant frequency and molecular analysis of Hprt mutations in rats treated with mutagenic car-cinr ens. Mutat Res 431, 389-395. 

Walker, V. E., Jones, I. M., Crippen, X L., Meng, Q., Walker, D. M., Bauer, M. J., Reilly, A. A., Tates, A. D., Nakamura, J., Upton, P. B., and Skopek, X. R. (1999). Relationships between exposure, cell loss and proliferation, and manifestation of Hprt mutant X cells following treatment of preweanUng, weanling, and adult male mice with (V-ethyl-iV-nitrosourea. Mutat Res 431, 371-388. 

Type II cell hyperplasia occurred after intratracheal instillation of quartz to F344 rats at 2.0 mg and 20 mg, but not at 0.2 mg (Driscoll et al. 1996). HPRT mutant frequencies determined 3 months after exposure were significantly increased after quartz with 20 6, 45 5, 56 6, and 106 15 mutants/10 epithehal cells detected for sahne, 0.2, 2.0 and 20 mg quartz treatment groups, respectively. 

Van Larabeke, N., Koppen, G., Nelen, V., Schoeters, G., Van Loon, H., Albering, H., et al., 2004. Differences in HPRT mutant frequency among middle-aged Flemish women in association with area of residence and blood lead levels. Biomarkers 9, 71—84. 

Cl. Cariello, N. F., Scott, J. K., et al, Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amphfication HPRT Munich. Am. J. Hum. Genet. 42(5), 726-734 (1988). 

Doetschman, T., Gregg, R G., Maeda, N., Hooper, M. L., Melton, D. W., Thompson, S., et al. (1987) Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576-578. 

Conversely, HPRT4 cells can be selected for in the presence of hypoxanthine-aminopterin-thymidine medium. It should be possible to select for HPRT cells in normal HPRT+ persons with one of the analogues, and this was one of the first systems used to detect spontaneously occurring mutants in freshly cultured cells from humans.88 388... 

Autoradiographic analysis is the only method which can determine the proportion of cells incorporating a radioactive precursor and the site of that incorporation. Thus, tritiated thymidine is incorporated into DNA in the nuclei of those cells in S-phase and tritiated hypoxanthine appears first in the nucleus and later in the cytoplasm of cells with HPRT but not in mutants lacking the enzyme (see 13.2). 

Although neither TK. cells nor HPRT- cells will grow separately in HAT medium it is found that mixed populations of these two mutant lines will grow in HAT medium (see Fig. 13.3). 

The assay was described by Clive and co-workers (Clive et al. 1972) as a mutational assay system using the TK locus in mouse lymphoma cells. In the following years, he and his collaborators undertook a large-scale of investigation of the potential and optimal conduct of the assay. This included the use of the above mentioned TFT to select tk mutants, a comparison of the hypoxanthine guanine phosphoribosyl transferase (hprt) and tk loci, an analysis of the best expression time for tk mutant selection and a description of distinct large and small colony tk mutants. 

CD-I mice were exposed to purified air or benzene by inhalation at 0.04, 0.1, or 1.0 ppm for 22 hours per day, 7 days per week for 6 weeks (Ward et al. 1992). The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period, lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The increased frequencies of mutant spleen lymphocytes were significant at the low and mid, but not the high dose, and the method does not take into account possible clonal expansion. Further evaluation of the induction of gene mutation at these dose levels seems warranted. 

Mutant HPRT Nucleotide change Amino acid change Clinical presentation... 

DNA single-strand break frequency was measured by alkaline elution [16] and sister chromatid exchanges (SCEs) were assayed by the method described by Wolff [32]. Hypoxanthine-guanine phosphoribosyl transferase (HPRT 6-thioguanine) and Na/K ATPase (ouabain) resistant mutants of CHO were determined by the methods described by Cleaver [7]. Repair replication after MMS treatment was measured in isopycnic gradients [8]. 

Cloned cDNA for HPRT (3) from a mouse neuroblastoma line was used as a probe against DNA from human fibroblasts and leukocytes digested with a variety of restriction enzymes and transferred to nitrocellulose by the method of Southern. The probe cross-hybridizes well with human genomic DNA (4). Fibroblasts were obtained from the Mutant Cell Repository, Camden, New Jersey, and from patients and their families. 

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