HPRT cells

Dimethylformamide inhibited intercellular communication (as measured by metabolic co-operation) between Chinese hamster V79 hprt cells. 

Mutations to loss of activity of this enzyme cause resistance to the guanine analogues 8-azaguanine and 6-thioguanine. Thus, wild-type HPRT " cells can be selected against by incorporation of one of these analogues. 

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... 

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). 

HPRT diploid human fibroblasts are very efficient at transferring the phosphorylated nucleoside of AG by cell-to-cell contact into HPRT" cells (metabolic cooperation),which renders the HPRT cells sensitive to killing by AG despite their genotype. Therefore, it is necessary to make use of large numbers of dishes to obtain sufficient surface area so that the cells will not be too close to each other at the end of the expression period. Thus, for the in situ technique, 500-1000 60-mm-diameter dishes were commonly used for a mutagenesis experiment involving a control and two or three experimental points, and each of these had to be exposed to UV radiation or carcinogen. Furthermore, since AG is unstable in the presence of calf serum,... 

Large-sized AG- or TG-resistant colonies randomly isolated at the end of the selection period were dispersed with trypsin, suspended in culture medium, and seeded into a 30-ml flask and into two 60-mm dishes, one of which contained a sterile coverslip. To avoid the possibility of culturing a minor fraction of HPRT" cells that might be transferred along with the AG- or TG-resistant colony (cf. Jacobs and DeMars 0 cells in the flask were grown to confluence in selective medium. Crude cell extracts were prepared according to the method of Milman et The level of HPRT enzyme activity, i.e.,... 

Table 1 presents the results of studies characterizing a representative sample of AG- or TG-resistant mutant colonies using the methods outlined in Section 2.4. The results show that the majority of the colonies tested had greatly reduced enzyme levels and that only the positive control cells were able to incorporate HX. One colony exhibited a low level of HPRT activity (6.5% of HPRT cells), yet cells from this colony, after growing for more than 10... 

Oiler, A.R. and Thilly, W.G. (1992). Mutational spectra in human jS-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene. J. Mol. Biol. 228, 813-826. 

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is insufficient, and the salvage pathway is essential for adequate nucleotide synthesis. In patients with Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent. People with this genetic deficiency have CNS deterioration, mental retardation, and spastic cerebral palsy associated with compulsive self-mutilation, Cells in the basal ganglia of the brain (fine motor control) normally have very high HPRT activity. These patients also all have hyperuricemia because purines cannot be salvaged. 

The drugs aUopurinol (used for gout) and 6-mercaptopurine (antineopiastic) also inhibit PRPP amidotransferase, These drugs are purine analogs that must be converted to their respective nucleotides by HPRT within cells. Also note that ... 

Cell transformation, RLV/Fiseher rat Cell transformation, SA7/Syrian hamster embryo eells Cell transformation, Syrian hamster embryo eells Unseheduled DNA synthesis, human hepatoeytes in vitro Gene mutation, human lymphoeytes, TK and HPRT loei in vitro Sister ehromatid exehange, human lymphoeytes in vitro Sister ehromatid exehange, human lymphoeytes (metabolie aetivation by eo-eultured with rat liver eells) in vitro Chromosomal aberrations, human lymphoeytes in vitro... 

Gene mutation, Hprt locus, ouabain resistance, Chinese hamster ovary cells in vitro... 

Gene mutation, mouse lymphoma L5178Y cells, Tk locus md Hprt locus in vitro... 

G9H, Gene mutation, Chinese hamster lung V79 cells, hprt locus in vitro (+) (+) 200 Lee Webber (198S)... 

GIH, Gene mutation, human lymphoblastoid AHH-1 cells hprt locus + NT 25 Crespi et al. (1985)... 

GVA, Gene mutation, B6C3F i mouse splenic T cells, hprt locus in vivo... 

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