Characterization of a V-79 mutant deficient in HPRT activity

A mutant Chinese hamster cell line, designated trMH-l9, is 99% deficient in the uptake of hypoxanthine when compared to the parent cell line, V-79. The aim of my thesis was to test the hypothesis that trMH-l9 had a genetically altered hypoxanthine phosphoribosyl transferase (HPRT) activity.

Plating efficiencies in media containing selective and counterselective inhibitors were performed. HAT medium contains hypoxanthine, aminopterin, and thymidine. In IAT medium inosine is substituted for the hypoxanthine of HAT medium. Cells viable in HAT and IAT media must be able to utilize exogenous purines via HPRT. V-79A cells exhibited normal viability in HAT and IAT media while trMH-l9 cells did not. I Eight-azaguanine is lethal to cells that can metabolize the compound via HPRT. V-79A cells were not viable in 8-azaguanine while trMH-l9 cells were. These results are consistent with the hypothesis that trMH-l9 does not have an in vivo functioning HPRT.

The metabolism of hypoxanthine was monitored in V-79A and trMH-l9 by incubating the cells with ³H-hypoxanthine and then extracting the ³H-products and separating them by thinlayer chromatography. The results of these experiments led to the conclusion that trMH-l9 cells did not convert hypoxanthine to any form of phosphorylated product. This was a strong indication of defective HPRT in trMH-l9.

HPRT enzyme from V-79A and trMH-l9 was assayed in cell extracts to determine the apparent K_Ms of the enzyme from each line for hypoxanthine and for phosphoribosyl pyrophosphate (PRPP). A twelve-fold difference in K_M for PRPP was found in the mutant enzyme. This difference in K_M indicates a structural change in the HPRT enzyme. This twelve-fold decrease in the affinity of HPRT from trMH-l9 for PRPP probably causes the 99% reduction in hypoxanthine uptake.