| Abstract: | Spontaneous phenotypic revertants of hypoxanthine phosphoribosyl-transferase (HPRT) temperature-sensitive V79 Chinese hamster cells were selected by plating a temperature-sensitive mutant in HAT medium at 39 degrees C. The incidence of such revertants was approximately 2 X 10(-4) per cell. The majority of the revertants examined had increases of between three- and tenfold in their specific activity of the enzyme, and they were able to grow continuously in the presence of HAT medium at 39 degrees C. When the revertants were cultivated in the absence of HAT, they recovered their HAT-sensitive phenotype and their lowered level of HPRT. Three of the revertants were examined for their temperature inactivation profiles, and all were found to have profiles identical to the ts parent, and quite different from the V79 wild type. The kinetic properties of the cell lines were studied: the Km for both PRPP and hypoxanthine was significantly different in the temperature-sensitive cells but was not significantly altered in the revertants with respect to the ts mutants. A specific antibody to Chinese hamster brain HPRT was employed in immunoprecipitation experiments. By measuring the point at which the immunoprecipitation of the antibody to HPRT was overcome by increasing concentrations of cell supernatant, it was possible to estimate the relative amount of enzyme molecules in the cell lines. From these data, it could be concluded that the revertants overproduced an enzyme with the same immunological properties as the ts line. Southern blots of the Hind III restricted DNA from the ts mutant and two revertant cell lines were examined with an HPRT cDNA probe. This established that the HPRT gene was amplified twofold in one of the revertants, and threefold in the other. However, if the revertants were reintroduced into nonselective medium, the gene copy number declined to one. Finally, northern blots of RNA extracted from the various cell lines demonstrated that the HPRT mRNA was augmented 1.5-fold in one revertant and 1.4-fold in the other. Reintroduction into non-selective medium resulted in a decline in mRNA level for the second mutant, whereas the first mutant appeared to be stabilized. We conclude that gene amplification and concomitant amplification of messenger RNA and enzyme levels are mechanisms of phenotypic reversion at the HPRT locus in Chinese hamster cells. |
