Gene amplification as a mechanism of reversion at the HPRT locus in V79 Chinese hamster cells.

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.

Bromodeoxyuridine resistance: thymidine transport and phosphorylation in Friend leukemia cells.

We have studied multiple step bromodeoxyuridine (BrdU) resistance in Friend leukemia cells. The mutation rate to 30 micrograms/ml resistance was 5.1 X 10(-5) per cell per generation, and to 100 micrograms/ml was 3.7 X 10(-7) per cell per generation. Resistant variants could not be obtained in a single step using BrdU concentrations higher than 100 micrograms/ml. Three clones isolated through multiple step selection were resistant to 640 micrograms/ml of BrdU and deficient in thymidine kinase, although their ability to transport radiolabeled thymidine was unimpaired relative to wild type. All three clones had low reversion frequencies, as judged by plating efficiencies in couterselective HAT medium. Two such revertant clones were isolated and tested for their forward mutation frequency in BrdU. No resistant clones were obtained when as many as 5 X 10(7) cells were tested. This observation argues against the hypothesis that the Friend cells possess two functional thymidine kinase loci and that the revertants represent a heterozygous condition. We conclude that the hypothesis of null mutations within a hemizygous or heterozygous thymidine kinase locus is sufficient to account for high-level BrdU resistance in Friend leukemia cells.

Action of human pancreas alanine aminopeptidase on biologically active peptides: kinin converting activity.

A group of 15 biologically active peptides were studied with respect to their susceptibility to chain shortening by human pancreas alanine aminopeptidase. Those susceptible were somatostatin, melanocyte stimulating hormone, fibrinopeptide A, eosinophilotactictetrapeptide, lysyl-bradykinin, and methionyl-lysyl-bradykinin. The latter two were selected for further study. Direct identification and determination of the reaction products, lysine and/or methionine, were undertaken to establish unequivocally the kinin-converting activity of human pancreas alanine aminopeptidase, which exhibited a pH optimum at pH 7.9. The Km and kcat values for this enzyme for lysyl-bradykinin were 57 mumol/l and 3000 min-1, respectively. The corresponding values for this enzyme for methionyl-lysyl-bradykinin were calculated to be 49 mumol/l and 16000 min-1, respectively. Bradykinin itself is extremely resistant to hydrolysis by this pancreatic enzyme.

Multiple molecular forms of human alanine aminopeptidase: immunochemical properties.

Human pancreas, kidney, and liver alanine aminopeptidases have similar if not identical antigenic determinants even though these three isoenzymes have distinctly different electrophoretic mobilities. Single precipitin lines without spur formation were obtained for all three enzymes with antisera obtained from rabbits immunized with these three purified enzymes. Treatment of these enzymes with neuraminidase eliminated the differences in their electrophoretic migration on polyacrylamide gels and on agarose immunoelectrophoresis gels. Treatment of the pancreas, kidney, and liver alanine aminopeptidases with their respective antibodies yielded enzymes that displayed non-competitive inhibition when the dependence of velocity upon substrate concentration was determined for each enzyme, i.e. the antibodies did not cause a change in the Km values obtained in the absence of the antibody whereas kcat was reduced to the same extent for each enzyme. The removal of sialic acid(s) from these enzymes did not alter their immunochemical characteristics or their kinetic characteristics.