The levels of
UMP synthase protein and
mRNA are increased in rat
hepatoma cells that have acquired resistance to
pyrazofurin, a potent inhibitor of
pyrimidine biosynthesis. A
cDNA plasmid library was prepared from partially purified
poly(A)+ mRNA isolated from the resistant cell line. Recombinant plasmids with inserts complementary to
UMP synthase mRNA were selected by differential hybridization with
cDNA prepared from wild type and resistant cell
mRNA and analysis of hybrid-selected
mRNA by in vitro translation reactions. One plasmid, pUMPS-2, contains a 850-base pair insert and was used to analyze
UMP synthase gene sequences in the wild type and resistant cell lines. Blot hybridization of restricted genomic
DNA demonstrated amplification of the
UMP synthase gene in the resistant cells. The number of
UMP synthase genes is increased 15-fold as determined by a modified dot hybridization procedure. Previous studies have shown that the resistant cells have a 16-fold increase in
UMP synthase mRNA but a 40-fold increase in synthase activity (Suttle, D.P. (1983) J. Biol. Chem. 258, 7707-7713). To further investigate this discrepancy between the amount of increase in
DNA and
mRNA versus the increase in
enzyme activity, we have determined the relative rate of synthesis and degradation of
UMP synthase. The rate of synthesis was 13-fold faster in the resistant cells. The degradation rate was not significantly different between the two cell lines. These data indicate that gene amplification is the major factor contributing to the
enzyme overproduction in the
pyrazofurin-resistant cells.