Rat
hepatoma cells that have undergone stepwise selection in increasing concentrations of
pyrazofurin have coordinately increased levels of both
orotate phosphoribosyltransferase (EC 2.4.2.10) and
orotidine-5'-phosphate decarboxylase (EC 4.1.1.23) activity. These two activities catalyze the conversion of
orotic acid to
UMP in de novo
pyrimidine biosynthesis. Cells selected in 50 microM
pyrazofurin have over 40 times the wild type level for both activities. A single
polypeptide of approximately 55,000 daltons is increased in the resistant cells in amounts corresponding to the increase in the two activities. Resistant cell lines that are grown for extended periods in the absence of
pyrazofurin are unstable, losing their elevated levels of both
enzyme activities and the increased specific
protein. Antibody prepared against a purified
protein containing both
enzyme activities binds specifically to this increased
protein. These results corroborate other evidence indicating the two
enzyme activities are contained within a single
polypeptide called
UMP synthase.
Poly(A+) mRNA isolated from wild type and resistant lines was analyzed by in vitro translation for production of
UMP synthase protein. Immunoprecipitation of the translation products shows the resistant cells have a 17-fold increase in translatable
mRNA activity coding for
UMP synthase. The synthase accounts for 0.24% of the total in vitro translation products synthesized with
poly(A+) mRNA from the
pyrazofurin-resistant cells as opposed to 0.014% with wild type
mRNA. A cloned
UMP synthase cDNA sequence hybridizes strongly to a 1.8-kilobase
mRNA in the resistant cells. This
mRNA is only barely detectable in equivalent preparations from wild type cells. Quantitation of the
mRNA by dot hybridization techniques indicates a 16-fold increase in
UMP synthase mRNA in the resistant cells. Although this increase in
mRNA for
UMP synthase could explain most of the increased
protein, it is not sufficient to totally account for the 40-fold increase in
UMP synthase.