Gene amplification and enzyme modification are responsible for the methotrexate-resistance of two carrot cell lines overproducing the bifunctional dihydrofolate reductase-thymidylate synthase

The molecular basis for the MTX-resistance of two
carrot cell lines that overproduce bifunctional dihydrofolate
reductase-thymidylate synthase (DHFR-TS)
was investigated using cDNA probes for carrot
DHFR-TS isolated in our laboratory. The results of
molecular and cytogenetic analyses suggest that only
one of the tested lines (E^C,) underwent an event of
gene amplification (10-fold) resulting in increased
levels of the target enzyme and its mRNA. The other
MTX-resistant line analysed (E^C,), which had a
normal content of the dhfr-ts gene and of the relevant
transcript, was found to contain a DHFR activity that
was less sensitive to MTX.
Southern blot analysis revealed variations in the
EcoRI restriction pattern for both resistant lines.
These results suggest that in carrot cells, as in
animal systems, MTX-resistance can be due to different
events such as gene amplification and gene modifications;
the latter appear to be responsible for the
increased production of a target enzyme with reduced
affinity for MTX.