GENE AMPLIFICATION AND ENZYME MODIFICATION ARE RESPONSIBLE FOR THE METHOTREXATE-RESISTANCE OF 2 CARROT CELL-LINES THAT OVERPRODUCE BIFUNCTIONAL DIHYDROFOLATE-REDUCTASE THYMIDYLATE SYNTHASE

The molecular basis for the MTX-resistance of two carrot cell lines th at overproduce bifunctional dihydrofolate reductase-thymidylate syntha se (DHFR-TS) was investigated using cDNA probes for carrot DHFR-TS iso lated in our laboratory. The results of molecular and cytogenetic anal yses suggest that only one of the tested lines (E(2)A(1)C(1)) underwen t an event of gene amplification (10-fold) resulting in increased leve ls of the target enzyme and its mRNA. The other MTX-resistant line ana lysed (E(2)A(1)C(6)), which had a normal content of the dhfr-ts gene a nd of the relevant transcript, was found to contain a DHFR activity th at was less sensitive to MIX. Southern blot analysis revealed variatio ns in the EcoRI restriction pattern for both resistant lines, These re sults suggest that in carrot cells, as in animal systems, MIX-resistan ce can be due to different events such as gene amplification and gene modifications; the latter appear to be responsible for the increased p roduction of a target enzyme with reduced affinity for MTX.