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.
Gene amplification and enzyme modification are responsible for the methotrexate-resistance of two carrot cell lines overproducing the bifunctional dihydrofolate reductase-thymidylate synthase
BALESTRAZZI, ALMA;CELLA, RINO
1997-01-01
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
