Characterization of dihydrofolate reductase-related DNA and RNA from human KB cell subclones containing different amounts of enzyme.

The present study was aimed to characterize dihydrofolate reductase (DHFR)-related DNA and RNA in a series of human KB cell sublines and to further understand the mechanisms of DHFR regulation. We used two probes, one which could recognize the 5' flanking sequence (p5') of the human DHFR gene and one derived from mouse complementary DNA (pDHFR 26) which contains the coding sequence for DHFR, to identify the DHFR-related DNA and RNA. Our results revealed no major differences in DNA gene structure as the copy number increases. The recognizable fragments of DHFR gene were similar including the 5' flanking sequence upstream from the first exon. We observed that all DHFR mRNA species identified were present in the subclones. The content of cytoplasmic DHFR mRNA does not always correlate with the relative gene copy number in these cell lines. Furthermore, we were able to detect high-molecular-weight RNA related to DHFR which might be the precursors of the DHFR mRNAs. Finally, using different RNA extraction procedures, we observed that different patterns of cytoplasmic DHFR mRNA can be obtained, which is probably due to differential breakdown of RNA species during extraction procedures.