Project Details


Antifolate-sensitive Chinese hamster lung (CHL) cells have been shown to
synthesize two molecular weight classes of dihydrofolate reductase (DHFR).
Each class contains two isoelectric forms of the protein and all four DHFRs
have been shown to be encoded by mRNA. A number of recent experiments have
now led to the conclusion that a pair of alleleic genes are responsible for
the synthesis of these different DHFRs and that the parental cell line,
DC-3F, is heterozygous at the DHFR locus. When challenged with antifolate
drugs, one or the other allele is amplified, thus accounting for the
over-production of one or the other of the two molecular weight classes of
DHFR and their associated isoelectric forms. These alleles display
different Southern patterns, and Northern blots show that their multiple
DHFR mRNA transcripts are present in substantially different abundancies.
DNA sequencing studies strongly suggest that the difference in transcript
abundancy is due to a polyadenylation site mutation in the 3' and of one of
the alleles. Additionally, it has been shown that, in the absence of
selection pressure, the DHFR gene amplification in these cells is unstable,
and that the chromosomally localized amplification units (HSRs) are lost at
the rate of about 150 kb of DNA per cell division. With the use of
recombinant DNA technology, DNA transfection techniques, restriction
endonuclease mapping, and DNA sequencing techniques, we propose: (1)\to
determine the molecular basis for the isoelectric forms of DHFR within each
molecular weight class; (2)\to characterize those structural features of
the different genes that are responsible for the observed abundant
differences in their multiple DHFR mRNAs; and (3)\to attempt to isolate
from reverting cell lines extrachromosomal copies of the DHFR gene and its
flanking regions. Characterization of these extrachromosomal sequences may
shed new light on the process of deamplification, i.e., reversion, at a
time when the overall regulatory implications of the amplification
phenomenon are being reevaluated in the context of malignant transformation
and differentiation functions. (I)
Effective start/end date12/31/8912/31/89


  • National Cancer Institute
  • National Cancer Institute


  • Genetics
  • Molecular Biology


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