Search

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5' untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.

We have examined in detail the RNA coding for dihydrofolate reductase (DHFR) in methotrexate-resistant mouse cells. We find four distinct DHFR messengers, ranging in size from 750 to 1600 nucleotides. All four are polyadenylated and polysomal and can be translated in vitro to produce a 21,000 dalton protein co-migrating with purified dihydrofolate reductase on SDS polyacrylamide gels. The major difference in these RNAs is the length of 3' untranslated regions, varying from about 80 nucleotides in the smallest mRNA to about 930 nucleotides in the largest. The RNAs are also present in methotrexate-sensitive murine cells and mouse liver. Multiple DHFR RNAs are found in the poly(A)+ RNA of methotrexate-resistant Chinese hamster ovary cells but are of different molecular weights than the mouse messengers. We discuss these results in terms of the function of 3' untranslated regions of eucaryotic mRNAs and the possible origin and significance of multiple messenger RNAs for a single protein.