Methotrexate-resistant Chinese hamster ovary cells have amplified a 135-kilobase-pair region that includes the dihydrofolate reductase gene.

Abstract

For the eventual purpose of isolating and studying a single animal cell replicon, a methotrexate-resistant Chinese hamster ovary cell line was developed that has amplified an early-replicating DNA sequence approximately 500 times; this sequence includes the gene coding for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). DHFR composes 30% of the cytoplasmic protein in this cell line, and DHFR mRNA represents 25% of the message translatable in vitro. After digestion of genomic DNA from resistant cells with restriction enzymes, a unique set of highly repetitive restriction fragments can be visualized on agarose gels by ethidium bromide staining. These bands are not present in digests of parental DNA. The total length of the unit repeated sequence is estimated to be 135 .+-. 15 kilobase pairs. Regardless of the restriction enzyme utilized, a subset of these repetitive fragments hybridizes to radioactive DHFR c[complementary]DNA. The homogeneously staining regions on mitotic chromosomes in which these amplified sequences are located are shown to be early-replicating, as are the highly repeated restriction fragments themselves. An early replicon can possibly be isolated from this region, and this entire, normally unique, genomic segment can possibly be cloned and mapped with respect to origins of DNA synthesis and promoters for transcription, as well as other genetic features of interest.