Molecular cloning of DNA sequences complementary to mouse thymidylate synthase messenger RNA

Abstract

We describe the isolation of recombinant cDNA clones containing sequences corresponding to mouse thymidylate synthase mRNA. Double-stranded cDNA was made from poly(A+) RNA isolated from the 5-fluoro-2'-deoxyuridine-resistant mouse cell line LU3 -7 that overproduces thymidylate synthase 50- to 100-fold as compared to the parental mouse 3T6 fibroblasts. The cDNA was inserted into pBR322 using the poly(dC)-poly(dG) tailing procedure and transformed into Escherichia coli HB101. A library of 30,000 colonies was screened for thymidylate synthase cDNA sequences by differential colony hybridization. Plasmid DNA was purified from a colony that gave a positive signal. RNA corresponding to this plasmid was isolated by hybridization and translated in vitro to yield a protein that coelectrophoresed with authentic thymidylate synthase. The identity of the translation product was confirmed by immunoprecipitation with thymidylate synthase antiserum and by peptide analysis. A restriction fragment from this plasmid was used to rescreen the library to give a collection of thymidylate synthase cDNA plasmids with overlapping restriction maps. Several plasmids contained cDNA inserts greater than 1 kilobase pair in length. The size and content of thymidylate synthase mRNA in the overproducing and parental cell lines was determined by RNA blot analysis. Multiple thymidylate synthase mRNA species were identified. The predominant species was 1.3 kilobase pairs in length. Thymidylate synthase mRNA was 50 times more abundant in LU3 -7 than in 3T6 cells.