Isolation and characterization of cDNA clones for human and bovine fibronectins.

Abstract

A bovine fibronectin (FN) c[complementary]DNA clone (pFB1) was isolated by screening a cDNA library of calf testis fibroblasts with a synthetic oligonucleotide probe. The probe was a mixture of 8 14-base-long oligonucleotides designed from the amino acid sequence Glu-Cys-Phe-Met-Pro present in the MW 3000 COOH-terminal fragment of bovine plasma FN. pFB1 contained a 1000 base-pair (bp) insert comprising the complete 3'' noncoding sequence (690 bp) and .apprxeq. 300 bp of the coding region. The clone pFB1 was used as a radioactive probe in the screening of a human cell line (Hs 578T) cDNA library. Positive cDNA clones (11) were detected, one of which, named pFH1, contained a 2000-bp insert comprising the complete 3'' noncoding region (693 bp) and .apprxeq. 1300 bp of the coding region of human FN. The sequences of the clone pFB1 insert and of the homologous region in clone pFH1 were determined. The nucleotide sequences are 90% homologous. Six amino acid changes were found, clustered in an area connecting 2 structural domains described in bovine plasma FN. The 204 COOH-terminal amino acid sequence of bovine FN was completed by overlapping 2 peptide fragments (MW 3000 and 23,000). Clone pFH1 was used in estimating the size of human fibronectin mRNA (7900 bases) through blot hybridization analysis. Southern blot studies suggest that human FN is coded by a unique gene.