Isolation and characterization of cDNA clones for human and bovine fibronectins

Abstract

A bovine fibronectin (FN) cDNA clone (pFB1) was isolated by screening a cDNA library of calf testis fibroblasts with a synthetic oligonucleotide probe. The probe was a mixture of eight 14-base-long oligonucleotides designed from the amino acid sequence Glu-Cys-Phe-Met-Pro present in the Mr 3,000 COOH-terminal fragment of bovine plasma FN [Petersen, T.E., Thøgersen, H.C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P., Sottrup-Jensen, L. & Magnusson. S. (1983) Proc. Natl. Acad. Sci. USA 80. 137-141]. pFB1 contained a 1,000 base-pair (bp) insert comprising the complete 3' noncoding sequence (690 bp) and approximately equal to 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. Eleven positive cDNA clones were detected, one of which, named pFH1, contained a 2,000-bp insert comprising the complete 3' noncoding region (693 bp) and approximately equal to 1,300 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 two structural domains described in bovine plasma FN. Furthermore, the 204 COOH-terminal amino acid sequence of bovine FN was completed by overlapping two peptide fragments (MrS 3,000 and 23,000). Clone pFH1 was used in estimating the size of human fibronectin mRNA (7,900 bases) through blot hybridization analysis. Southern blot studies suggest that human FN is coded by a unique gene.