Isolation of genomic DNA clones spanning the entire fibronectin gene.

Abstract

Overlapping recombinant clones that appear toencompass the entire fibronectin gene have been isolated by stepwise screening of a library of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned fibronectin cDNA hybridization probe. The remaining clones were obtained by using defined fragments of this and successive genomic clones as probes. Their relationships and overlaps were determined by electron microscopy, restriction mapping, and heteroduplex analysis. Based on electron microscopic analysis of hybrids between these clones and fibronectin mRNA, the gene is approximately 48 kilobases long, more than 5 times larger than the corresponding mRNA. This large gene contains at least 48 exons interrupted by introns of highly variable size. The total exon size as estimated by R-loop analysis is 8 kilobases, similar to the mRNA for fibronectin. With the exception of the 3'-and 5'-terminal exons, the exons are small and roughly similar in size. The average exon size is 147 ± 37 base pairs, corresponding to a protein unit of50 amino acids. The nucleotide sequence of one of these exons was determined. The deduced amino acid sequence has marked homologies with one type of repetitive protein sequence unit known to exist in bovine fibronectin. These results suggest that the gene for fibronectin may have arisen by multiple gene duplications of a primordial gene or genes 150 base pairs long.Cellular fibronectin is a glycoprotein of subunit Mr 220,000 present on the cell surface of many cells; a similar but not identical protein termed plasma fibronectin is a major glycoprotein in plasma. The fibronectins are involved in a number of biological events, including cell adhesion, migration, and differentiation-(reviewed in refs. 1-4).. The fibronectin molecule is composed of a series of structural and functional domains mediating.adhesive interactions with the cell surface, collagen, fibrin, and several other-macromolecules (1-4).Quantities of fibronectin are altered in many diseases', including malignancy (reviewed-in ref. 5). To examine the regulation of fibronectin, we previously isolated the mRNA for chicken cellular fibronectin (6, 7) and constructed plasmids containing cDNA for fibronectin (7-9). Using a cloned cDNA probe, we found that levels of mRNA for fibronectin were decreased to 10-13% of normal levels after oncogenic transformation, accounting for much of the known decrease in fibronectin in these cells after transformation (9).We now describe the isolation and partial characterization of a series of overlapping DNA clones from a phage library of genomic DNA fragments. These clones span approximately 59 kilobases (kb) ofcontiguous DNA sequence and appear to cover the entire 48-kb gene. The gene for cellular fibronectin has an unusual structure consisting ofa series ofat least 48 exons, most of which are close to 150 base pairs (bp) long. MATERIALS AND METHODSHybridization Probes. A 600-bp cDNA fragment carried in, pBR322 (pFN600) has been characterized previously and correspon...

Expression of a human alpha-globin/fibronectin gene hybrid generates two mRNAs by alternative splicing.

“…XFN4 and XFN5 overlap by 2.7 kb and together span a central 28-kb portion of the FN gene. Although the size of the human gene has not been determined, it is likely to be similar to that of the chicken FN gene (-50 kb) (Hirano et al, 1983). The approximate position of the exons -3, -2 and + 2, + 3 on the gene map ( Figure 1B (pSVED-2511 …”

“…The sequences of the exon/intron boundaries are shown in Figure 2. The sequence of the exons (not shown) and that of the corresponding area in the cDNA clones (Kornblihtt et al, 1984a) (Hirano et al, 1983). The approximate position of the exons -3, -2 and + 2, + 3 on the gene map ( Figure 1B Flg.…”

“…Based on electron microscopy R loop analysis, Hirano et al (1983) have reported the overall structure of the chicken FN gene, which is -48 kb long and contains at least 48 exons. When counting the exons from the 3' end of the chicken gene, assuming exon 48 to be the 3' non-coding region and allowing one exon for type I homology, two exons per type III homology and one exon for the IIICS region (see Figure 4A), exon 35 is in an analogous position to the human ED exon.…”

Complete primary structure of the collagen-binding domain of bovine fibronectin

“…This is roughly compatible with the sizes of both type I and type I1 homologies, suggesting that in general one exon codes for one domain. That this is actually the case has been shown for exon number 12 [29], which codes for the sequence corresponding to the fourth finger in the 45-kDa fragment (residues 528 -571) (Fig. 4B).…”

“…Hirano et al [29] have found that the chicken fibronectin gene contains at least 48 exons, with a general exon size of about 150 base pairs, corresponding to approximately 50 amino acid residues. This is roughly compatible with the sizes of both type I and type I1 homologies, suggesting that in general one exon codes for one domain.…”