We report the isolation and characterization of four overlapping cDNA clones coding for human cellular fibronectin which continuously cover more than 3 kilobases in length. The nucleotide sequence of these cDNAs has been determined, thus elucidating the amino acid sequence of the C-terminal 794 residues of human fibronectin, which cover the edge of cellular-, heparin-, and fibrin-binding domains of this protein. Comparisons of the nucleotide sequences and the deduced amino acid sequences with those of rat [Schwarzbauer, J. E., Tamkun, J. W., Lemischka, I. R., & Hynes, R. O. (1983) Cell (Cambridge, Mass.) 35, 421] indicate a high degree of conservation at both nucleotide and amino acid levels. Comparison with previously published data on amino acid sequences of bovine fibronectin made it possible to identify structurally important features of the protein during the evolution of human, calf, and rat. The deduced human amino acid sequences contain five type III and three type I repeats of internal homologies. The interspecies conservation in amino acids is more pronounced in regions containing the internal repeats and within each functional domain. The implications of these interspecies conservation and divergence are discussed.

The development of capillary basement membrane thickening has been linked to microvascular changes known to occur in tissues of patients with type II diabetes. Previous evidence has suggested that capillary basement membrane thickening is due to increased basement membrane synthesis. In this study, skin samples from 8 diabetic patients with confirmed capillary basement membrane thickening and 7 non-diabetic controls were used to assess steady state levels of mRNAs coding for several basement components including pro alpha 1(IV) collagen, laminin and fibronectin. Total RNA was extracted from abdominal skin samples and levels of mRNAs coding for the basement membrane components laminin, fibronectin and pro alpha 1(IV) collagen, a fibrillar collagenous protein, pro alpha 1(I) collagen and an intracellular polypeptide, gamma-actin, were determined by dot blot hybridization analysis. While there were no changes of steady state levels of pro alpha 1(I) collagen mRNA and laminin mRNA, a significant reduction was noted in the quantitative recovery of mRNA levels for pro alpha 1(IV) collagen, gamma-actin and fibronectin in total RNA isolated from the skin of diabetic patients. This reduction in levels of mRNAs coding for basement membrane components contrasts with pathological confirmation of an accumulation of endothelial capillary basement membrane in skin from diabetic patients and suggests that basement membrane thickening arises more as a consequence of reduced basement membrane degradation than elevated synthesis of basement membrane components.

A 0.9-kilobase DNA fragment from the genome of Moloney murine leukemia virus, including the viral long terminal repeat, was covalently linked to the herpes simplex virus I thymidine kinase (tk) gene whose promoter was previously removed. The hybrid DNA structure was introduced into the chromosome of tkmouse cells at single copy numbers, via transfection procedures. Cells expressing the newly introduced tk gene were identified by the HAT selection procedure and analyzed for tk-and moloney murine leukemia virus-specific DNA and RNA sequences by blot hybridization procedures. Expression of the tk gene is dependent on function(s) provided in cis by the viral DNA fragment. Vectors derived from this region are termed rGag (rG) vectors.

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