Effects of transformation on fibronectin gene expression using cloned fibronectin cDNA.

Abstract

Recombinant plasmids containing cellular fibronectin (CFN) cDNA sequences have been constructed and cloned in Escherichia coli The cloned sequences from one plasmid were shown to be complementary to CFN mRNA by linking this DNA to diazobenzyloxyniethylcellulose, incubating with total mRNA from chick embryo fibroblasts (CEF) under DNA-RNA hybridization conditions, and demonstrating that the RNA which hybridized specifically to the DNA-cellulose was enriched 24-fold in CFN mRNA translational activity.The sequence of this cloned cDNA has been partially determined and has characteristics common to the 3' noncoding regions of other eukaryotic mRNAs.Cloned CFN cDNA was used as a hybridization probe to quantitate CFN mRNA levels in normal and avian sarcoma virus (ASV)-transformed CEF.It was found that CFN mRNA is decreased to 10 to 13% of normal in ASV-transformed CEF.When CEF transformed with ASV mutants temperature-sensitive for transformation were cultured at the permissive temperature and then transferred to the nonpermissive temperature, there was a 10-h lag following which CFN mRNA levels increased to normal over a period of 15 h.The time course of these changes was indentical with that observed for the changes in the pro-a1 and pro-a2 collagen mRNAs but was much slower than the changes in morphology, CAMP levels, and glucose transport that occur in CEF transformed by the same temperature-sensitive ASV mutants.These findings are consistent with the possibility that the levels of the CFN and procollagen mRNAs are coordinately regulated as part of a pleotrophic differentiation program specific for CEF.The slow rise in mRNA levels following temperature shift of CEF infected with temperature-sensitive ASV is consistent with an indirect action of the ASV src gene product on regulation of cellular genes.Transformation of cells in culture with RNA tumor viruses not only results in the expression of viral genetic information but also alters the expression of cellular genes.The work presented here is directed towards understanding the mechanisms by which viral transformation influences cellular gene expression.Cellular fibronectin, the major glycoprotein found on the surface of chick embryo fibroblasts (CEF) and other fibroblasts in culture, constitutes 1 to 3% of total cellular protein (1).The function of fibronectin is primarily adhesive, contributing to both cell-cell and cell-substratum interactions (2-5).