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

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 at 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.