Modulation of fibronectin gene activity in chick embryo fibroblasts transformed by a temperature-sensitive strain (ts68) of Rous sarcoma virus

Abstract

Transcriptional regulation of the fibronectin gene is a major mechanism for lowering steady-state levels of fibronectin mRNA in chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) (1). In the present study, we have measured the change of transcriptional activity of the fibronectin gene in CEF transformed by a temperature-sensitive strain of RSV (ts68). Ts68-CEF maintained at either 35 degrees C or 41 degrees C were shifted to 41 degrees C or 35 degrees C, respectively, at 5-hour intervals, and isolated nuclei were used in runoff transcription assays. Nuclear RNA labeled with [alpha-32P]UTP was hybridized to DNA fragments encoding the src gene, the beta-actin gene and the fibronectin gene. In shift-up (35 degrees C----41 degrees C) and shift-down (41 degrees C----35 degrees C) experiments, src gene and beta-actin gene activities in ts68-CEF nuclei remained relatively unchanged. In ts68-CEF shifted to the nonpermissive temperature (41 degrees C), a lag time of at least 5 hours was followed by a 4- to 5-fold increase in fibronectin specific RNA 15 hours after the shift. When cells were shifted to the permissive temperature (35 degrees C), a 4- to 5-fold decrease in fibronectin RNA was apparent within 5 hours of the temperature shift and a 17- to 18-fold decrease was observed 15 hours after the shift. The relatively slow rates of changes of fibronectin gene activity in shift-up experiments suggest that the effect of p60src on fibronectin gene activity is indirect.