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Nucleic Acids Research, 1985, Vol. 13, No. 22 8275-8284
© 1985

Articles

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

Jaya Sivaswami Tyagi1, Hideyasu Hirano2 and Ira Pastan

Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health Bethesda, MD 20892, USA

Received May 20, 1985. Revised October 9, 1985. Accepted October 15, 1985.

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°C or 41°C were shifted to 41°C or 35°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 ß-actin gene and the fibronectin gene. In shift-up (35°C -> 41°C) and shift-down (41°C -> 35°C) experiments, src gene and ß-actin gene activities in ts68-CEF nuclei remained relatively unchanged. In ts68-CEF shifted to the nonpermissive temperature (41°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°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 p60srcon fibronectin gene activity is indirect.

1Present address: CSIR Centre for Biochemicals, Delhi University Campus, Delhi-110007, India

2Present address: Department of Biochemistry, University Of Occupational and Environmental Health, Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan


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