Changes in synthesis of DNA binding proteins during the onset of transformation in NRK cells transformed by a temperature sensitive mutant of Rous sarcoma virus

Synthesis of cytoplasmic DNA-binding proteins was investigated after a shift from the nonpermissive to the permissive temperature in NRK cells (a rat cell line) transformed by a temperature-sensitive mutant of Rous sarcoma virus [ts339(RSV)]. Cells were labeled for several generations in [³H]leucine and were pulse-labeled with [³⁵S]methionine for 1 h at the nonpermissive temperature (39°C) and at the permissive temperature (33°C, 5 h after shift from 39°C). Proteins binding to sequential columns of double-stranded and single-stranded DNA-cellulose were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the ³⁵S/³H ratios were obtained for each column fraction and for individual polypeptides. The protein fractions binding to single-stranded, but not double-stranded, DNA and eluting at high salt concentrations (greater than 0.60 M NaCl) showed elevated ³⁵S/³H ratios. This indicated increased synthesis of these proteins within 5 h after the onset of transformation. The majority of the polypeptides in these fractions showed increased synthesis as a consequence of transformation. One prominent polypeptide among them constituted 0.1% of the cytosol protein and had a molecular weight of 93,000. It is concluded that the synthesis of proteins binding tightly to single-stranded DNA is increased early after the onset of transformation.