Previous studies in this laboratory have shown that the nonhistone chromatin proteins from rat liver and calf thymus contain a replicative type D N A polymerase (1, 2). It was subsequently found that isolated cell nuclei from solid Walker tumor are especially rich in this enzymic activity. Investigation of the acidic chromosomal proteins from Walker 256 carcinosarcoma has resulted in the partial purification of a DNA polymerase. The method of the enzyme purification is described in the present report. I t will be shown that the purified DNA polymerase requires only native D N A as template and a cumplete supplement of all four deoxyriboside triphosphates as substrates. The enzymic reaction also depends on the presence of M e + , monovalent cations being inhibitory. Materials and Methods. Walker 256 carcinosarcomas were developed for 7 days after tumor cells were injected intramuscularly into the hind legs of rats. The tumor cells had a mitotic cycle of 22-25 days and * Supported by grants from the American Cancer Society (E-44) and the Public Health Service (GM-11698).showed well-organized endoplasmic reticulum network. All the following operations to be described were carried out at 2 4 ' .Cell nuclei were isolated from the tumor tissue by the method of Chauveau et al. (
) .The tumor nuclei were extracted with 100 vol of 0.05 M Tris-HC1, pH 7.4, containing 5 mM MgC12, followed by repeating the buffer extraction three times. The nonhistone chromosomal proteins were prepared from the washed nuclei according to previously described procedure (4).The isolated acidic chromosomal proteins were dialyzed overnight against 0.05 M Tris-HC1, pH 8.5, containing 1 mM 2-mercaptoethanol. To the dialyzed acidic protein solution, saturated ammonium sulfate (AS) solution (adjusted to pH 8.5 with NH,OH) was added slowly with stirring to 40% saturation with respect to ammonium sulfate. After standing for 15 min, the mixture was centrifuged at 10,OOg for 15 min and the precipitate was discarded. To the clear supernate enough ammonium sulfate was added to a final 607% saturation of ammonium sulfate. The precipitate (4&60% AS) thus formed at UNSW Library on July 11, 2015 ebm.sagepub.com Downloaded from