Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities

Abstract We report on the discovery and isolation of DNA‐ and RNA‐containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of ∼ 90 and ∼ 25kbp. The deoxyribonucleoprotein‐ribonucleoprotein complexes conatin RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large‐cell lymphoma cells by restriction digestion with Msp ‐1, gentle extraction with solutions containing MgCl 2 , but without chelating agents, and low ionic strength gel electrophoresis. Two‐dimensional (isoelectric focusing/M r ) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of ∼ 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific acticvites. Using the DNP/RNP complexes a discrete DNA polymerase α product of ∼ 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase α inhibitor aphidicolin. RNA polymerase assays in the presence of excess α‐amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex‐associated enzyme activities to be cell‐cycle dependent. The DNA and RNA polymerase‐related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors. Hybridization sutides showed that the complexes contained the abl oncogene which is expressed in RAW117 cells, but not the β‐casein gene which is not expressed in this cell system. © 1992 Wiley‐Liss, Inc.