NUCLEOPROTEIN COMPLEXES FROM METASTATIC CELLS CONTAINING ONCOGENES AND TISSUE-SPECIFIC GENES - A NOVEL METHOD TO TRACK GENES ASSOCIATED WITH SPECIFIC NUCLEOPROTEINS

Intact nuclei derived from murine metastatic large-cell lymphoma and human chronic myelogenous leukemia cells were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein precursor complexes by treatment with Msp-I. The resultant complexes were composed of nucleoproteins (NPs) that were isolated and purified by two-dimensional isoelectric focusing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D-SDS-PAGE), electroelution from the gel, and removal of SDS by extractigel chromatography. Various NPs purified by 2D-SDS-PAGE were examined for the presence of oncogenes and tissue-specific genes using a dot-blot hybridization technique. The RNA polymerase products of NPs were labelled, purified, rind subsequently used in a back-hybridization assay to identify transcripts for particular genes. By utilizing a 2D-SDS-PAGE Southwestern technique in parallel with the dot-blot and RNA back-hybridization assays, we assessed whether it is possible to ''track'' a gene and its associations in particular NPs. In patients with chronic myelogenous leukemia, we screened similar to 1000 NPs for bcl-2 sequences and found them present in a single NP of apparent M(r) similar to 19,000, pI similar to 5.5. In murine RAW117-H10 cells transformed by the abl oncogene, we found by Western analysis that an antigen cross-reacting with abl antigen was localized to a p53 gene-containing NP of apparent M(r) similar to 22,000, pI similar to 7.2. A coincident Southwestern experiment using the same blot showed that the abl gene was bound by the same NP. The techniques described present the basis for ''tracking'' a particular gene to individual NPs and studying its relationship to other genes, their respective gene products, and its binding properties with particular NPs.