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 labeled, purified, and 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 approximately 1000 NPs for bcl-2 sequences and found them present in a single NP of apparent M(r) approximately 19,000, pI approximately 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) approximately 22,000, pI approximately 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.
Rosenberg-Nicolson
N L
NL
Department of Tumor Biology, University of Texas-M. D. Anderson Cancer Center, Houston 77030.
Nicolson
G L
GL
D002364:Caseins / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D016313:Genes, abl; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D008970:Molecular Weight; D009362:Neoplasm Metastasis; D009698:Nucleoproteins / Q000032:analysis* / Q000302:isolation & purification; D009857:Oncogenes; D009928:Organ Specificity; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2
D002364:Caseins; D009698:Nucleoproteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2