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Nucleoprotein complexes from metastatic cells containing oncogenes and tissue-specific genes: a novel method to track
N L Rosenberg-Nicolson1, G L Nicolson
1Department of Tumor Biology, University of Texas-M. D. Anderson Cancer Center, Houston 77030.
Abstract:
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.
Insights
Researchers developed novel methods to isolate and analyze nucleoprotein complexes (NPs), enabling the tracking of specific genes within these structures. This technique allows for the study of gene associations and their functional roles in diseases like leukemia.
Area of Science:
- Molecular Biology
- Cancer Research
- Genomics
Background:
- Nucleoprotein complexes (NPs) play a crucial role in gene regulation and cellular processes.
- Understanding the composition and function of NPs is essential for deciphering gene expression in health and disease.
Purpose of the Study:
- To develop and validate a methodology for isolating and characterizing discrete nucleoprotein complexes.
- To investigate the presence and association of specific genes and oncogenes within these purified NPs.
- To establish a framework for tracking gene localization and interactions within subchromatin structures.
Main Methods:
- Digestion of nuclei from lymphoma and leukemia cells to yield subchromatin complexes.
- Purification of nucleoproteins (NPs) using two-dimensional SDS-PAGE and electroelution.
- Dot-blot hybridization and RNA back-hybridization assays to identify genes and transcripts.
- 2D-SDS-PAGE Southwestern technique to assess gene-binding properties within NPs.
Main Results:
- Successfully isolated and purified specific NPs containing oncogenes (bcl-2, abl) and tumor suppressor genes (p53).
- Identified a single NP containing bcl-2 sequences in chronic myelogenous leukemia patients.
- Localized an abl-cross-reacting antigen to a p53 gene-containing NP in murine cancer cells, with the abl gene bound to the same NP.
Conclusions:
- The described techniques enable the precise tracking of individual genes within specific NPs.
- This approach facilitates the study of gene relationships, their products, and binding interactions within subchromatin structures.
- Provides a foundation for understanding gene behavior in complex cellular environments and disease states.