Nucleoproteins derived from subnuclear RNA polymerase complexes of metastatic large‐cell lymphoma cells possess transcription activities and regulatory properties in vitro

Abstract

Intact nuclei derived from poorly or highly liver-metastatic murine large-cell lymphoma cell line RAW117 were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein (DNP/RNP) complexes with Msp-I. The DNP/RNP complexes were composed of DNP/RNPs which were derived from the DNP/RNP complexes by incubation in the presence or absence of DNase-I and subsequent isolation by two-dimensional [isoelectric focusing/sodium dodecylsulfate (SDS)] polyacrylamide gel electrophoresis (PAGE), electroelution from the gel, and removal of SDS. Approximately 450 DNP/RNPs in the two-dimensional gels corresponding to discrete spots or in some cases streaks were analyzed for the presence of v-abl, p53, c-neu, c-H-ras, beta-casein, 18s rDNA, and mu-chain immunoglobulin genes using a hybridization technique. Ten DNP/RNP complexes contained tightly associated p53 DNA, whereas six contained c- or v-abl, four contained mu-chain gene, two contained c-H-ras, one contained dot-blot beta-casein, two contained 18s rDNA, and c-neu was found in one of the DNP/RNPs. The DNP/RNPs were also analyzed for in vitro RNA polymerase and primase activities. To assess the potential transcription abilities of the isolated DNP/RNPs, individual DNP/RNPs or DNP/RNP mixtures (reconstituted after SDS-PAGE separation) were examined for RNA polymerase initiation and synthesis. When RNA products were formed, these were purified by extracellulose chromatography and used as back-hybridization probes for the genes of interest. The RNA products were also analyzed by RNA gel electrophoresis. RNA formation was inhibitable by actinomycin D, and the RNAs formed ranged in size from approximately 80 kbp to approximately 1 kbp. By mixing various DNP/RNP complexes together, different patterns of RNA synthesis were found. For example, one DNP/RNP of M(r) approximately 140,000, isoelectric point (pl) approximately 5.8 synthesized a high molecular weight RNA in vitro that hybridized with beta-casein cDNA, but beta-casein is not expressed in RAW117 cells, suggesting that the silencing of the beta-casein gene was negated by isolation of the DNP/RNP. Mixing this DNP/RNP with two other specific DNP/RNPs again inhibited the synthesis of beta-casein RNA, suggesting that interactions between DNP/RNP complexes can result in differential RNA expression or regulation of RNA polymerases in vitro.

Nucleoprotein gene tracking: Localization of specific HIV-1 genes to subchromatin nucleoprotein complexes containing endonuclease activity in HIV-1-infected human cells

“…NPGT has been used to analyze NPC fractions from various cell systems for the presence of specific genes as well as enzymatic activity. For example, we have screened NPC from metastatic variants of the murine large cell lymphoma for the presence of abl, p53, c-neu, c-H-ras, ␤-casein, 18S rDNA, and µ-chain immunoglobulin genes and found that these genes were differentially expressed in the highly metastatic variants [Rosenberg-Nicolson and Nicolson 1992b;Nicolson and Nicolson, 1992b, 1993,1994aNicolson et al, 1995;1996]. We also used RNA back-hybridization assays to determine whether individual nuclei-derived NPC are capable of synthesizing mRNA in vitro that could back-hybridize to specific genes, indicating the presence of enzymatic activities capable of producing RNA with specific sequences capable of hybridizing back to the gene sequences from which they were derived.…”