Preferential association of a transcriptionally active gene with the nuclear matrix of rat fibroblasts transformed by a simian-virus-40–pBR322 recombinant plasmid

Abstract

To study the relationship between the structural organization and function of the eukaryotic genome, DNA associated with nuclear matrix was analysed by using a transformed rat fibroblast cell line. The nuclear matrices were prepared from the isolated nuclei of pSV1-FR, a rat fibroblast cell line transformed by a pBR322-based recombinant plasmid containing an early gene region, which codes for large T-antigen, of simian virus 40. This transformed cell contained a single copy of the plasmid sequence integrated into the chromosomal DNA of the host cell. The early gene of this plasmid was constitutively expressed, as demonstrated by positive immunofluorescence staining of the cell for large T-antigen and by RNA-blot analysis for its specific mRNA. DNAs were extracted from whole isolated nuclei and nuclear-matrix preparations of the cells, and the relative amounts of the sequence similar to that of the plasmid were compared between these DNA preparations. By employing dot hybridization and Southern-blot analyses we found that the plasmid sequence was more enriched in the DNA extracted from the nuclear matrices than in the DNA extracted from the whole nuclei. When an albumin gene sequence that was not transcribed in this cell line was compared similarly as a control, we found no significant enrichment of this sequence in the DNA associated with the nuclear matrix. Our results strongly support the concept that a transcriptionally active gene is preferentially associated with the nuclear matrix.

What they are, How they Work and Why they do What they do? The Story of SV40-derived Gene Therapy Vectors and What They Have to Offer

“…Rather, they appear to integrate more frequently than does wtSV40. The mechanism(s) of SV40 (and, by extension, rSV40) integration is unknown: the degree of homology between viral and cellular DNA sequences at the integration sites is low [Gilbert and Cohen, 1990;Ogata, 1990]. SV40 DNA is known to associate with nuclear matrix, which brings it into proximity with open chromatin.…”

Sequence-specific DNA-binding proteins are components of a nuclear matrix-attachment site.

“…There are also data demonstrating that actively transcribed genes are preferentially associated with the nuclear matrix (21)(22)(23)(24)(25). However, information describing the sequence specificity and the protein components of these MARs is limited.…”

Nuclear matrix proteins in human coloncancer.

“…(ii) Since colon tumor cell lines are all derived from epithelial adenocarcinoma and not stromal cells, the nuclear matrix proteins from cell lines help to rule out significant stromal protein contamination of the nuclear matrix preparation. Each of the tumorspecific proteins CC-1 through CC-6 can be detected in one or more colon tumor cell lines ( (1994) matrix protein structures are associated with actively transcribed genes (29,30) and serve as sites for processing and transport of heterogeneous nuclear RNA (31,32). Tissuespecific nuclear matrix proteins have been identified in the rat, mouse, and human (21)(22)(23), and changes in the presence or absence of specific nuclear matrix proteins have been associated with cellular transformation and differentiation (29,33,34).…”