Screening of isolated DNA for sequences released from anchorage sites in nuclear matrix.
Isolated chromosomal DNA is associated with polypeptides that are not released from DNA by several methods designed to purify DNA, e.g. treatment with sodium dodecyl sulphate. DNA fragments associated with these very tight DNA/protein complexes show high affinity to nitrocellulose filters in the presence of salt concentrations of 500 mM or greater. Consequently, a fraction of AluI-fragmented native DNA comprising the complexes and 0.2 to 0.3 micron of vicinal DNA can be isolated by one filtration step. This fraction of DNA shows characteristics of residual DNA sequences retained in nuclei after extraction with nucleases and high salt (nuclear matrix). The DNA fragments retained on filters are highly enriched in replicative DNA; and their degree of hybridization with poly(A)+ RNA points to enrichment in actively transcribed sequences. The results support previous work indicating that the very tight DNA/polypeptide complexes co-isolating with DNA under conditions that release other peptide materials from DNA may be anchorage sites of DNA in the nuclear matrix. Moreover, the method described here allows isolation of replicating and actively transcribed DNA sequences directly from isolated total genomic DNA by skipping artefact-prone isolations of the nuclear matrix.
D000818:Animals; D001483:Base Sequence; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis; D002467:Cell Nucleus / Q000032:analysis; D002478:Cells, Cultured; D003101:Collodion; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004261:DNA Replication; D005374:Filtration / Q000295:instrumentation; D009693:Nucleic Acid Hybridization; D010455:Peptides / Q000032:analysis; D014158:Transcription, Genetic
D010455:Peptides; D003101:Collodion; D004247:DNA