Neuer B, Werner D
J Mol Biol. 1985 Jan 5;181(1):15-25. doi: 10.1016/0022-2836(85)90321-3.
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.
分离的染色体DNA与一些多肽相关联，这些多肽不能通过多种旨在纯化DNA的方法（如用十二烷基硫酸钠处理）从DNA上释放下来。与这些紧密的DNA/蛋白质复合物相关的DNA片段在盐浓度为500 mM或更高时，对硝酸纤维素滤膜表现出高亲和力。因此，通过一步过滤可以分离出一部分包含这些复合物和0.2至0.3微米邻近DNA的AluI酶切的天然DNA。这部分DNA显示出在用核酸酶和高盐（核基质）提取后保留在细胞核中的残余DNA序列的特征。保留在滤膜上的DNA片段在复制性DNA中高度富集；并且它们与聚（A）+ RNA的杂交程度表明在活跃转录序列中富集。这些结果支持了先前的工作，即表明在能从DNA释放其他肽物质的条件下与DNA共同分离的紧密DNA/多肽复合物可能是DNA在核基质中的锚定位点。此外，这里描述的方法通过跳过容易产生假象的核基质分离步骤，允许直接从分离的总基因组DNA中分离出正在复制和活跃转录的DNA序列。
