Ascoli C A, Link M R, Venturo N, Kuchler R J, Mandeles S
Department of Biochemistry, Faculty of Arts and Sciences, Rutgers-State University of New Jersey, Piscataway 08854.
Arch Biochem Biophys. 1988 Jun;263(2):334-48. doi: 10.1016/0003-9861(88)90644-3.
We have characterized two components of DNA isolated from mouse L-M cell nuclei. These components, designated as HMW (high molecular weight) and VHMW (very high molecular weight) DNA, were characterized by rate zonal sedimentation, agarose gel electrophoresis, and for protein content. Our electron micrographs revealed that HMW-DNA contained mainly linear molecules with few single rosette structures, while the VHMW-DNA was enriched in rosettes, many of which were significantly larger and linked together in multimeric structural forms. The VHMW-DNA component was also enriched for residual protein, which we believe represents the core of the rosette. The characteristics of this residual protein are consistent with reported findings of the most tightly bound proteins. The rosette conformation does not appear to be an artifact of microscopy or of an aggregate nature for several reasons: (i) rosettes are preferentially found in the VHMW-DNA component; (ii) further manipulation or purification of the DNA disrupts the rosette structure and produces linear fragments; (iii) the amount of proteinaceous material at the core of the rosette is diminished when the DNA is further purified; and (iv) treatment of intact nuclei with a novel bisamine reagent putatively crosslinks DNA in vivo and minimizes the disruption of rosettes by shear. We believe this separation of chromatin is critical to establish the architectural forms of euchromatin and heterochromatin of interphase DNA in the eucaryotic system. Once established, fractionated chromatin can be used to identify specifically expressed or repressed genes with linear form DNA and rosette form DNA. We discuss rosettes as derivatives of chromosomal domains that retain structural features because of residual peptide elements.
我们已对从小鼠L-M细胞核中分离出的两种DNA成分进行了表征。这些成分，分别命名为HMW（高分子量）和VHMW（极高分子量）DNA，通过速率区带沉降、琼脂糖凝胶电泳以及蛋白质含量分析进行了表征。我们的电子显微镜照片显示，HMW-DNA主要包含线性分子，只有少量单花环状结构，而VHMW-DNA富含花环状结构，其中许多明显更大，并以多聚体结构形式连接在一起。VHMW-DNA成分还富含残留蛋白质，我们认为这些蛋白质代表了花环状结构的核心。这种残留蛋白质的特性与已报道的最紧密结合蛋白质的研究结果一致。花环状构象似乎并非显微镜观察的假象或聚集性质，原因如下：（i）花环状结构优先在VHMW-DNA成分中发现；（ii）对DNA进行进一步操作或纯化会破坏花环状结构并产生线性片段；（iii）DNA进一步纯化时，花环状结构核心的蛋白质物质数量会减少；（iv）用一种新型双胺试剂处理完整细胞核，推测该试剂可在体内交联DNA，并最大程度减少剪切对花环状结构的破坏。我们认为这种染色质的分离对于在真核系统中建立间期DNA常染色质和异染色质的结构形式至关重要。一旦确定，分级分离的染色质可用于通过线性形式DNA和花环状形式DNA鉴定特异性表达或抑制的基因。我们将花环状结构视为染色体结构域的衍生物，由于残留的肽元素而保留了结构特征。
