Palmer D K, Margolis R L
Mol Cell Biol. 1985 Jan;5(1):173-86. doi: 10.1128/mcb.5.1.173-186.1985.
We have developed a competitive enzyme-linked immunosorbent assay for solubilized kinetochore components, using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) scleroderma autoimmune antibodies specific for these kinetochore elements. Using this quantitative assay, we found interphase persistent or "pre-kinetochore" components in low- and moderately high-salt (375 mM salt) extracts of micrococcal nuclease-digested rat liver and chicken erythrocyte nuclei. The release of antigen activity from nuclei under these conditions has been correlated with loss of pre-kinetochore foci as determined by immunofluorescence microscopy. Combined biochemical and competition assay analysis of chicken erythrocyte nuclear extracts indicates that pre-kinetochore components are tightly bound to chromatin of mononucleosome size. The conclusions based on competition assay data are supported by a direct binding assay, which confirms that antigens recognized by CREST sera are present on chromatin. These results raise the possibility that the kinetochore-specific chromosomal antigen(s) we have detected substitutes for "standard" mononucleosome components, such as histone H1. Furthermore, they suggest approaches to the isolation of kinetochore-specific DNA sequences from higher eucaryotes.
我们利用针对这些动粒成分的人CREST（钙质沉着、雷诺现象、食管功能障碍、指端硬化、毛细血管扩张）硬皮病自身抗体，开发了一种用于溶解动粒成分的竞争性酶联免疫吸附测定法。通过这种定量测定法，我们在微球菌核酸酶消化的大鼠肝脏和鸡红细胞核的低盐和中等高盐（375 mM盐）提取物中发现了间期持续存在的或“前动粒”成分。在这些条件下，核中抗原活性的释放与免疫荧光显微镜确定的前动粒焦点的丧失相关。对鸡红细胞核提取物的生化和竞争测定联合分析表明，前动粒成分与单核小体大小的染色质紧密结合。基于竞争测定数据得出的结论得到了直接结合测定法的支持，该测定法证实了CREST血清识别的抗原存在于染色质上。这些结果增加了一种可能性，即我们检测到的动粒特异性染色体抗原替代了“标准”单核小体成分，如组蛋白H1。此外，它们还提示了从高等真核生物中分离动粒特异性DNA序列的方法。
