Lica L M, Narayanswami S, Hamkalo B A
J Cell Biol. 1986 Oct;103(4):1145-51. doi: 10.1083/jcb.103.4.1145.
The experiments described were directed toward understanding relationships between mouse satellite DNA, sister chromatid pairing, and centromere function. Electron microscopy of a large mouse L929 marker chromosome shows that each of its multiple constrictions is coincident with a site of sister chromatid contact and the presence of mouse satellite DNA. However, only one of these sites, the central one, possesses kinetochores. This observation suggests either that satellite DNA alone is not sufficient for kinetochore formation or that when one kinetochore forms, other potential sites are suppressed. In the second set of experiments, we show that highly extended chromosomes from Hoechst 33258-treated cells (Hilwig, I., and A. Gropp, 1973, Exp. Cell Res., 81:474-477) lack kinetochores. Kinetochores are not seen in Miller spreads of these chromosomes, and at least one kinetochore antigen is not associated with these chromosomes when they were subjected to immunofluorescent analysis using anti-kinetochore scleroderma serum. These data suggest that kinetochore formation at centromeric heterochromatin may require a higher order chromatin structure which is altered by Hoechst binding. Finally, when metaphase chromosomes are subjected to digestion by restriction enzymes that degrade the bulk of mouse satellite DNA, contact between sister chromatids appears to be disrupted. Electron microscopy of digested chromosomes shows that there is a significant loss of heterochromatin between the sister chromatids at paired sites. In addition, fluorescence microscopy using anti-kinetochore serum reveals a greater inter-kinetochore distance than in controls or chromosomes digested with enzymes that spare satellite. We conclude that the presence of mouse satellite DNA in these regions is necessary for maintenance of contact between the sister chromatids of mouse mitotic chromosomes.
所描述的实验旨在了解小鼠卫星DNA、姐妹染色单体配对和着丝粒功能之间的关系。对一条大型小鼠L929标记染色体进行电子显微镜观察发现，其多个缢痕中的每一个都与姐妹染色单体接触位点以及小鼠卫星DNA的存在位置重合。然而，这些位点中只有位于中央的那个位点拥有动粒。这一观察结果表明，要么单独的卫星DNA不足以形成动粒，要么当一个动粒形成时，其他潜在位点会受到抑制。在第二组实验中，我们发现经Hoechst 33258处理的细胞（希尔维希，I.，和A. 格罗普，1973年，《实验细胞研究》，81:474 - 477）中高度伸展的染色体缺乏动粒。在这些染色体的米勒铺展片中看不到动粒，并且当使用抗动粒硬皮病血清对这些染色体进行免疫荧光分析时，至少有一种动粒抗原不与这些染色体相关联。这些数据表明，着丝粒异染色质处的动粒形成可能需要一种因Hoechst结合而改变的高级染色质结构。最后，当中期染色体用能够降解大部分小鼠卫星DNA的限制性酶进行消化时，姐妹染色单体之间的接触似乎会被破坏。对消化后的染色体进行电子显微镜观察显示，配对位点处姐妹染色单体之间的异染色质大量丢失。此外，使用抗动粒血清进行荧光显微镜观察发现，与对照或用不切割卫星DNA的酶消化的染色体相比，动粒间距离更大。我们得出结论，这些区域中存在小鼠卫星DNA对于维持小鼠有丝分裂染色体姐妹染色单体之间的接触是必要的。
