Hempel K, Strätling W H
Institut für Physiologische Chemie, Universitäts-Krankenhaus Eppendorf, Hamburg, FR Germany.
J Cell Sci. 1996 Jun;109 ( Pt 6):1459-69. doi: 10.1242/jcs.109.6.1459.
Cultured chicken cells were encapsulated in agarose microbeads, lysed in a near-physiological buffer and resulting encapsulated nuclei were digested with a restriction enzyme and electroeluted. After removal of approximately 97% of the chromatin, the nuclear lamina, residual nucleoli and an internal nuclear network remained. The majority of nascent RNA was also recovered in digested and electroeluted nuclei. Surprisingly, however, the chicken lysozyme gene 5' MAR was quantitatively electroeluted from digested nuclei of expressing and non-expressing cells, as well as the promoter region and the coding sequence. When encapsulated nuclei were digested partially, the proportion of elutable 5' MAR chromatin was comparable to that of elutable bulk chromatin. Furthermore, after digestion of encapsulated nuclei from Drosophila Kc cells, the histone SAR was electroeluted to the same extent as bulk chromatin. We conclude that the lysozyme gene 5' MAR and the histone SAR are not permanently attached to a nuclear matrix or scaffold.
将培养的鸡细胞封装在琼脂糖微珠中，在接近生理条件的缓冲液中裂解，然后用限制性内切酶消化得到的封装细胞核并进行电洗脱。去除约97%的染色质后，核纤层、残留的核仁及一个内部核网络保留了下来。大多数新生RNA也在消化和电洗脱的细胞核中回收。然而，令人惊讶的是，鸡溶菌酶基因5' MAR以及启动子区域和编码序列从表达和非表达细胞的消化细胞核中被定量电洗脱。当部分消化封装的细胞核时，可洗脱的5' MAR染色质的比例与可洗脱的整体染色质的比例相当。此外，消化果蝇Kc细胞的封装细胞核后，组蛋白SAR被电洗脱的程度与整体染色质相同。我们得出结论，溶菌酶基因5' MAR和组蛋白SAR并非永久附着于核基质或支架。
