Demin S Iu
Institute of Cytology, Russian Academy of Sciences, St. Petersburg.
Tsitologiia. 1999;41(1):66-86.
Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.
表面伸展的无膜细胞核和有丝分裂图像的制备用于揭示高阶核结构和染色体结构。通过将无膜细胞核和有丝分裂图像悬浮液滴加在湿润的超净载玻片上的甲醇 - 冰醋酸混合物（3:1）中来获得制备物。无膜细胞核和有丝分裂图像是通过在条件培养基中用新鲜制备的0.018 - 0.06% Triton X - 100溶液对完整的小鼠和人类细胞（L1210、SK - UT - 1B细胞系、PHA刺激的淋巴细胞）进行1 - 5分钟的预固定毛细管移液分离得到的。伸展的无膜细胞核和有丝分裂图像没有诱导染色质分散和浓缩的特征。伸展的间期无膜细胞核显示出空间上分离的单个结构（细染色质纤维、核仁、核内体）、核仁周围染色质聚集的多态模式以及偶尔出现的串珠状粗染色质纤维和一个染色中心。有丝分裂图像中伸展染色体的染色粒模式与G带染色的模式非常相似但并不完全相同。在所有测试的细胞类型中，伸展的前中期有丝分裂图像总是包含松散的“残留”核仁，就其形状和每个细胞（有丝分裂图像）中的数量而言，看起来像典型的前期核仁。伸展的有丝分裂图像和前期无膜细胞核的大多数染色体都附着在核仁物质上。所有测试的细胞系在前期无膜细胞核和前中期有丝分裂图像中，附着在核仁上的染色体数量几乎都有相同的变化。伸展的有丝分裂图像中的一些染色体与“残留”核仁共定位，看起来缩短且高度浓缩。其他局部与“残留”核仁相关的染色体是直的，并径向指向这些核仁。涂片上有丝分裂细胞和伸展的有丝分裂图像中的染色体相互排列是相似的。用PBS洗涤的SK - UT - 1B细胞的赤道板比未处理细胞的赤道板显示出更好的伸展能力。在前一种情况下，中期染色体在GTG带染色程序后更均匀地伸展且易于识别。每个伸展的有丝分裂图像（或每个伸展的前期无膜细胞核）中染色体间（主要是端粒 - 端粒和端粒 - 着丝粒）连接的数量在晚前中期最少，在前期和早前中期最多，在中期居中。根据有丝分裂染色体的拓扑结构和纵向异质性对所得数据进行了讨论。
