FESEM IMAGING OF NUCLEAR STRUCT IN TRANSCRIPTION SPLICING & RNA TRANSPORT

我们感兴趣的是了解功能和结构 哺乳动物细胞核的组织。由于高度紧凑， 原子核的性质，很难分析不溶解的 通过检查无树脂中最佳固定的细胞来观察细胞核的部分 切片使用高分辨率扫描电子显微镜。这个 样品的无树脂制备可能会显示出不同的情况 难以区分的结构特征。这些物质的免疫标记 结构将加深我们对功能的理解 原子核的组织。斯佩克特博士和黄女士 调查组织的结构和功能 哺乳动物的细胞核。我们的目标是确定大分子 核内与RNA聚合酶转录相关的结构域 I和II，剪接因子的组装位置，前mRNA的位置 剪接，以及将mRNA运输到核膜上。之后 化学固定和EPON包埋或快速冷冻固定 然后冷冻置换和包埋，薄片 由传输EM成像。不幸的是，薄片的显微照片 通常很难解释。因此，我们应该准备更厚的 部分(200 Nary)，然后使用开发的方法移除EPON Ris和Malecki(1993)，并使用LOW制作立体显微照片 电压-场强扫描电子显微镜(日立S-900型)。进度报告：收到的数据块 到目前为止，黄女士的保存情况并不令人满意 细胞结构。1997年7月，黄女士将担任教职员工。 在芝加哥西北大学医学院的职位。我们 将继续这一项目。

We are interested in understanding the functional and structural organization of the mammalian cell nucleus. Due to the highly compact nature of the nucleus, it is diff'cult to analyze the insoluble portion of the nucleus by examining optimally fixed cells in resinless sections using high resolution scanning electron microscopy. The resinless preparation of the specimen may reveal otherwise indistinguishable structural features. Immunolabeling of these structures will further our understanding of the functional organization of the nucleus. Dr. Spector and Ms Huang are investigating the structural and functional organization of the mammalian cell nucleus. The goal is to determine the macromolecular domains in the nucleus associated with transcription by RNA polymerase I and II, sites of assembly of splicing factor, sites of pre-mRNA splicing, and of transport of mRNA to the nuclear envelope. After either chemical fixation and epon embedding, or rapid cryo-fixation followed by freeze-substitution and embedding, thin sections were imaged by transmission EM. Unfortunately micrographs of thin sections are often difficult to interpret. We shall therefore prepare thicker sections (200 nary), then remove the epon with the method developed by Ris and Malecki (1993), and prepare stereo micrographs using low voltage-FESEM (Hitachi S-900). Progress repon: The blocks received so far from Ms Huang suffered from unsatisfactory preservation of cellular structure. In July 1997 Ms Huang will assume a faculty position at Northwestem University Medical School in Chicago. We shall then continue this project.