Jenkins H, Hölman T, Lyon C, Lane B, Stick R, Hutchison C
Department of Biological Sciences, The University, Dundee, UK.
J Cell Sci. 1993 Sep;106 ( Pt 1):275-85. doi: 10.1242/jcs.106.1.275.
Xenopus egg extracts, which support nuclear assembly and DNA replication in vitro, were physically depleted of lamin B3 using monoclonal antibodies linked to magnetic beads. Depleted extracts were still able to support nuclear envelope assembly around demembranated sperm heads but the resulting pronuclei lacked a lamina and were unable to initiate semiconservative DNA replication or to assemble replicases, confirming previous data. Immunoblotting analysis of isolated nuclei and nuclear matrix fractions indicated that lamin-depleted nuclei still accumulated nucleoporins and PCNA. Furthermore, the rate of PCNA uptake was identical in lamin-depleted and control nuclei. However, neither the nucleoporins nor the PCNA was associated with nuclear matrix fractions. The major protein components of sperm pronuclear matrix fractions were characterized by two-dimensional gel electrophoresis. Of these proteins only three out of 22 species, other than the lamins, were significantly reduced in lamin-depleted nuclei, indicating that these nuclei do assemble a nuclear matrix.
非洲爪蟾卵提取物能够在体外支持细胞核组装和DNA复制，利用与磁珠相连的单克隆抗体从物理上耗尽其中的核纤层蛋白B3。耗尽核纤层蛋白B3的提取物仍然能够支持围绕去膜精子头部的核膜组装，但产生的原核缺乏核纤层，无法启动半保留DNA复制或组装复制酶，这证实了先前的数据。对分离出的细胞核和核基质组分进行免疫印迹分析表明，耗尽核纤层蛋白的细胞核仍然积累核孔蛋白和增殖细胞核抗原（PCNA）。此外，在耗尽核纤层蛋白的细胞核和对照细胞核中，PCNA摄取速率相同。然而，核孔蛋白和PCNA均未与核基质组分相关联。通过二维凝胶电泳对精子原核基质组分的主要蛋白质成分进行了表征。在这些蛋白质中，除核纤层蛋白外，22种蛋白质中只有3种在耗尽核纤层蛋白的细胞核中显著减少，这表明这些细胞核确实组装了核基质。
