Isolation and characterization of the nuclear matrix in higher plant cells

用含有非离子洗涤剂的缓冲体系从胡萝卜体细胞胚胎中分离出细胞核。为了制备核基质，纯化的细胞核用DNA酶消化，然后用高浓度的氯化钠提取。一种针对胡萝卜核基质蛋白的单抗CML-1，在共聚焦显微镜和电子显微镜下显示时，选择性地标记胡萝卜原生质体的核外围。该抗体几乎不标记间期细胞的胞浆和核浆。CML-1识别从残核基质中尿素提取的表观分子质量为96-130 kDa的几种蛋白质。为了鉴定与脊椎动物核层结构同源的高等植物细胞的组成蛋白，从lambdagt11胡萝卜胚核表达文库中克隆了编码CML-1识别蛋白的cDNA，并测定了其序列。该蛋白被命名为NMCP1。当将推导出的氨基酸序列与主要数据库中包含的其他已知蛋白质序列进行比较时，没有发现NMCP1整个区域的蛋白质具有很高的序列同源性。然而，该氨基酸序列与肌球蛋白重链、原肌球蛋白以及包括脊椎动物蛋白在内的一些中间丝蛋白有很强的相似性。对NMCP1蛋白的结构分析表明，它含有一个长的α-螺旋区域，显示出非极性残基的Haptad重复，与丝状形成蛋白的结构非常相似。此外，还存在潜在的核定位信号序列基序和不同类型蛋白激酶的底物。这些结果表明，NMCP1蛋白形成卷曲的丝状结构，是高等植物细胞核外围结构的组成部分。

Nuclei were isolated from somatic embryos of carrot using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified nuclei were digested with DNase, followed by extraction with a high concentration of NaCl. A monoclonal antibody, CML-1, raised against carrot nuclear-matrix proteins, selectively labeled the nuclear periphery of carrot protoplasts when visualized by confocal and electron microscopy. The antibody scarcely labeled the cytoplasm and nucleoplasm of interphase cells. CML-1 recognized several proteins with apparent molecular masses of 96-130 kDa that were urea-extracted from the residual nuclear matrices. To identify the constituent proteins of higher plant cells structurally homologous to the vertebrate nuclear lamina, wer cloned a cDNA encoding a CML-1-recognized protein from a lambdagt11 carrot embryo cDNA expression library and determined its sequence. The protein was designated NMCP1. When the deduced amino acid sequence wascompared with other known protein sequences contained in major databases, no protein was found to show high sequence identity across the whole region of NMCP1. However, the amino acid sequence showed partial strong similarities with myosin heavy chain, tropomyoshin and some intermediate filament proteins including vertebrate lamins. Structural analysis of the NMCP1 protein indicated that it contained a long alpha-helical region exhibiting haptad repeats of apolar residues, demonstrating significant structural similarity with those of filament-forming proteins. In addition, there were potential sequence motifs for nuclear locating signals and substrates for different types of protein kinases. These results suggest that NMCP1 protein forms coiled-coil filaments and is a constituent of the peripheral architecture of the higher plant cell nucleus.