Isolation and characterization of the nuclear matrix in higher plant cells

高等植物细胞核基质的分离和表征

基本信息

批准号：
05640738
负责人：
MASUDA Kiyoshi
金额：
$ 1.22万
依托单位：
HOKKAIDO UNIVERSITY (1995)Akita Prefectural College of Agriculture (1993-1994)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

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.

使用含有非离子洗涤剂的缓冲系统从胡萝卜的体细胞中分离核。为了制备核基质，用DNase消化了纯化的核，然后用高浓度的NaCl提取。一种单克隆抗体CML-1，针对胡萝卜核矩阵蛋白提出，当通过共聚焦和电子显微镜可视化时，选择性地标记了胡萝卜原生质体的核外围。抗体几乎没有标记相间细胞的细胞质和核质。 CML-1识别几种蛋白质，具有96-130 kDa的明显分子质量，这些蛋白是从残留核基质中提取尿素的。为了鉴定与脊椎动物核薄片在结构同源的较高植物细胞的组成蛋白，用lambdagt11胡萝卜胚胎cDNA表达文库编码CML-1-识别蛋白的cDNA，并确定其序列。该蛋白被指定为NMCP1。当推断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相比时，未发现在整个NMCP1区域中显示高序列同一性。然而，氨基酸序列与肌球蛋白重链，tropomyoshin和一些中间丝蛋白（包括脊椎动物层粘连蛋白）显示出部分强相似性。 NMCP1蛋白的结构分析表明，它包含一个长的α-螺旋区域，表现出触觉残基的触觉重复序列，显示出与形成细丝蛋白的结构相似性。此外，对于不同类型的蛋白激酶，还有用于核定位信号和底物的潜在序列基序。这些结果表明NMCP1蛋白会形成卷曲的纤维丝，并且是较高植物细胞核的外围结构的组成部分。