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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05640738/
• 关键词: Nuclear matrix; Nuclear protein; Carrot (nuclear structure); somatic embryo; 核タンパク; 中間経フィラメント

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.

采用含有非离子去污剂的缓冲体系从胡萝卜体细胞胚中分离细胞核。为了制备核基质，用DNA酶消化纯化的核，然后用高浓度NaCl提取。一种单克隆抗体，CML-1，提出了对胡萝卜核基质蛋白，选择性地标记胡萝卜原生质体的核周边时，通过共聚焦和电子显微镜可视化。抗体几乎不标记间期细胞的胞质和核质。CML-1识别从残留核基质中尿素提取的表观分子量为96-130 kDa的几种蛋白质。为了鉴定与脊椎动物核纤层结构同源的高等植物细胞的组成蛋白，我们从胡萝卜胚cDNA表达文库中克隆了一个编码CML-1识别蛋白的cDNA，并测定了其序列。该蛋白被命名为NMCP 1。将推导的氨基酸序列与主要数据库中的其他已知蛋白质序列进行比较，没有发现在NMCP 1的整个区域内显示出高度序列同一性的蛋白质。然而，其氨基酸序列与肌球蛋白重链、原肌球蛋白和一些中间丝蛋白包括脊椎动物核纤层蛋白显示出部分强相似性。NMCP 1蛋白的结构分析表明，它含有一个长的α-螺旋区域，表现出半抗原重复的非极性残基，表现出显着的结构相似性与那些形成蛋白质。此外，还存在潜在的核定位信号和不同类型蛋白激酶底物的序列基序。这些结果表明，NMCP 1蛋白形成卷曲螺旋丝，是高等植物细胞核的外周结构的组成部分。

项目成果

K. Masuda, S. Takahashi, K. Nomura, M. Arimoto, M.Inoue: "Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L." Planta. 191. 532-540 (1993)

K. Masuda、S. Takahashi、K. Nomura、M. Arimoto、M.Inoue：“胡萝卜体细胞胚细胞核外围框架的残留结构和组成蛋白”。

Kiyoshi Masuda, Seiko Takahashi, Koji Nomura, Mitue Arimoto, Masyasu Inoue: "Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L." Planta. 191. 532-540 (1993)

Kiyoshi Masuda、Seiko Takahashi、Koji Nomura、Mitue Arimoto、Masyasu Inoue：“胡萝卜体细胞胚细胞核外围框架的残留结构和组成蛋白。”

Masuda etal.: "Residual Structure and constitnent proteins of the peripheral framewark of the cell nucleus in somatic embryos from Doucus carotaL" Planta. 191. 532-540 (1993)

Masuda 等人：“Doucus carotaL 体细胞胚胎中细胞核外围框架的残余结构和组成蛋白”Planta。