GAMMA-TUBULIN IN ARABIDOPSIS - GENE SEQUENCE, IMMUNOBLOT, AND IMMUNOFLUORESCENCE STUDIES
GAMMA-TUBULIN IN ARABIDOPSIS - GENE SEQUENCE, IMMUNOBLOT, AND IMMUNOFLUORESCENCE STUDIES
复制标题
DOI：
10.1105/tpc.6.2.303
复制
发表时间：
1994-02-01
期刊：
影响因子：
11.6
通讯作者：
SNUSTAD, DP
中科院分区：
文献类型：
--
作者：
LIU, B;JOSHI, HC;SNUSTAD, DP
γ-微管蛋白是与多种真核细胞中的微管（Mt）组织中心相关的蛋白质。不幸的是，人们对植物中的这种中心知之甚少。基因组和部分cDNA克隆编码两个γ-微管蛋白的拟南芥分离和测序。基因组和cDNA序列的比较表明，这两个基因，TubG 1和TubG 2，含有9个内含子在保守的位置。这两个基因的序列都预测蛋白质含有474个氨基酸，分子量分别为53，250和53，280 D。预测的γ 1-和γ 2-微管蛋白表现出彼此98%的氨基酸同一性，并且与动物和真菌的γ-微管蛋白具有类似于70%的氨基酸同一性。RNA凝胶印迹结果表明，这两个基因的转录在悬浮培养细胞，幼苗，根和花的成熟植物。使用对γ-微管蛋白的保守肽具有特异性的抗体对拟南芥蛋白质进行免疫印迹，显示出M（r）为58，000的主要交叉反应多肽。相同的抗体染色该物种的组织和悬浮培养细胞中的所有Mt阵列。结合被两个拟南芥基因序列预测的γ-微管蛋白中的同源寡肽抑制。抗体染色避免了在动粒和细胞板的MTS的正端，但与动物细胞中的情况不同，似乎定位于广泛延伸的动粒纤维和成膜体朝向负端。我们的结论是，至少有两个γ-微管蛋白的蛋白质同源物存在于拟南芥，其中至少有一个是本地化的沿着吨阵列。它的分布与植物中线粒体组织的独特特征有关，并可能有助于解释这些特征。
gamma-Tubulin is a protein associated with microtubule (Mt)-organizing centers in a variety of eukaryotic cells. Unfortunately, little is known about such centers in plants. Genomic and partial cDNA clones encoding two gamma-tubulins of Arabidopsis were isolated and sequenced. Comparisons of genomic and cDNA sequences showed that both genes, TubG1 and TubG2, contain nine introns at conserved locations. The sequences of the two genes both predict proteins containing 474 amino acids, with molecular masses of 53,250 and 53,280 D, respectively. The predicted gamma 1- and gamma 2-tubulins exhibit 98% amino acid identity with each other and similar to 70% amino acid identity with the gamma-tubulins of animals and fungi. RNA gel blot results demonstrated that both genes are transcribed in suspension culture cells, seedlings, and roots and flowers of mature plants. Immunoblots of Arabidopsis proteins using an antibody specific to a conserved peptide of gamma-tubulin showed a major cross-reacting polypeptide with an M(r) of 58,000. The same antibody stained all Mt arrays in tissue and suspension culture cells of this species. Binding was inhibited by the homologous oligopeptide in the gamma-tubulins predicted by the two Arabidopsis gene sequences. Antibody staining avoided the plus ends of Mts at the kinetochores and cell plate, but unlike the case in animal cells, seemed to be localized over broad stretches of the kinetochore fibers and phragmoplast toward the minus ends. We concluded that at least two gamma-tubulin protein homologs are present in Arabidopsis and that at least one of them is localized along Mt arrays. Its distribution is correlated with and may help explain unique characteristics of Mt organization in plants.