Lee V D, Huang B
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Plant Cell. 1990 Nov;2(11):1051-7. doi: 10.1105/tpc.2.11.1051.
Two beta-tubulin mutants of Chlamydomonas reinhardtii, colR4 and colR15, were previously isolated in our laboratory. Each mutant expressed an acidic beta-tubulin variant as a result of an alteration in the coding sequence of one of the two beta-tubulin genes in C. reinhardtii, which in the wild type encode identical proteins. In this report, we describe the identity of the specific beta-tubulin altered in the colR mutants and the precise nature of the genetic lesions. Hybrid selection of mutant poly(A)+ RNA with cDNA probes specific for the two beta-tubulins in C. reinhardtii indicated that both mutations resided in the beta 2-tubulin gene. cDNA libraries were constructed with mutant poly(A)+ RNA, and beta 2-tubulin cDNA clones were isolated. Results of in vitro transcription of cloned beta 2-tubulin cDNAs confirmed the identity of the altered genes. Sequencing of the entire coding regions of the beta 2-tubulin cDNA clones revealed that the mutants carried different single-base substitutions in the same codon for the amino acid at position 350 in the beta 2-tubulin sequence, effecting a change from a lysine to a glutamic acid in the colR4 variant and to a methionine in the colR15 variant. These changes in amino acids are consistent with the difference in the charge of the two variant polypeptides observed in isoelectric focusing. Because both the colR4 and colR15 mutations confer an altered sensitivity to a number of different microtubule inhibitors and herbicides, lysine 350 appears to be of functional importance in the structure of the tubulin molecule.
莱茵衣藻的两个β-微管蛋白突变体colR4和colR15，先前已在我们实验室中分离得到。每个突变体都表达一种酸性β-微管蛋白变体，这是由于莱茵衣藻两个β-微管蛋白基因之一的编码序列发生改变所致，在野生型中这两个基因编码相同的蛋白质。在本报告中，我们描述了colR突变体中发生改变的特定β-微管蛋白的身份以及遗传损伤的确切性质。用莱茵衣藻中两个β-微管蛋白特异的cDNA探针进行突变体poly(A)+ RNA的杂交筛选，表明这两个突变都位于β2-微管蛋白基因中。用突变体poly(A)+ RNA构建cDNA文库，并分离出β2-微管蛋白cDNA克隆。克隆的β2-微管蛋白cDNA的体外转录结果证实了发生改变的基因的身份。对β2-微管蛋白cDNA克隆的整个编码区进行测序，结果显示突变体在β2-微管蛋白序列中第350位氨基酸的同一个密码子上发生了不同的单碱基替换，在colR4变体中导致赖氨酸变为谷氨酸，在colR15变体中变为甲硫氨酸。这些氨基酸的变化与在等电聚焦中观察到的两种变体多肽电荷的差异一致。由于colR4和colR15突变都使细胞对多种不同的微管抑制剂和除草剂的敏感性发生改变，因此赖氨酸350在微管蛋白分子结构中似乎具有重要功能。
