Department of Biological Sciences, Faculty of Science and Engineering, Chuo University, Tokyo, Japan.
Biological Science Course, Graduate School of Science and Engineering, Chuo University, Tokyo, Japan.
PLoS One. 2020 Nov 23;15(11):e0242694. doi: 10.1371/journal.pone.0242694. eCollection 2020.
The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two β-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we screened an insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. Most of the disruptants did not exhibit major defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate tubulin-mutants resistant to the anti-tubulin agents propyzamide (pronamide) or oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 12 strains bearing 7 different β-tubulin mutations. One of the mutations is at a residue similar to that of a mutation site known to confer drug resistance in human cancer cells. Some strains had the same amino acid substitutions as those reported previously in C. reinhardtii; however, the mutants with single tubulin genes showed slightly stronger drug-resistance than the previous mutants that express the mutated tubulin in addition to the wild-type tubulin. Such increased drug-resistance may have facilitated sensitive detection of tubulin mutation. Single-tubulin-gene disruptants are thus an efficient background of generating tubulin mutants for the study of the structure-function relationship of tubulin.
单细胞绿藻莱茵衣藻（Chlamydomonas reinhardtii）拥有两个α-微管蛋白基因（tua1 和 tua2）和两个β-微管蛋白基因（tub1 和 tub2），每对中的两个基因编码相同的氨基酸序列。在这里，我们筛选了一个插入文库，建立了 8 个 tua2、tub1 或 tub2 表达缺陷的突变体。大多数突变体在细胞生长、鞭毛长度或截肢后鞭毛再生方面没有表现出主要缺陷。由于迄今为止报道的莱茵衣藻微管蛋白突变体很少，我们随后使用我们的突变体，以及来自 Chlamydomonas Library Project（CLiP）的 tua1 突变体，分离对抗微管蛋白药物丙嗪酰胺（pronamide）或oryzalin 具有抗性的微管蛋白突变体。经过几次尝试，我们获得了 8 株带有 7 种不同α-微管蛋白突变的菌株和 12 株带有 7 种不同β-微管蛋白突变的菌株。其中一个突变位于与已知赋予人类癌细胞耐药性的突变位点相似的残基上。一些菌株的氨基酸取代与之前在莱茵衣藻中报道的相同；然而，与表达突变型微管蛋白和野生型微管蛋白的先前突变体相比，带有单个微管蛋白基因的突变体显示出稍强的耐药性。这种增强的耐药性可能有助于敏感地检测微管蛋白突变。因此，单微管蛋白基因敲除突变体是生成微管蛋白突变体的有效背景，可用于研究微管蛋白的结构-功能关系。
