Adams G M, Huang B, Piperno G, Luck D J
J Cell Biol. 1981 Oct;91(1):69-76. doi: 10.1083/jcb.91.1.69.
Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.
四种莱茵衣藻突变体代表了独立的基因位点，已证实在分离的轴丝制剂中，它们完全缺乏（pf - 18、pf - 19和pf - 15）或几乎完全缺乏（pf - 20）中央微管对复合体。使用两种电泳方法对35S标记的轴丝蛋白进行分析，结果显示所有这四种突变体缺乏或显著缺乏18种多肽，其分子量范围从360,000到20,000，这些多肽在野生型轴丝中正常存在。对在32P标记培养基上通过细胞生长标记的轴丝蛋白进行分析表明，这18种多肽中的8种亚群被磷酸化。使用高分辨率二维电泳系统结合含有从衣藻微管蛋白制备的抗α或抗β微管蛋白血清的琼脂糖凝胶覆盖物，对突变体和野生型轴丝及鞭毛的微管蛋白亚基含量进行了分析。免疫沉淀鉴定出两种主要的α微管蛋白、一种主要的β微管蛋白以及一种也能被抗β血清沉淀的次要成分。这些微管蛋白在突变体轴丝中均未显示出特定缺陷，并且微管蛋白多肽在突变体鞭毛中的二维图谱位置也未改变。这18种多肽为鉴定影响中央对微管复合体的其他突变体提供了有用的特征。此类突变体可能有助于确定这些多肽在中央微管中的结构或功能作用。基于此处分析的四种突变体的运动表型来获得其他中央对突变体的努力，已产生了与这四种突变体中的三种等位的突变体。
