FLAGELLAR MUTANTS OF CHLAMYDOMONAS - STUDIES OF RADIAL SPOKE-DEFECTIVE STRAINS BY DIKARYON AND REVERTANT ANALYSIS
FLAGELLAR MUTANTS OF CHLAMYDOMONAS - STUDIES OF RADIAL SPOKE-DEFECTIVE STRAINS BY DIKARYON AND REVERTANT ANALYSIS
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DOI：
10.1073/pnas.74.8.3456
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发表时间：
1977-01-01
影响因子：
11.1
通讯作者：
HUANG, B
中科院分区：
文献类型：
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作者：
LUCK, D;PIPERNO, G;HUANG, B
C.莱茵衣藻Pf14鞭毛轴丝中缺乏放射状辐条结构，野生型中存在的12种蛋白质在其35S标记的鞭毛蛋白质的二维图谱（等电聚焦/十二烷基硫酸钠电泳）中缺失。这些相同的蛋白质中有六个在pf1中缺失，pf1缺少辐头。为了确定任何缺失的蛋白质是否代表突变基因产物，应用了2种实验方法。第一个利用的事实，即任一突变株的配子与野生型配子融合形成四鞭毛双核体时，鞭毛功能恢复。通过用35S预标记突变蛋白并允许在不存在蛋白质合成的情况下发生恢复来监测分子水平的恢复。预期突变基因产物不会恢复为放射性蛋白，并且恢复将取决于未标记的野生型对应物的组装。第二种技术利用紫外线照射诱导的回复突变体。在pf14的情况下，双核体拯救导致11种放射性成分的恢复;只有蛋白质3不能作为放射性斑点出现。对于pf1，仅2种放射性蛋白被恢复;蛋白4、6、9和10不具有放射性。对pf1的回复突变体的分析给出了证据（改变的图谱位置），即蛋白质4是突变基因产物。在pf14的情况下，对22个回复突变体的分析没有提供类似的阳性证据表明蛋白3是基因产物。
The motility mutant of C. reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a 2-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its 35S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spokeheads. To determine whether any of the missing proteins represent the mutant gene product 2 experimental approaches were applied. The first makes use of the fact that gametes of either mutant strain when fused with a wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with 35S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The 2nd technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only 2 radioactive proteins are restored; proteins 4, 6, 9 and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.