Forward and reverse genetic analysis of microtubule motors in Chlamydomonas
Forward and reverse genetic analysis of microtubule motors in Chlamydomonas
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DOI：
10.1006/meth.2000.1081
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发表时间：
2000-12-01
期刊：
影响因子：
4.8
通讯作者：
Witman, GB
中科院分区：
文献类型：
--
作者：
Pazour, GJ;Witman, GB
整合生物化学，细胞生物学和遗传学方法的能力，使衣原体reinhardtii的真核生物鞭毛及其相关的分子马达的研究的首要模式生物。在衣原体中已经发现了数百种运动性突变，包括许多影响动力蛋白和驱动蛋白的突变。这些突变产生了许多关于马达的结构和功能以及马达内单个亚基的作用的信息。插入突变的发展为衣原体的基因分析打开了一扇大门。插入突变体是通过用含有DNA的选择性标记转化细胞来产生的。DNA在整个基因组中随机整合，并且通常在插入位点删除部分染色体，从而产生由整合的DNA标记的突变。这些突变可用于正向遗传方法，其中人们表征突变表型，然后使用整合的DNA作为标签克隆相关基因。插入突变体也可用于反向遗传方法，其中通过DNA杂交鉴定缺乏目的基因的突变体。我们描述的方法来产生和表征插入突变体，使用影响外动力蛋白臂的突变为例。(C)北京大学出版社.
The ability to integrate biochemical, cell biological, and genetic approaches makes Chlamydomonas reinhardtii the premier model organism for studies of the eukaryotic flagellum and its associated molecular motors. Hundreds of motility mutations have been identified in Chlamydomonas, including many that affect dyneins and kinesins. These mutations have yielded much information on the structure and function of the motors as well as the roles of individual subunits within the motors. The development of insertional mutagenesis has opened the door to powerful new approaches for genetic analysis in Chlamydomonas. Insertional mutants are created by transforming cells with DNA-containing selectable markers. The DNA is randomly integrated throughout the genome and usually deletes part of the chromosome at the site of insertion, thereby creating mutations that are marked by the integrated DNA. These mutations can be used for forward genetic approaches where one characterizes a mutant phenotype and then clones the relevant gene using the integrated DNA as a tab. The insertional mutants also may be used in a reverse genetic approach in which mutants lacking a gene of interest ave identified by DNA hybridization. We describe methods to generate and characterize insertional mutants, using mutations that affect the outer dynein arm as examples. (C) 2000 Academic Press.