Chapter 74 Insertional Mutagenesis and Isolation of Tagged Genes in Chlamydomonas

Recently, the popularity of Chlamydomonas reinhardtii for molecular characterization of cellular mechanisms has greatly increased because of the development of DNA insertional mutagenesis for tagging and cloning genes. The success of this approach derives from the development of techniques for efficient introduction of DNA into Chlamydomonas cells and the almost random integration of the transforming DNA into nonhomologous sites in the nuclear genome. Using this new approach, a large number of mutants with defects in different cellular processes such as cell motility, phototaxis, chloroplast RNA splicing, sulfur and acetate metabolism, nitrogen assimilation, mating, and cell division have been identified. Moreover, this approach has proven to be particularly useful for isolating genes involved in flagellar function and assembly. There are many advantages to using transforming DNA to disrupt genes in Chlamydomonas. First, integration of the transforming DNA into the nuclear genome appears to be effective in producing random mutations. Second, gene tagging efficiency is very high. Third, with some precautions in generating mutants, the copy number of the integrated plasmid can be kept low so that many of the tagged genes can readily be cloned. Fourth, cloned fragments from mutants of interest can readily be placed on the molecular map of the Chlamydomonas genome, so that insertional mutations can be mapped without the extensive strain construction and crosses required for standard genetic mapping.