XVI International Botanical Congess

The ability to transform the nuclear genome of Chlamydomonas has opened the door to the powerful methodologies of gene tagging and reverse genetics. When Chlamydomonas is transformed, the transforming DNA inserts at random into the genome, disrupting or deleting genes at the site of insertion. The result is a mutation that is tagged by the exogenous DNA. The tag can be used to isolate DNA flanking the site of insertion, which in turn can be used to clone the wild-type gene. Conversely, if a gene has been cloned but no mutations are known, insertional mutants can be screened by DNA hybridization to identify those in which the gene is deleted. These approaches will be illustrated for genes involved in flagellar assembly. Because the reverse genetics approach is rapid and can be applied on a large scale, it should be feasible to construct and screen a collection of insertional mutants extensive enough to contain knockouts of virtually all non-essential genes.