Gene Hunting by Complementation of Pooled Chlamydomonas Mutants

Abstract

We have generated a library of 34 photosynthesis mutants of Chlamydomonas reinhardtii, using insertional mutagenesis with the antibiotics resistance markers ble and aphVIII. To clone the affected genes, we transform the mutants by electroporation with an indexed cosmid library. Because the recipient strain has been selected for high efficiency transformation, we are able to perform transformations of mutants in pools of 4. Once a cosmid pool has been identified that yields phototrophic transformants, the individual mutant is identified. The individual cosmid is then pooled out by transforming with preparations from rows and columns of the 96-well cosmid plate. Using this strategy, we have analyzed a first pool of four mutants (one ATPase mutant and three ac mutants with no fluorescence phenotype). One gene has been cloned, and two others are at the stage of de-pooling. We are now embarking on a full-size screen of the library, with the aim of identifying new genes involved in photosynthesis or biogenesis of the photosynthetic apparatus.