Abstract

Although Chlamydomonas reinhardtii is a widely used model organism for studies of a variety of cell biological processes, the identification and cloning of genes known by mutations is still arduous. Current methods are inefficient for many of the existing mutations as their mutant alleles can either spontaneously revert or produce no selectable phenotype. With the sequencing and the annotation of the C. reinhardtii nuclear genome and mapping of available molecular markers, positional cloning is now possible. This thesis explored the application of bulked segregant analysis (BSA) with two types of molecular markers, amplified fragment length polymorphisms and single nucleotide polymorphisms, in order to identify and characterize the nuclear TBC1 gene and its predicted product. TBC1 was previously shown to functionally interact with specific regions and structures in the 5' untranslated region of chloroplast psbC mRNA to promote its translation and the synthesis of its product, the CP43 subunit polypeptide of PSII. Using BSA, TBC1 was mapped to an 8 map-unit region of Linkage Group VI. Complementation analysis narrowed down its position to a 41 kb region. Analyses of predicted genes in the region identified an exonuclease II orthologue as the best candidate for being TBC1