IDENTIFICATION OF THE GENE ENCODING THE TRYPTOPHAN SYNTHASE BETA-SUBUNIT FROM CHLAMYDOMONAS-REINHARDTII

We report the isolation of a Chlamydomonas reinhardfii cDNA that encod es the beta-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Esche richia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest i dentity to TSB polypeptides from other photosynthetic organisms. With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that confer red resistance to 5-fluoroindole. These mutations fell into three comp lementation groups, MAA2, MAA7 and TAR1. In vitro assays showed that m utations at each of these loci affected TSB activity. Restriction frag ment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA 2 and TAR1 may act to regulate the activity of MAA7 or its protein pro duct.