Identification of the Gene Encoding the Tryptophan Synthase β-Subunit from Chlamydomonas reinhardtii1

Abstract

Abstract We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the β-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of atrp-operon-deleted strain of Escherichia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest identity 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 conferred resistance to 5-fluoroindole. These mutations fell into three complementation groups, MAA2, MAA7, andTAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested thatMAA7 encodes TSB. MAA2 andTAR1 may act to regulate the activity ofMAA7 or its protein product.