Identification of the gene encoding the tryptophan synthase beta-subunit from Chlamydomonas reinhardtii.
We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the beta-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-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, and TAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product.
Palombella
A L
AL
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA.
Dutcher
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005799:Genes, Dominant; D017343:Genes, Plant; D005808:Genes, Recessive; D005816:Genetic Complementation Test; D008040:Genetic Linkage; D005819:Genetic Markers; D007211:Indoles / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009876:Operon; D010788:Photosynthesis; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014367:Tryptophan Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D004791:Enzyme Inhibitors; D005819:Genetic Markers; D007211:Indoles; D046911:Macromolecular Substances; C095066:5-fluoroindole; D014367:Tryptophan Synthase