The DNA sequence of a tryptophan synthase gene and the flanking 5' and 3' regions has been determined for Arabidopsis thaliana. The sequence encodes only the g3 subunit domain, indicating that a and (3 subunits are specified by separate genes. The gene contains four introns and encodes 470 amino acid residues. The plant amino acid sequence is highly conserved with respect to corresponding microbial sequences. The NH2-terminal amino acid sequence is characteristic of chloroplast transit peptides. Identity of the sequences of the genomic exons and a cDNA clone and the presence of cellular RNA corresponding in size and 5' sequence to the gene indicate that the gene is expressed. Analysis of Arabidopsis genomic DNA suggests the presence of a second gene for the 13 subunit.Our understanding of the regulation of the tryptophan biosynthetic pathway comes primarily from the analysis of the expression of tryptophan synthase genes in bacteria and fungi. Tryptophan synthase [L-serine hydro-lyase (adding indoleglycerol-phosphate); EC 4.2.1.20] catalyzes the last step of the pathway, conversion of indoleglycerol phosphate to tryptophan. The enzyme contains two functional domains: tryptophan synthase a (TRPA) catalyzes the conversion of indole glycerol phosphate to indole and tryptophan synthase 13 (TRPB) catalyzes the conversion of indole plus serine to tryptophan. In bacteria each of these domains is encoded by a separate gene, whereas in yeast and Neurospora both domains are encoded by a single gene (1-5). The domains of the two bacterial genes can be aligned with the single yeast gene: the NH2-terminal domain of TRP5 is homologous to the trpA gene and the CO2 terminal is homologous to the trpB gene. These homologies have prompted the speculation that the fungal gene evolved by fusion of trpA to trpB. Gene fusions have also been proposed to account for other bifunctional enzymes of the tryptophan pathway (6, 7).Although tryptophan has a special role in plants as a precursor of the auxin indoleacetic acid, the biochemistry of tryptophan synthesis and its regulation are not well understood because the biosynthetic enzymes and the genes encoding them have not been isolated from plants. Biochemical studies on plant tryptophan synthase were not sufficient to establish whether TRPA and TRPB activities are encoded by one or two genes. The individual activities were separated during purification steps (8-10), but, as in Neurospora and yeast fractionations, the low activities may represent results of proteolysis, and attenuated protein fragments may provide partial activity (11-13).We report the cloning and sequencing of an Arabidopsis gene for TRPB. (17). The plasmid pMBT2 was constructed by inserting a 4.2-kb EcoRI fragment from the strongly hybridizing genomic phage clone AMlg into the EcoRI site of pUC18 (14). Plasmid pMBT3 was constructed by ligation of the 2.1-kilobase (kb) cDNA insert from AGT1O phage ACD2 into the EcoRI site of pUC118 (15).Hybridization Probes and Conditions. The yeast fragment used to probe the ge...

Ultracentrifugation in sucrose density gradients was employed to estimate the molecular weights and to determine possible physical aggregation of the five enzymes catalyzing steps two to six in the prechorismic acid portion of the polyaromatic synthetic pathway in six species of bacteria: Escherichia coli, Salmonella typhimurium, Aerobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor. The five enzymes were not aggregated in extracts of any of the species examined, nor are the genes encoding these enzymes clustered in those bacterial species for which genetic evidence exists. (An initial examination of the blue-green alga Anabaena variabilis indicates nonaggregation in this species also.) This situation in bacteria is in marked contrast to that found in Neurospora crassa and other fungi in which the same five enzymes are associated as an aggregate encoded (at least in the case of N. crassa) by a cluster of five genes. In addition, also in contrast to N. crassa, no evidence was obtained for more than one kind of dehydroquinase activity in any of the bacteria examined. These comparative results are discussed in relation to the origin, evolution, and functional significance of the gene-enzyme relationships existing in the early steps of aromatic biosynthesis in bacteria and fungi. other bacteria. The coliform bacteria and P. aeruginosa were also grown in minimal medium supplemented with 500 jsg of quinic acid per ml. Log-phase cells were harvested by centrifugation, washed in 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochlo-222

Haploid tobacco (Nicotiana tabacum L.) cell cultures derived from quite different cultivars have been grown photoautotrophically in medium lacking sucrose and with 1.6 pM naphtbaleneacetic acid and 1.5 AM isopentenylamino.purine. Cells were grown for 5 months on agar medium in Petri plates in air with dry weight increases of 1.5-to 3-fold per month. Callus cells were also grown photoautotrophically for at least three consecutive transfers 3 weeks apart in shallow liquid medium in horizontally placed gaswashing bottles where they were gassed continuously with air or air enriched with CO2. Raising the CO2 level in the air surrounding the cells increased the growth rate, and after about 3 weeks in 1% CO2 the dry weight was approximately 3-fold greater than the inoculum. Growth rates remained about the same after each consecutive transfer. Autotrophic growth with this regime is not restricted to specific clones or cultivars.Photosynthetic measurements in an atmosphere containing "4CO2 established that rates of CO2 assimilation in the callus cells at high CO2 levels were similar to those of leaves on a chlorophyll basis, but were much slower on a fresh weight basis. Photosynthetic light saturation was achieved at an irradiation of about 125 ,ueinsteins m-2 sec-1 (400-700 nm). The availability of photosynthetically dependent haploid cells provides an opportunity to select photosynthetic mutations which can be expressed in plants regenerated from these cells.Despite the widely held view that plant cell cultures are not photosynthetically self-sufficient a few cases of photoautotrophically grown plant cells have been reported (2,6,7,10 Leaves of 918.11 had a fast photorespiration in the "4C-assay (19) and a net photosynthesis of only 16.7 mg CO2 per dm2 -hr. Leaves of 918.23 had a slower photorespiration and a net photosynthesis of 23.6 mg CO2 per dm2 -hr. These cell cultures were isolated and generously provided by our colleagues P. R. Day and S. Anagnostakis.Culture Media. Cultures were maintained on Linsmaier and Skoog medium (14) with substitution of the Fe-EDTA solution of Kasperbauer and Collins (12) and with 16 jAM naphthaleneacetic acid (3 mg/l), 1.5 ;AM isopentenylaminopurine (0.3 mg/l), and 2% sucrose. The other organic constituents added to the medium were inositol (100 mg/l) and thiamin (1 mg/l). Photoautotrophic cultures were grown on this medium lacking sucrose and with 1.6 jAM naphthaleneacetic acid (0.3 mg/l). In some experiments, where indicated, photoautotrophic cultures were grown without any organic supplements.One per cent agar was used for solid media and Petri plates were sealed with 1-inch rubber bands. Experiments in which cultures were aerated with air or air-CO2 mixtures at flow rates of 6 to 10 ml/min were conducted in horizontally placed 500-ml gas washing bottles with small volumes of liquid media forming a shallow liquid phase (e.g., 1 g of tissue in 10 ml of medium). The incoming gas mixture passed through a cotton filter anda 0.20-,um membrane filter in an in-line filter holder. ...

The contents of the E. coli Genetic Stock Center database and the availability in electronic form of the subset of information most relevant to sequence databases are described. The database uses the long-standing Stock Center records (developed and curated by Dr B.J.Bachmann) in describing genotypes of mutant derivatives of E.coli K-12 in terms of alleles, structural mutations, mating type, and plasmids as well as the derivation, names and originators of the strain, and references. The database includes descriptions of mutations, mutation properties, genes, gene properties, and gene products, with EC number identifiers for enzymes. Sequence information is not included, but entries refer to sequence database accession numbers for sequenced regions. A gene is described as a subtype of a more general category of chromosome interval called Site. Since sites are used to describe any chromosomal interval, mapping information is associated with sites. Alleles are described as mutations of those sites and they are not primary map objects, but inherit map position information from the corresponding site description. The database design is intended to preserve richness of detail where it is known and uncertainty of measurements or information as it occurs in order to represent the stock center records as accurately as possible.