Laboratory studies were performed to characterize the lepidopteran toxicity of cotton plants expressing two different toxin proteins from Bacillus thuringiensis (Bt), in order to assess insect resistance management implications of a commercial, two‐toxin transgenic cotton. An independent and additive interactive effect of two Bt δ‐endotoxins expressed by the transgenic cotton variety 15985 was demonstrated by examining the responses of Heliothis virescens (F.), Helicoverpa zea (Boddie), and Spodoptera frugiperda (J.E. Smith) larvae to field‐ or greenhouse‐grown tissue from genetic near‐isolines, which expressed Cry1A only, Cry2Ab only, or both toxins. In all cases, the Cry2Ab component was the larger contributor to total toxicity in the two‐toxin isoline. Toxin‐specific, quantitative enzyme‐linked immunosorbent assay (ELISA) tests confirmed that the levels of each toxin in tissues of the two‐toxin isoline were not statistically different (P > 0.05) from the levels found in the corresponding tissues of the respective single‐toxin isoline. Resistance management considerations were discussed. Considering the additive interaction of toxins, a relatively simple insect resistance‐monitoring procedure was proposed for the monitoring of commercial cotton varieties expressing both toxins.

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To better understand the evolution of a key metabolic pathway, we have sequenced the trpCFBA gene cluster of the hyperthermophilic bacterium Thermotoga maritima. The genes were cloned by complementation in vivo of trp deletion strains of Escherichia coli. The new sequences, together with earlier findings, establish that the trp operon of T.maritima has the order trpE(G.D)CFBA, which might represent the ancestral organization of the tryptophan operon. Heterologous expression of the trp(G.D) and trpC genes in E.coli and N‐terminal sequencing of their polypeptide products showed that their translation is initiated at the rate start codons TTG and ATC, respectively. Consequently, the N‐terminus of the trp(G.D) fusion protein is 43 residues shorter than previously postulated. Amino acid composition and sequence analyses of the protein products of T.maritima trpC (indoleglycerol phosphate synthase), trpF (phosphoribosyl anthranilate isomerase) and trpA (alpha‐subunit of tryptophan synthase) suggest that these thermostable (beta alpha)8‐barrel proteins may be stabilized by additional salt bridges, compared with the mesostable forms. Another notable feature is the predicted lack of the N‐terminal helix alpha 0 in the alpha‐subunit of tryptophan synthase.

The role of the extra helix do in the N-terminal extension of the eight-fold ßa barrel of indoleglycerol phosphate synthase was probed by point mutation and truncation. Replacing invariant leucine 5 by valine of the enzyme from Escherichia coli affected neither k cat nor K M , but deletion of 8 N-terminal residues decreased solubility strongly. The similarly truncated variant from the hyperthermophile Sulfolohus solfataricus was soluble, and had the same k cat value as the wild-type protein but a 220-fold greater K M value. These results suggest that the Nterminal portion of helix Oo provides for strong binding of the substrate, but is not essential for stabilizing the bound transition state. Thus, three enzymes of tryptophan biosynthesis operate essentially as canonical eight-fold ßa barrels, as required for their divergent evolution.