Acetolactate synthase (ALS) inhibitors are among the most commonly used herbicides. They fall into four distinct families of compounds: sulfonylureas, imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We have investigated the molecular basis of imidazolinone tolerance of two field isolates of cocklebur (Xanthium sp.) from Mississippi and Missouri. In both cases, tolerance was conferred by a form of ALS that was less sensitive to inhibitors than the wild type. The insensitivity pattern of the Mississippi isolate was similar to that of a commercial mutant of corn generated in the laboratory: ICI 8532 IT. Sequencing revealed that the same residue (Ala57-->Thr) was mutated in both Mississippi cocklebur and ICI 8532 IT corn. ALS from the Missouri isolate was highly insensitive to all the ALS herbicide families, similar in this respect to another commercial corn mutant: Pioneer 3180 IR corn. Sequencing of ALS from both plants revealed a common mutation that changed Trp552 to Leu. The sensitive cocklebur ALS cDNA, fused with a glutathione S-transferase, was functionally expressed in Escherichia coli. The recombinant protein had enzymatic properties similar to those of the plant enzyme. All the possible point mutations affecting Trp552 were investigated by site-directed mutagenesis. Only the Trp-->Leu mutation yielded an active enzyme. This mutation conferred a dramatically reduced sensitivity toward representatives of all four chemical families, demonstrating its role in herbicide tolerance. This study indicates that mutations conferring herbicide tolerance, obtained in an artificial environment, also occur in nature, where the selection pressure is much lower. Thus, this study validates the use of laboratory models to predict mutations that may develop in natural populations.

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We have isolated a cDNA clone encoding a precursor form of the diuretic hormone from the tobacco hornworm Manduca sexta (Mas-DH). Translation of the cDNA revealed a 138-amino acid precursor consisting of the Mas-DH amino acid sequence bounded by dibasic amino acid processing sites, a putative signal sequence, and additional peptide sequence on either side of the Mas-DH coding sequence. The region of the precursor upstream of the mature Mas-DH sequence shows limited (28%) homology to the cryptic region of the ovine corticotropin-releasing factor precursor. The Mas-DH RNA is 1.5-1.6 kilobases long; it is present in both the heads and bodies of adult and larval insects. In prewandering fifth stadium larvae, Mas-DH mRNA is expressed in brain, nerve cord, gut, and Malpighian tubules, but not in the fat body. There is a single genomic copy of the Mas-DH gene; the message is multiply spliced.Manduca sexta diuretic hormone (Mas-DH) is an amidated 41-amino acid peptide that was isolated from pharate adult tobacco hornworm heads on the basis of induction of fluid loss in beheaded adult cabbageworms (1). This peptide shows homology to a family of vertebrate neuropeptides, including sauvagine, corticotropin-releasing factor, and urotensin I (1). Since the isolation of Mas-DH, other peptides of related structure, which function in regulating fluid balance in insects, have been isolated from Manduca (2) and from two orthopteran genera: Locusta (3, 4) and Acheta (5). Mas-DH itself has been shown to induce water loss in both lepidopteran (1, 6) and orthopteran (7) species. The structural and biological properties of these related insect diuretic hormones have been reviewed recently (8).Due to the importance of Mas-DH and to the possibility of improving the utility for insect control of recombinant baculoviruses expressing this insect hormone (9), we have determined a precursor sequence for Mas-DH after isolation of a cDNA clone for the peptide.t In addition to the coding sequence for the mature diuretic hormone and a signal sequence, the precursor peptide encodes additional peptide sequences on either side of the coding region and is bounded by processing sites similar to processing sites extensively characterized in mammalian systems (10). Mas-DH is expressed in both heads and bodies of pharate adult and prewandering fifth stadium larvae; in these larval bodies, Mas-DH mRNA is observed in nerve cord, gut, and Malpighian tubules. The gene for the hormone appears to be present as a single copy in the M. sexta genome; multiple splicing events are required for mRNA assembly. MATERIALS AND METHODSDNA Synthesis and Sequencing. Degenerate oligonucleotides were purchased from Clontech. Nondegenerate oligonucleotides were synthesized by using an Applied Biosystems model 380A DNA synthesizer. Plasmid DNA was sequenced using the chain-termination method (11) and the Sequenase kit (United States Biochemical).Reverse Transcriptase-PCR. Two sets of degenerate oligonucleotides were used to reverse transcribe RNA for later amplificati...

Anthranilate synthase is involved in tryptophan (Tip) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione Stransferase (CST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-' mg-'), the apparent K, for chorismate (180 &M), and the feedback inhibition by Trp (complete inhibition by 10 f i~ Trp) of the purified fusion product (CST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion protein product and purified by affinity chromatography on a CST-ASAl-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. CST-ASA1 cleavage by thrombin, as well as sitedirected mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.