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Structural differences in dihydrofolate reductases from different species have been exploited to develop specific inhibitory molecules, such as chemotherapeutic agents, antibiotics or antihelminthics, that show species specificity or selectivity. As dihydrofolate reductase (DHFR) is a crucial enzyme for the synthesis of purines, pyrimidines and some amino acids, and also because developing insects show a remarkably rapid rate of cell division, DHFR is a potentially promising target for the discovery of novel insecticides. We have thus isolated and characterized the enzyme from a serious agricultural pest, Heliothis (Helicoverpa) virescens, the tobacco budworm. Sequencing tryptic peptides of the 35 000-fold purified DHFR allowed the subsequent isolation of a partial cDNA, with the full Dhfr gene sequence obtained from a genomic library. The H. virescens Dhfr spans 4 kb, with three introns, and encodes 185 amino acids. The enzyme shows an overall similarity of ≈ 68% with DHFR from other metazoans, which has facilitated the molecular modeling of the protein. DHFRs from insects appear to have strikingly reduced sensitivity to inhibition by methotrexate, compared with the vertebrate enzymes, and this reduction was also reflected in the total binding energy seen after modeling experiments. Four residues that may be characteristic of insect DHFR, as well as a unique cysteine in the H. virescens DHFR active site, offer insight into the nature of inhibitor selectivity and provide suitable target sites for insecticide discovery.

An indirect, competitive, enzyme-linked immunosorbent assay (ELISA) was developed for the measurement, in soil, of clomazone [FMC 57020, 2-[(2-chlorophenyl)methyl]-4,4-dimethyl-3-isoxazolidinone], the active ingredient of Command herbicide. The p-aminophenyl analogue of clomazone was used to prepare a bovine serum albumin conjugate for immunization of rabbits. With soil, the sensivity limit is about 10 ppb and the detection limit is about 5 ppb. The detection range in soil extends from 10 ppb to 1.25 ppm. The method described here uses simple aqueous extraction of soil (...)