Nelfinavir mesylate (formerly AG1343) is a potent and selective inhibitor of human immunodeficiency virus (HIV) protease approved for the treatment of individuals infected with HIV. Nucleotide sequence analysis of protease genes from plasma HIV type 1 (HIV-1) RNA revealed a unique aspartic acid (D)-to-asparagine (N) substitution at residue 30 (D30N) in 25 of 55 patients treated with nelfinavir for a median of 13 weeks. Although the appearance of D30N was occasionally associated with concurrent or sequential emergence of other changes (e.g., at residues 35, 36, 46, 71, 77, and 88), genotypic changes associated with phenotypic resistance to other protease inhibitors were not observed (e.g., at residues 48, 50, 82, and 84) or were only rarely observed (e.g., at residue 90). In phenotypic assays, viral isolates with high-level resistance to nelfinavir remained susceptible to indinavir, saquinavir, ritonavir, and amprenavir (formerly VX-478/141W94). Similar results were observed in phenotypic assays utilizing HIV-1 NL4-3, which contained the D30N substitution alone or in combination with substitutions at other residues (e.g., residues 46, 71, and 88). These data indicate that the initial pathway of resistance to nelfinavir is unique and suggest that individuals failing short courses of nelfinavir-containing regimens may respond to regimens containing other protease inhibitors.

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In an attempt to increase the insecticidal effect of the ␦-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 g/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 g of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 g of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.

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Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis ␦-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific f lanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.Insecticidal Cry proteins, produced as protoxins in parasporal crystals of Bacillus thuringiensis (Bt), are active as selective entomocidal agents. The crystalline Bt protoxins are solubilized and activated in the midgut of insects by proteolysis. The activated toxins (60-70 kDa) bind to the membrane of midgut columnar cells and form ion channels, inducing osmotic lysis of the epithelium (1-3). Engineering of insect resistance in maize, rice, cotton, tomato, potato, and tobacco shows that a significant modification of the bacterial cry coding sequences is essential to express these Bt toxin genes in plants (4-11). Efficient transcription of recombinant cry genes in plant cell nuclei was achieved by the removal of AϩT-rich sequences that may cause mRNA instability (8-11) or aberrant splicing (12), and the translation of cry mRNAs is enhanced by modification of their codon usage to make it more similar to that of the host plant (8). Expression of bacterial cry genes in chloroplasts may overcome these technological demands; however, to date chloroplast transformation is only available in tobacco (13).The insecticidal spectrum of Bt toxins thus far expressed in transgenic plants is limited. Therefore, the engineering of Bt toxins with novel specificity is essential for the biological control of recalcitrant plague insects, such as Spodoptera. Members of the Spodoptera genus (Lepidoptera, Noctuidae) feed on over 40 different plant families world-wide, including at least 87 species of economic importance (14). Studies of Spodoptera-specific Bt isolates indicated that many of them synthesize CryIC ␦-endotoxins that are much more effective against these insects than other CryI proteins (15). Here we describe the chemical synthesi...