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A partially palindromic 15-nt. sequence upstream from a juvenile hormone-regulated gene (jhp21) was previously identified in the African migratory locust, Locusta migratoria. This sequence was proposed as a juvenile hormone (JH) response element (JHRE), and a protein that bound to it, as a transcription factor (TF). A yeast strain was constructed containing four tandem copies of the JHRE and after transfection with a cDNA library made to fat bodies from vitellogenic females, yeast one-hybrid experiments yielded sequences for four putative binding proteins. One of these sequences, corresponding to a transcript that was present in fat body irrespective of JH stimulation, encodes a 35kDa protein. This was designated tfp1 and appears to have a leucine zipper motif and a lipid-binding motif. Recombinant tfp1 bound to JHRE in electrophoretic mobility shift experiments and addition of tfp1 antibody in the binding reaction resulted in the disappearance or shift of TF. We suggest that JH induces the association of pre-existing proteins, including tfp1, to form an active complex, which binds to the JHRE upstream from jhp21 and regulates its transcription.

Not surprisingly, in the spruce budworm, Choristoneura fumiferana, antifreeze protein (AFP) gene expression is most abundant in the second instar, overwintering stage. However, low level RNA and protein expression was also found in the sixth instar larvae, a summer stage. In situ hybridization further confirmed the presence of AFP mRNA in sixth instar midgut tissues. Sequencing of cDNAs corresponding to ‘‘summer-expressed’’ transcripts revealed an isoform that was not apparent in a cDNA library made to second instar larvae. Although similar to AFP cDNAs obtained from overwintering larvae, this AFP-like isoform (CfAFP6) has two Cys substitutions. Since AFPs from this species fold into a b-helix that is stabilized by disulfide bonds, it was of interest to determine if this summerexpressed isoform had AFP activity. No thermal hysteresis activity was found when CfAFP6 was cloned and expressed in E. coli, even after in vitro denaturation and refolding. As well, there was no activity detected when the sequence of a known, active isoform was changed to mimic the Cys substitutions in CfAFP6. Since CfAFP6 does not appear to contribute to freeze resistance, its apparent absence in the overwintering second instar should not in itself be considered curious. r 2006 Elsevier Ltd. All rights reserved.

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

The enzyme triosephosphate isomerase (TPI) was purified to homogeneity from the mosquito Culex tarsalis. Anti-C. tarsalis TPI antibodies cross-reacted with TPIs from other organisms but bands on western blots were most intense with proteins from closely related Dipterans. Using a degenerate primer corresponding to the amino-terminal sequence of the protein in a polymerase chain reaction (PCR), a cDNA corresponding to the TPI gene (Tpi) was isolated and sequenced. Subsequently, a genomic sequence including 305 bp to the 5'-end of the coding sequence was obtained. Comparison of C. tarsalis Tpi to that of Drosophila melanogaster revealed that although the two genes had little similarity in the intron and 5' flanking sequences, they were highly similar (73% identity) in their coding sequence. The rate of synonymous substitution in insect genes may be slower than that of vertebrates, but the nonsynonymous substitution rate, and hence the rate of TPI evolution, appears to be faster in insects than in vertebrates.

Malathion resistance in a strain of Culex tarsalis mosquitoes is due primarily to the activity of a malathion carboxylesterase (MCE). The resistant strain was 150 times more resistant to malathion than the susceptible strain and was weakly resistant to malaoxon and carbaryl, but not to any other insecticide tested. The phenotype could be reversed with the carboxylesterase inhibitor triphenylphosphate, but no synergism was observed with either the phosphatase or polysubstrate monooxygenase inhibitors, NaF and piperonyl butoxide. MCE is expressed throughout development and is most concentrated in the gut tissues of the larvae. Subcellular fractionation indicated that MCE was localized primarily in the mitochondria of resistant insects and the cytoplasm of susceptible insects. The enzyme was purified to homogeneity from both strains, and has a molecular weight of 59,000. However, chromatofocusing indicated that resistant insects have two MCEs with pIs of 6.8 and 6.2, while susceptible insects possessed only one MCE with a pI of 6.8. The MCE unique to the resistant strain hydrolysed malathion 18 times faster than the MCE common to both strains, suggesting that malathion resistance in C. tarsalis is due to the presence of a qualitatively different esterase in the resistant strain.