Molecular cloning and expression of the dihydrofolate reductase ( DHFR ) gene from adult buffalo fly ( Haematobia irritans exigua ): effects of antifolates

Abstract

The folate analogues methotrexate, aminopterin and pyrimethamine were toxic when fed in a blood meal to adult buffalo flies (Haematobia irritans exigua), but aminopterin caused greater mortality than methotrexate, while trimethoprim was not toxic to adult flies. This is the first recorded instance of mortality in adult insects caused by ingestion of folate analogues. In order to investigate the mechanism of this toxicity, the dihydrofolate reductase (DHFR) gene was cloned from adult buffalo fly cDNA using a PCR-based approach. The full-length DHFR coding sequence (BF-DHFR) was 887 bp and contained an open reading frame encoding a protein of 188 amino acids. The deduced protein sequence identities between BF-DHFR and the other known insect DHFR sequences were: Drosophila melanogaster, 75%; Aedes albopictus, 54%; Heliothis virescens, 43%. The BF-DHFR gene has a single 52 bp intron, an organization more similar to Dipteran species (Drosophila and Aedes).The cDNA encoding BF-DHFR was inserted into an Escherichia coli expression vector and the recombinant protein was expressed to levels representing about 25% of total cell protein. The active enzyme was purified by affinity chromatography on methotrexate-agarose and displayed a relatively low affinity (IC50 = 30 nM) for methotrexate.