THE MOSQUITO DIHYDROFOLATE-REDUCTASE AMPLICON CONTAINS A TRUNCATED SYNAPTIC VESICLE PROTEIN GENE

When maintained under continuous selection with the folate inhibitor, methotrexate, cultured Aedes albopictus mosquito cells amplify an simi lar to 200 kb region of DNA containing the dihydrofolate reductase gen e, To determine whether the amplicon contained additional coding regio ns, Southern blots of cosmid clones containing amplicon DNA were probe d separately with reverse-transcribed mRNA from methotrexate-sensitive and methotrexate-resistant cells. Cosmid pWED118 contained five EcoRI fragments (A, B, C, F, G) ranging in size from 2 to 5 kb that hybridi zed with cDNA from resistant cells. Of these, fragments B and F also h ybridized to probe representing mRNA from sensitive cells, and all but fragment G hybridized to repetitive DNA from wild-type cells. Fragmen t G, which appeared to encode a low copy number gene in wild-type cell s that subsequently became part of the dihydrofolate reductase amplico n in methotrexate-resistant cells, hybridized strongly to a 7 kb band and more weakly to bands measuring 9 and 3 kb on Northern blots contai ning RNA from resistant cells. Fragment G contained a 1203 bp open rea ding frame, encoding 401 amino acids homologous to synaptic vesicle pr otein SV2, a member of a transmembrane transporter family expressed in neural and endocrine cells. The region of homology included the six N -terminal transmembrane domains, an internal cytoplasmic loop, a seven th transmembrane domain, and most of an intravesicular loop. This part ial sequence, which appears to correspond to a truncated gene generate d during formation of the dihydrofolate reductase amplicon, provides a useful basis for more extensive characterization of an important gene family that may be the target of novel insecticides.