An amplified mosquito dihydrofolate reductase gene: amplicon size and chromosomal distribution

Abstract

The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate-resistant cells. Subsequent to amplification, an additional polymorphism at a ten base-pair XmnI recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed-field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase amplicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx-5011-128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.