A cosmid library containing genomic DNA from mosquito cells in which the dihydrofolate reductase gene had become amplified in response to methotrexate selection was used to define the structure of the amplified DNA region (amplicon). A series of overlapping cosmids identified more than 200 kb of amplicon DNA, which was mapped relative to BssHII fragments from genomic DNA separated by transverse alternating field electrophoresis. These analyses indicated that, in Mtx-5011-256 mosquito cells, dhfr genes occur in at least two types of amplicon. The predominant Type I amplicon measures at least 215 kb and contains most of the dhfr genes. In addition, a small proportion of dhfr genes reside in a Type II amplicon, which is arranged in head-to-tail tandem repeats measuring 162 kb. Both amplicons share at least 70 kb of DNA sequence at their 5' ends. Approximately 20 fragments containing repeated sequence elements have been identified in the cloned amplicon DNA. Hybridization of amplicon DNA fragments containing repeated sequences to genomic DNA detected polymorphisms between wild type and methotrexate- resistant cells, suggesting that recombination may generate the divergence observed at the 3'-ends of Type I and Type II amplicons. This first detailed analysis of an insect dhfr amplicon provides an essential basis for ongoing investigation of repeated sequences, transcribed units and replication origins within the amplicon DNA.
- Mosquito cells
- dhfr genes