Abstract
To extend our understanding of amplicon structure in methotrexate- resistant Mtx-5011-256 Aedes albopictus mosquito cells, we examined a series of cosmids containing genomic DNA corresponding to the unique 3'-end of the Type I dihydrofolate reductase amplicon. Cosmid pWED118 contained five EcoRI fragments ranging from 2 to 5 kb (A, B, C, F, G) that hybridized to cDNA from methotrexate-resistant cells. Of these, fragments B and F hybridized weakly to first-strand cDNA from sensitive cells and shared considerable sequence identity. Fragment G occurred twice in the map of pWED118; one copy mapped within a 10 kb BssHII core fragment from the Type I amplicon and a second copy mapped downstream in the 48 kb BssHII core fragment. Hybridization signals among fragments contained in overlapping cosmids suggested that a branch point defining two or more subtypes of the Type I amplicon occurs within or near the 10 kb BssHII genomic DNA fragment. A 1.8 kb sequence common to fragments B and F included an ~0.4 kb region that shared sequence similarities with a LINE element from Aedes aegypti and with a repeated sequence from Anopheles gambiae. In addition, these elements shared amino acid similarity to a reverse transcriptase from the nematode, Caenorhabditis elegans. Shared sequence between Aedes and Anopheles elements supports the hypothesis that an ancestral LINE-like element was active in mosquito genomes prior to the divergence of the subfamilies Culicinae and Anophelinae. The presence of homologies to LINE-like elements near a branch point in the dihydrofolate reductase amplicon is consistent with a possible role of repeated sequences in amplicon shortening.
Original language | English (US) |
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Pages (from-to) | 613-623 |
Number of pages | 11 |
Journal | Insect Biochemistry and Molecular Biology |
Volume | 28 |
Issue number | 8 |
DOIs | |
State | Published - Aug 1998 |
Bibliographical note
Funding Information:This work (contribution # 98-1-17-0010) was supported by grants AI 20385 and HD 24869 from the NIH and by the University of Minnesota Experiment Station, St. Paul, MN.
Keywords
- Cell line
- Gene amplification
- LINE elements
- Methotrexate
- Zebedee