Early-replicating DNA from mosquito cells is associated with a distinct EcoRI fragment

In an effort to define an origin of bi-directional DNA replication (OBR) in mosquito genomic DNA, we applied methods that take advantage of characteri stic features of single-stranded DNA to methotrexate-resistant Aedes albopi cus cells. The Mtx-5011-256 cells contained approximately 1000 copies of a 200 kb amplicon containing the dihydrofolate reductase locus, which likely contained one or more replication origins. When Mtx-5011-256 cells were syn chronized by treatment with hydroxyurea, released into the S phase of the c ell cycle, and labeled in vivo with tritiated DNA precursors, a 1.9 kb EcoR I fragment was preferentially labeled in EcoRI-digested genomic DNA. Simila rly, we detected a 1.9 lib EcoRI fragment in DNA from wild type cells after cell cycle synchronization and in vivo labeling. In a complementary method , unlabeled single-stranded DNA was isolated from Mtx-5011-256 cells, label ed in vitro, and hybridized to EcoRI-digested genomic DNA from mosquito cel ls. The labeled probe hybridized preferentially to a 1.9 kb fragment. Final ly, a 1.9 kb EcoRI fragment was detected when nascent DNA was recovered fro m unsynchronized cells, made double-stranded by in vitro labeling, and dige sted with EcoRI. Taken together, these results suggest that in Aedes albopi ctus mosquito cells, many replication origins used at different times durin g S are flanked by EcoRI sites that define a 1.9 kb fragment, which has bec ome more abundant in Mtx-5011-256 cells because it occurs in the dhfr ampli con. Tentative mapping of this origin to amplicon DNA remains ambiguous, fu rther suggesting that a repeated sequence element occurs at or near the ori gin of replication. (C) 1999 Elsevier Science Ltd. All rights reserved.