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.