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

Zhong Hui Wang, Ann M Fallon

Research output: Contribution to journalArticlepeer-review

Abstract

In an effort to define an origin of bi-directional DNA replication (OBR) in mosquito genomic DNA, we applied methods that take advantage of characteristic features of single-stranded DNA to methotrexate-resistant Aedes albopictus 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 synchronized by treatment with hydroxyurea, released into the S phase of the cell cycle, and labeled in vivo with tritiated DNA precursors, a 1.9 kb EcoRI fragment was preferentially labeled in EcoRI-digested genomic DNA. Similarly, we detected a 1.9 kb 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 Mtx5011-256 cells, labeled in vitro, and hybridized to EcoRI-digested genomic DNA from mosquito cells. The labeled probe hybridized preferentially to a 1.9 kb fragment. Finally, a 1.9 kb EcoRI fragment was detected when nascent DNA was recovered from unsynchronized cells, made double-stranded by in vitro labeling, and digested with EcoRI. Taken together, these results suggest that in Aedes albopictus mosquito cells, many replication origins used at different times during S are flanked by EcoRI sites that define a 1.9 kb fragment, which has become more abundant in Mtx-5011-256 cells because it occurs in the dhfr amplicon. Tentative mapping of this origin to amplicon DNA remains ambiguous, further suggesting that a repeated sequence element occurs at or near the origin of replication.

Original languageEnglish (US)
Pages (from-to)53-61
Number of pages9
JournalInsect Biochemistry and Molecular Biology
Volume29
Issue number1
DOIs
StatePublished - Jan 1999

Bibliographical note

Funding Information:
This work was supported by grant AI 20385 from the National Institutes of Health and by the University of Minnesota Agricultural Experiment Station (publication # 981170010), St Paul, MN.

Keywords

  • Amplicon
  • Cell cycle synchronization
  • Dihydrofolate reductase
  • Hydroxyurea
  • Origin of replication

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