In vitro replication directed by a cloned adenovirus origin

George D. Pearson, Kuan Chih Chow, Robert E. Enns, Kevin G. Ahern, Jeffry L. Corden, Jerry A. Harpst

Research output: Contribution to journalArticlepeer-review

Abstract

A 5.7-kb recombinant plasmid, called XD-7, contains the terminal XbaI-E fragment from the left end of type 2 adenovirus cloned into the EcoRI site of pBR322. An average of 9% ± 1% of input supercoiled, protein-free XD-7 DNA replicated as rolling circles with single-stranded tails ranging up to unit length and longer in reaction mixtures containing nuclear and cytoplasmic extracts from adenovirus-infected, but not uninfected, HeLa cells. The adenovirus origin was mapped on XD-7 by electron microscopy at the left boundary of the cloned adenovirus segment. Since replication proceeded rightwards, we conclude that the adenovirus l strand was displaced during replication. No origin was located at or near the EcoRI site on pBR322. Reversing the orientation of the adenovirus origin reversed the direction of replication, and deletion of the adenovirus origin abolished replication.

Original languageEnglish (US)
Pages (from-to)293-305
Number of pages13
JournalGene
Volume23
Issue number3
DOIs
StatePublished - Sep 1983
Externally publishedYes

Keywords

  • Recombinant DNA
  • deletion mutants
  • plasmid vector pBR322
  • rolling circles
  • strand displacement

ASJC Scopus subject areas

  • Genetics

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