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PMID: 1316616 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

In vitro replication of adeno-associated virus DNA.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 89 ·No. 10 ·1992-05-15 ·Pages 4673-7

Hong G, Ward P, Berns KI

Abstract

An in vitro assay for adeno-associated virus (AAV) DNA replication has been developed. The substrate is a plasmid containing the duplex form of AAV DNA in pBR322. The AAV insert is excised or rescued from the plasmid by extracts of uninfected cells. Replication was assayed by production of full-length excised AAV DNA resistant to Dpn I digestion. The following results were obtained. (i) Only extracts of cells coinfected with AAV and adenovirus replicated the excised insert. (ii) Density label experiments showed semiconservative replication. (iii) Only the excised AAV insert was replicated; pBR322 sequences were not. (iv) Replication was dependent on the presence of the AAV terminal repeat. (v) If the terminal 55 bases were deleted from both ends of the AAV insert, no rescue took place: replication occurred and both AAV and pBR322 sequences were replicated. We conclude that the AAV terminal repeat is essential for DNA replication but that under some conditions an initiation mechanism that does not involve hairpin priming may be used.

MeSH Terms
Adenoviruses, Human/genetics Blotting, Southern Centrifugation, Density Gradient DNA Replication DNA, Viral/biosynthesis,genetics,isolation & purification Dependovirus/genetics HeLa Cells Humans Models, Genetic Plasmids Repetitive Sequences, Nucleic Acid Transfection
Chemicals
DNA, Viral
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Hong G
Department of Microbiology, Cornell University Medical College, New York, NY 10021.
Ward P
Berns K I
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.89.10.4673
Published
1992-05-15
Pages
4673-7
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC49145
Subset
IM
Grants
NIAID NIH HHS · AI22251 · United States
NIAID NIH HHS · AI26122 · United States
External Links
PubMed source DOI: 10.1073/pnas.89.10.4673 PMC Full Text View journal details
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