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

doi:10.1016/0378-1119(83)90019-7

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 journal › Article › peer-review

9 Scopus citations

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 language	English (US)
Pages (from-to)	293-305
Number of pages	13
Journal	Gene
Volume	23
Issue number	3
DOIs	https://doi.org/10.1016/0378-1119(83)90019-7
State	Published - Sep 1983
Externally published	Yes

Keywords

Recombinant DNA
deletion mutants
plasmid vector pBR322
rolling circles
strand displacement

ASJC Scopus subject areas

Genetics

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10.1016/0378-1119(83)90019-7

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title = "In vitro replication directed by a cloned adenovirus origin",

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.",

keywords = "Recombinant DNA, deletion mutants, plasmid vector pBR322, rolling circles, strand displacement",

author = "Pearson, {George D.} and Chow, {Kuan Chih} and Enns, {Robert E.} and Ahern, {Kevin G.} and Corden, {Jeffry L.} and Harpst, {Jerry A.}",

note = "Funding Information: We thank Kate Mathews for excellent technical assistance, Forest Ziemer for technical advice and assistance with certain experiments, Dr. Andrew Taylor for instruction in electron microscopy, James Downing for assistance with cloning experiments and electron microscopic length measurements, Dr. Lyle Brown for supplying Ml3 cloning vectors, and Dr. Jon Beaty for instruction in dideoxy sequencing techniques. G.D.P. was the recipient of a Faculty Research Award (FRA-236) from the American Cancer Society. This work was supported by a grant from the Oregon Medical Research Foundation, a Biomedical Sciences Support grant RR07079 administered through the Oregon State University Research Council, and a National Cancer Institute grant CA17699 awarded to G.D.P., and a National Institute of General Medical Sciences grant GM29828 awarded to J.A.H.",

year = "1983",

month = sep,

doi = "10.1016/0378-1119(83)90019-7",

language = "English (US)",

volume = "23",

pages = "293--305",

journal = "Gene",

issn = "0378-1119",

publisher = "Elsevier B.V.",

number = "3",

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T1 - In vitro replication directed by a cloned adenovirus origin

AU - Pearson, George D.

AU - Chow, Kuan Chih

AU - Enns, Robert E.

AU - Ahern, Kevin G.

AU - Corden, Jeffry L.

AU - Harpst, Jerry A.

N1 - Funding Information: We thank Kate Mathews for excellent technical assistance, Forest Ziemer for technical advice and assistance with certain experiments, Dr. Andrew Taylor for instruction in electron microscopy, James Downing for assistance with cloning experiments and electron microscopic length measurements, Dr. Lyle Brown for supplying Ml3 cloning vectors, and Dr. Jon Beaty for instruction in dideoxy sequencing techniques. G.D.P. was the recipient of a Faculty Research Award (FRA-236) from the American Cancer Society. This work was supported by a grant from the Oregon Medical Research Foundation, a Biomedical Sciences Support grant RR07079 administered through the Oregon State University Research Council, and a National Cancer Institute grant CA17699 awarded to G.D.P., and a National Institute of General Medical Sciences grant GM29828 awarded to J.A.H.

PY - 1983/9

Y1 - 1983/9

N2 - 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.

AB - 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.

KW - Recombinant DNA

KW - deletion mutants

KW - plasmid vector pBR322

KW - rolling circles

KW - strand displacement

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ER -

In vitro replication directed by a cloned adenovirus origin

Abstract

Keywords

ASJC Scopus subject areas

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