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PMID: 2985958 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

Molecular and cellular biology ·Vol. 5 ·No. 3 ·1985-03-00 ·Pages 563-8

Ariga H, Tsuchihashi Z, Naruto M, Yamada M

Abstract

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.

MeSH Terms
Animals Antigens, Polyomavirus Transforming Antigens, Viral, Tumor/physiology Cell Line DNA Replication DNA, Recombinant/metabolism DNA, Viral/biosynthesis Genetic Vectors Haplorhini Humans Mice Mice, Inbred C3H/genetics Replicon Saccharomyces cerevisiae/genetics Simian virus 40/physiology Viral Proteins/physiology
Chemicals
Antigens, Polyomavirus Transforming Antigens, Viral, Tumor DNA, Recombinant DNA, Viral Viral Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Ariga H
Tsuchihashi Z
Naruto M
Yamada M
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42 references, click to expand
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.5.3.563-568.1985
Published
1985-03-00
Pages
563-8
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC366749
Subset
IM
External Links
PubMed source DOI: 10.1128/mcb.5.3.563-568.1985 PMC Full Text View journal details
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