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Dissection of mammalian replicators by a novel plasmid stability assay
Author(s) -
Hashizume Toshihiko,
Shimizu Noriaki
Publication year - 2007
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.21210
Subject(s) - plasmid , biology , genetics , transcription (linguistics) , gene , autonomously replicating sequence , microbiology and biotechnology , origin of replication , dna , dna replication , linguistics , philosophy
A plasmid, bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR) was previously shown to be efficiently amplified to high copy number in mammalian cells and to generate chromosomal homogeneously staining regions (HSRs). The amplification mechanism was suggested to entail a head‐on collision at the MAR between the transcription machinery and the hypothetical replication fork arriving from the IR, leading to double strand breakage (DSB) that triggered HSR formation. The experiments described here show that such plasmids are stabilized if collisions involving not only promoter‐driven transcription but also promoter‐independent transcription are avoided, and stable plasmids appeared to persist as submicroscopic episomes. These findings suggest that the IR sequence that promotes HSR generation may correspond to the sequence that supports replication initiation (replicator). Thus, we developed a “plasmid stability assay” that sensitively detects the activity of HSR generation in a test sequence. The assay was used to dissect two replicator regions, derived from the c‐ myc and DHFR ori ‐ β loci. Consequently, minimum sequences that efficiently promoted HSR generation were identified. They included several sequence elements, most of which coincided with reported replicator elements. These data and this assay will benefit studies of replication initiation and applications that depend on plasmid amplification. J. Cell. Biochem. 101: 552–565, 2007. © 2007 Wiley‐Liss, Inc.