Article
- The EMBO Journal (2005) 24, 190 - 198
- doi:10.1038/sj.emboj.7600503
Published online: 16 December 2004
Subject Category:
A novel gene amplification system in yeast based on double rolling-circle replication
Takaaki Watanabe1 and Takashi Horiuchi2,3
- Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies (Sokendai), Myodaiji, Okazaki, Japan
- Department of Biosystems Science, School of Advanced Sciences, The Graduate University of Advanced Studies (Sokendai), Shonan Village, Hayama, Kanagawa, Japan
- National Institute for Basic Biology, Nishigonaka, Myodaiji, Okazaki, Japan
Correspondence to:
Takashi Horiuchi, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Japan. Tel./Fax: +81 564 55 7690; E-mail: kishori@nibb.ac.jp
Received 29 June 2004; Accepted 11 November 2004
Abstract
Gene amplification is involved in various biological phenomena such as cancer development and drug resistance. However, the mechanism is largely unknown because of the complexity of the amplification process. We describe a gene amplification system in Saccharomyces cerevisiae that is based on double rolling-circle replication utilizing break-induced replication. This system produced three types of amplification products. Type-1 products contain 5–7 inverted copies of the amplification marker, leu2d. Type-2 products contain 13 to 
100 copies of leu2d (up to 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies. Type-3 products are acentric multicopy minichromosomes carrying leu2d. Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively. Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage. These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.
Keywords:
- break-induced replication (BIR),
- double rolling-circle replication (DRCR),
- gene amplification,
- homogeneously staining region (HSR),
- yeast
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