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A novel gene amplification system in yeast based on double rolling-circle replication : implications for amplification in higher eukaryotes
https://ir.soken.ac.jp/records/1391
https://ir.soken.ac.jp/records/1391a8bf2ba4-e424-4ccb-bf8c-89a1ce66010e
| 名前 / ファイル | ライセンス | アクション |
|---|---|---|
要旨・審査要旨 (297.9 kB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2010-02-22 | |||||
| タイトル | ||||||
| タイトル | A novel gene amplification system in yeast based on double rolling-circle replication : implications for amplification in higher eukaryotes | |||||
| タイトル | ||||||
| タイトル | A novel gene amplification system in yeast based on double rolling-circle replication : implications for amplification in higher eukaryotes | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
| 資源タイプ | thesis | |||||
| 著者名 |
渡邊, 孝明
× 渡邊, 孝明 |
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| フリガナ |
ワタナベ, タカアキ
× ワタナベ, タカアキ |
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| 著者 |
WATANABE, Takaaki
× WATANABE, Takaaki |
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| 学位授与機関 | ||||||
| 学位授与機関名 | 総合研究大学院大学 | |||||
| 学位名 | ||||||
| 学位名 | 博士（理学） | |||||
| 学位記番号 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | 総研大甲第874号 | |||||
| 研究科 | ||||||
| 値 | 生命科学研究科 | |||||
| 専攻 | ||||||
| 値 | X2 分子生物機構論専攻 | |||||
| 学位授与年月日 | ||||||
| 学位授与年月日 | 2005-03-24 | |||||
| 学位授与年度 | ||||||
| 値 | 2004 | |||||
| 要旨 | ||||||
| 内容記述タイプ | Other | |||||
| 内容記述 | Gene amplification occurs in numerous organisms and has probably played an important role in genome evolution. The ribosomal RNA genes (rDNA) arc tandemly repeated in most eukaryotes and must have been amplified at some point during evolution. Oncogene amplification is frequently observed in the process of cancer development. Gene amplification also occurs when cultured cells respond to selection for drug-resistance and when insects and plants acquire resistance to agricultural chemicals. In bacteria, amplification can be an essential intermediate in the adaptive mutation phenomenology. Gene amplification has been applied to the industrial production of therapeutic protein.<br /> Models proposed to explain the amplification process include; unequal sister-chromatid exchange, localized over-replication, rolling-circle and double rolling-circle replication, extrachromosomal amplification and reintegration, and breakage-fusion-bridge (BFB) cycles. Despite great effort, the molecular mechanism responsible for the majority of amplification events remains unknown. For example, the gene amplification occurring in tissue-cultured cells during selection for resistance to methotrexate (MTX) is believed to be initiated by the BFB cycles. However rather complicated products are produced which may require additional kinds of amplification processes. The complexity of these structures has made it difficult to analyze the mechanism at a molecular level. <br /> The mechanism responsible for amplification of rDNA is fairly well understood. In S. cerevisiae, rDNA amplification is initiated by a double-strand break (DSB) produced when a DNA replication fork encounters a replication fork barrier (RFB) present in each rDNA unit. This double-strand end enhances unequal sister chromatid recombination, resulting in rDNA amplification. The blocking of replication forks also causes gene amplification in E. coli at a recombinational hotspot, named Hot DNA. However, known chromosomal amplifications in E. coli and yeast are invariably direct tandem copies, while mammalian cells show a mixture of direct and inverted repeats. Thus, understanding of amplification in higher eukaryotes might be enhanced by development of a microbial system that generates products analogous to those observed in tumors and cultured cells. <br /> The mechanisms underlying these gene amplifications in yeast and bacteria appear to be quite different. While yeast rDNA amplification occurs through unequal sister-chromatid recombination, bacterial Hot amplification seems to proceed through rolling-circle replication. However, it is possible that both processes are initiated by break-induced replication (BIR). While inter-chromatid unequal BIR could cause rDNA amplification in yeast, a similar intra-chromatid BIR could cause the rolling-circle type amplification of Hot DNA in E. coli.<br /> The double rolling-circle replication (DRCR) is a continuous replication process in which two replication forks synthesize a circular DNA. This model was first proposed by Futcher for amplification of yeast circular 2μ plasmid and experimentally confirmed by Volkert and Broach. Later a similar model was proposed by Hyrien et al. to explain a case of drug-resistance gene amplification. However, the former DRCR depends on a 2μ-specific site-specific recombination system (FLP1/FRT) and the latter model has not been examined experimentally. Thus far, no amplification system has shown to rely on DRCR. <br /> Here, he uses double-strand breaks to study the sequences, structures and reactions required for in vivo gene amplification in S. cerevisiae. This system is based on the action of break-induced replication (BIR) and double rolling-circle replication (DRCR). BIR is a recombination-dependent replication process and starts as a one-ended recombination event. A broken chromosome end finds a homologous sequence, invades it to form a new replication fork, and initiate replication. In the system described here, a single chromosome is cut by HO endonuclease to generate two fragments designed to amplify a sequence by DRCR. Each fragment can act both as end donor and end recipient for BIR. If the two BIR forks start simultaneously, double rolling-circle replication (DRCR) should highly amplify the regions flanked by the replication forks within a single cell cycle. The DRCR process can terminate by recombination between leu2d genes on each bi-directionally elongated arm. <br /> The system formed a single type of product containing 3-5 inverted copies of the amplification marker, leu2d, on a plasmid-derived artificial mini-chromosome, and it successfully produced three types of amplification products on a resident chromosome. Type- 1 products contain 5-7 inverted copies of leu2d. Type-2 products contain 13-〓100 copies of leu2d (up to 〓730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2ds copies. Type-3 products are acentric multi-copy mini-chromosomes carrying leu2ds. Structures of type-2 and -3 products resemble those of homogeneously staining region (HSR) and double minutes (DMs) 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. Utilization of this amplification system in mammalian cells has the potential to provide great savings of time and energy in the overproduction of recombinant proteins for medical uses. | |||||
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| 値 | 有 | |||||
