2005 Fiscal Year Final Research Report Summary
Yeast genome engineering and applications
| Project/Area Number |
15380064
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | Osaka University |
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Principal Investigator |
HARASHIMA Satoshi Osaka University, Graduate School of Engineering, Professor, 大学院・工学研究科, 教授 (70116086)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIZAWA Masafumi Keio University, School of Medicine, Lecturer, 医学部, 講師 (20218150)
KANEKO Yoshinobu Osaka University, Graduate School of Engineering, Associate Professor, 大学院・工学研究科, 助教授 (90161182)
SUGIYAMA Minetaka Osaka University, Graduate School of Engineering, Research Assistant, 大学院・工学研究科, 助手
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| Project Period (FY) |
2003 – 2005
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| Keywords | Yeast / Genome engineering / Chromosome engineering / Chromosome splitting / Minimal genome yeast / Ribosome DNA / Life span / Silencing |
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| Research Abstract |
Chromosome engineering is playing an increasingly important role in functional analysis of genomes. A simple and efficient technology for manipulating large chromosomal segments is key to advancing these analyses. In this study, we developed a simple but innovative method to split chromosomes in Saccharomyces cerevisiae, which we call PCR-mediated chromosome splitting (PCS). Using this novel method, chromosomes I (230 kb) and XV (1091 kb) of a haploid cell were split collectively into 10 minichromosomes ranging in size from 29 to 631 kb with high efficiency (routinely 80%) that occasionally were lost during mitotic growth in various combinations.
We also successfully applied this method to shuffle selected regions of chromosomes from two strains in S.cerevisiae. This novel technique, which we call 'chromosome shuffling', could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also in
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dustrial strains of S.cerevisiae. The PCR method was further improved by conferring a yeast ARS (autonomously replicating sequenc) element so that chromosome region without ARS can be converte into artificial chromosome.
We have further employed this method to explore the function of ribosomal DNA (rDNA). The rDNA cluster in S.cerevisiae is located 450 kb from the left end and 610 kb from the right end of chromosome XII and consists of ca.150 tandemly repeated copies of a 9.1 -kb rDNA unit. Chromosome XII was split at both sides of the rDNA cluster and strains harboring deleted variants of chromosome XII consisting of 450-kb, 1,500-kb (rDNA cluster only) and 610-kb were created. In the strain harboring the 1,500 kb variant of chromosome XII consisting solely of rDNA, the size of the rDNA cluster was found to decrease. The frequency of silencing within the rDNA locus was found to be greater than in a wild -type strain. The localization and morphology of the nucleolus was also affected such that a single and occasionally two foci for Nop1p and a rounded nucleolus were observed. Notably, strains harboring split chromosome consisting solely of rDNA cluster had shorter life spans than wild -type. These observations suggest that the context of chromosome XII plays an important role in maintaining a constant rDNA copy number and in physiological processes related to rDNA function in S.cerevisiae. In conclusion, this novel technique could provide a useful tool not only to elucidate the genome function but also for biotechnology. Less
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