| Project/Area Number |
15370077
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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 |
Molecular biology
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| Research Institution | Kyoto University |
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Principal Investigator |
TAKEYASU Kunio Kyoto Univ., Graduate School of Biostudies, Professor, 生命科学研究科, 教授 (40135695)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIMURA Shigehiro Kyoto Univ., Graduate School of Biostudies, As.Professor, 生命科学研究科, 助教授 (90346106)
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| Project Period (FY) |
2003 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥12,600,000 (Direct Cost: ¥12,600,000)
Fiscal Year 2006: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2005: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2004: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2003: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | Atomic Force Microscopy / Nano-scale imaging / Genome Organization / Nucleoid / Chromosome / Staphylococcus aureus / Clostridium perfringens / Bioinformatics / ナノテクノロジー / 原子間力顕微鏡(AFM) / Dps / テロメア / TRF2 |
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| Research Abstract |
In order to elucidate the molecular mechanisms of chromatin folding, we have taken two independent but comparative approaches ; one from biochemical reconstitution and the other from molecular analytical point of views. First, we established a highly efficient chromatin reconstitution procedure and analyzed the resultant structures using atomic force microscopy (AFM). Second, we developed a 'nucleus on-substrate-lysis' method and investigated the semi-intact inner nuclear structures under AFM. Third, we compared the genome architectures among prokaryotes and eukaryotes. From these, we propose a structural model that assumes a fundamental mechanism of genome packing is common in both prokaryotes and eukaryotes. A series of reconstitution experiments showed that the efficiency of the chromatin reconstitution by salt-dialysis method was drastically increased simply by using longer (>100 kb) and supercoiled DNA. This suggests that the physical properties of DNA are critical for the higher-
… More
order chromatin folding. Since double-stranded DNA, like other polymer chains, carries certain elasticity and flexibility, the length of DNA could affect the stability of nucleosome and chromatin fiber. This notion is well supported by the fact that in eukaryotic chromosome, the averaged length of a single chromatin loop is approximately 100 kb. On the other hand, the large-scale structure of chromatin fiber could be drastically affected by a local protein binding. Indeed, histone H1 was found to be essential for the reconstitution of 30 nm fibers. Depending on the salt environment, the thickness of the fiber was dynamically changed between 20 nm and 30-40 nm. As the higher-order structure than 30 nm fiber,・0 nm beaded structures could be reconstituted by the addition of 4 times amount of H1 into the reconstituted nucleosomes. The higher-order architectures of the Escherichia coli genome (nucleoid) is also achieved via step-wise genome folding from 40 nm to "80-nm beaded structure". When HeLa cells on a cover glass were successively treated with a detergent and a high-salt solution to remove the cytoplasmic and nucleoplasmic materials, the interphase chromosome was found to be composed of a fuzzy granular unit. After a brief treatment with MNase, the granular units could be observed enough clear to measure the size with〜90 nm in diameter. These observations suggest that the higher-order architectures of both prokaryotic and eukaryotic genomes may be similarly governed by simple physical natures of chromatin as well as specific nuclear proteins. Less
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