All-or-none switching of transcriptional activity on single DNA molecules caused by a discrete conformational transition

Recently, it has been confirmed that long duplex DNA molecules with sizes larger than several tens of kilo-base pairs (kbp), exhibit a discrete conformational transition from an elongated coil state to a compact globule state upon the addition of various kinds of chemical species that usually induce DNA condensation. In this study, we performed a single-molecule observation on a large DNA, Lambda ZAP II DNA (ca. 41 kbp), in a solution containing RNA polymerase and substrates along with spermine, a tetravalent cation, at different concentrations, by use of fluorescence staining of both DNA and RNA. We found that transcription, or RNA production, is completely inhibited in the compact state, but is actively performed in the unfolded coil state. Such an all-or-none effect on transcriptional activity induced by the discrete conformational transition of single DNA molecules is discussed in relation to the mechanism of the regulation of large-scale genetic activity.Comment: 14 pages, 2 figure

10. Phase Transition and Segregation in Chromatin Reconstituted from Giant DNA(poster presentation,Soft Matter as Structured Materials)

この論文は国立情報学研究所の電子図書館事業により電子化されました。長鎖DNA(35μm)とコアヒストンから再構成したクロマチンが、ヌクレオソーム密度に応じて急激な凝縮転移を起こし、ヌクレオソームの高密度な相と低密度な相に分離することを発見した。原子間力顕微鏡観察によりヌクレオソーム間の実効的相互作用ポテンシャルを求め、この凝縮転移を記述するモデルを提案した

キョダイ イオン ソンザイカ デ ノ DNA 1ブンシ ノ レンゾク フレンゾク オリタタミ テンイ

京都大学0048新制・課程博士博士(理学)甲第12804号理博第3114号新制||理||1463(附属図書館)UT51-2007-H77京都大学大学院理学研究科物理学・宇宙物理学専攻(主査)教授 吉川 研一, 教授 小貫 明, 教授 太田 隆夫学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDA

5. Single-chain Characteristics in Giant DNA : Larger Hydrodynamic Radius in Circular than that in Linear(poster presentation,Soft Matter as Structured Materials)

この論文は国立情報学研究所の電子図書館事業により電子化されました。環状高分子の広がり(慣性半径や末端間距離の2乗平均)が同じ全長の線状高分子のものより小さいことはよく知られている。しかし環状高分子の合成は難しく、実験的な検証が困難なため環状高分子に関しては不明な点が多い。一方天然高分子DNAでは生体内にウィルスのプラスミドなど環状DNAが存在し、制限酵素を用いて一箇所切断することにより、容易に全長が単分散な環状および線状高分子を得ることができる。そこで本研究では106キロ塩基対(全長約36μm)の線状および環状DNAの流体力学的半径を蛍光顕微鏡を用いた単分子直接観察によって測定した。その結果、長鎖環状DNAの流体力学的半径は線状のものよりも25%以上大きいことを見出した

Hydrodynamic radius of circular DNA is larger than that of linear DNA

It is generally accepted that linear polymers are larger, such as with regard to the end-to-end distance and gyration radius, than the corresponding circular polymers. We measured the Brownian motion of individual linear and circular 106-kbp DNA molecules using fluorescence microscopy. Unexpectedly, the hydrodynamic radius of linear DNA was markedly smaller than that of circular DNA

Removal of histone tails from nucleosome dissects the physical mechanisms of salt-induced aggregation, linker histone H1-induced compaction, and 30-nm fiber formation of the nucleosome array

In order to reveal the roles of histone tails in the formation of higher-order chromatin structures, we employed atomic force microscopy (AFM), and an in vitro reconstitution system to examine the properties of reconstituted chromatin composed of tail-less histones and a long DNA (106-kb plasmid) template. The tail-less nucleosomes did not aggregate at high salt concentrations or with an excess amount of core histones, in contrast with the behavior of nucleosomal arrays composed of nucleosomes containing normal, N-terminal tails. Analysis of our nucleosome distributions reveals that the attractive interaction between tail-less nucleosomes is weakened. Addition of linker histone H1 into the tail-less nucleosomal array failed to promote the formation of 30 nm chromatin fibers that are usually formed in the normal nucleosomal array. These results demonstrate that the attractive interaction between nucleosomes via histone tails plays a critical role in the formation of the uniform 30-nm chromatin fiber

In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence.

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT)(n)(ACTGGGAAGGGAC)(n)-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA