Kaneko Shinya, Akioka Manami, Tsuge Kenji, Itaya Mitsuhiro
Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan.
J Mol Biol. 2005 Jun 24;349(5):1036-44. doi: 10.1016/j.jmb.2005.04.041.
The combined use of the contemporary vector systems, the bacterial artificial chromosome (BAC) vector and the Bacillus subtilis genome (BGM) vector, makes possible the handling of giant-length DNA (above 100 kb). Our newly constructed BGM vector efficiently integrated DNA prepared in the BAC vector. A BAC library comprised of 18 independent clones prepared from mitochondrial DNA (mtDNA) of Arabidopsis thaliana was converted to a parallel BGM library using the new BGM vector. The effectiveness of the combined use of the vector systems was confirmed by the stable recovery of all 18 DNAs as BAC clones from the respective BGM clones. We show that DNA in BGM was stably preserved at room temperature after spore formation of the host B.subtilis. Rapid and stable shuttling between Escherichiacoli and the B. subtilis host, combined with spore-mediated DNA storage, may facilitate the long-term and low-cost preservation and the transportation of DNA resources.
当代载体系统,即细菌人工染色体(BAC)载体和枯草芽孢杆菌基因组(BGM)载体的联合使用,使得处理超长DNA(超过100 kb)成为可能。我们新构建的BGM载体能够有效地整合在BAC载体中制备的DNA。由拟南芥线粒体DNA(mtDNA)制备的18个独立克隆组成的BAC文库,利用新的BGM载体转化为平行的BGM文库。通过从各个BGM克隆中稳定回收所有18个DNA作为BAC克隆,证实了载体系统联合使用的有效性。我们表明,在宿主枯草芽孢杆菌形成孢子后,BGM中的DNA在室温下能够稳定保存。大肠杆菌和枯草芽孢杆菌宿主之间快速稳定的穿梭,再加上孢子介导的DNA储存,可能有助于DNA资源的长期低成本保存和运输。