Defining Components of the Chromosomal Origin of Replication of the Hyperthermophilic Archaeon Pyrococcus furiosus Needed for Construction of a Stable Replicating Shuttle Vector
Defining Components of the Chromosomal Origin of Replication of the Hyperthermophilic Archaeon Pyrococcus furiosus Needed for Construction of a Stable Replicating Shuttle Vector
复制标题
DOI：
10.1128/aem.05057-11
复制
发表时间：
2011-09-01
影响因子：
4.4
通讯作者：
Westpheling, Janet
中科院分区：
文献类型：
--
作者：
Farkas, Joel;Chung, Daehwan;Westpheling, Janet
我们报告了一系列复制穿梭载体的构建，该载体由大肠杆菌的低拷贝数克隆载体和超嗜热古菌Pyrococcus furiosus染色体的复制起点（oriC）的功能组分组成。在确定P. furiosus中自主质粒复制所需的最小复制起点序列的过程中，我们发现通过生物信息学分析和体外结合研究预测的起点的几个特征对于稳定的自主质粒复制不是必需的。鉴定了促进质粒DNA复制所需的最小区域，并且基于该序列的质粒容易转化激烈毕赤酵母。质粒自主复制，以单拷贝存在。与基于来自密切相关的超嗜热菌深海火球菌的质粒的穿梭载体相比，基于激烈毕赤酵母染色体来源的质粒在转化后结构不变，并且在没有选择的情况下稳定超过100代。
We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus, plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.