Pritchard A E, Herron L M, Cummings D J
Gene. 1980 Oct;11(1-2):43-52. doi: 10.1016/0378-1119(80)90085-2.
Fragments containing the replication-initiation region of mitochondrial (mt) DNA from four species of Paramecium aurelia were ligated to the pBR322 plasmid and used to transform Escherichia coli. The criteria for identifying the desired mitochondrial DNA restriction fragments were based on the palindromic dimer structure of the replicative intermediate of this DNA. The nature of the cloned sequences was verified by hybridization to mitochondrial DNA fragments containing the initiation region, by determining the size of snapback renaturation products, and by determining the symmetry of restriction-endonuclease sites within the clones. Heterologous hybridization experiments showed that one species' initiation region is not homologous to the other three. Cleavage patterns of each of the clones by 18 different restriction enzymes were determined. There are no sites within approx. 250 bp of the center of the insert of any clone although there are numerous cleavages in more distal regions. These sites are generally symmetric about the center of the insert as expected for a palindromic structure. The characteristics of the cloned sequences support a proposed replication model for all four species studied.
将来自四种双小核草履虫的含有线粒体（mt）DNA复制起始区域的片段连接到pBR322质粒上，并用于转化大肠杆菌。鉴定所需线粒体DNA限制性片段的标准基于该DNA复制中间体的回文二聚体结构。通过与含有起始区域的线粒体DNA片段杂交、测定快速复性产物的大小以及测定克隆内限制性内切酶位点的对称性，验证了克隆序列的性质。异源杂交实验表明，一个物种的起始区域与其他三个物种的起始区域不同源。测定了18种不同限制性酶对每个克隆的切割模式。尽管在更远端区域有许多切割，但在任何克隆插入片段中心约250 bp范围内没有位点。如回文结构所预期的那样，这些位点通常关于插入片段的中心对称。克隆序列的特征支持了所研究的所有四个物种的一个提出的复制模型。
