Holst A, Müller F, Zastrow G, Zentgraf H, Schwender S, Dinkl E, Grummt F
Institute of Biochemistry, University of Würzburg, Federal Republic of Germany.
Cell. 1988 Feb 12;52(3):355-65. doi: 10.1016/s0092-8674(88)80028-x.
Plasmids that replicate autonomously in mouse L cells were constructed by inserting random genomic DNA fragments from Ltk- cells into a plasmid containing the HSV-1 thymidine kinase gene with a truncated low-efficiency promoter. HAT resistance was used as a selective marker. The presence of free plasmids in the DNA of transformants was demonstrated by hybridization with a specific plasmid probe, by electron microscopic visualization of circular DNA, and by recovering these plasmids by E. coli transformation. Nineteen different DNA fragments were isolated. They were characterized as murine autonomously replicating sequences by Mbol restriction endonuclease sensitivity, by bromodeoxyuridine substitution, by copy number determination, and by segregation analysis. Sequence analysis of the inserts of nine plasmids revealed a conserved element of 12 bp (CTCATGAGAGGCCAA) in five out of nine autonomously replicating sequences.
通过将来自Ltk-细胞的随机基因组DNA片段插入到含有截短的低效率启动子的单纯疱疹病毒1型胸苷激酶基因的质粒中，构建了能在小鼠L细胞中自主复制的质粒。以对氨基蝶呤、氨基蝶呤和胸腺嘧啶核苷（HAT）的抗性作为选择标记。通过与特异性质粒探针杂交、对环状DNA进行电子显微镜观察以及通过大肠杆菌转化回收这些质粒，证明了转化体DNA中游离质粒的存在。分离出了19个不同的DNA片段。通过Mbol限制性内切酶敏感性、溴脱氧尿苷取代、拷贝数测定和分离分析，将它们鉴定为小鼠自主复制序列。对9个质粒插入片段的序列分析显示，在9个自主复制序列中的5个序列中存在一个12 bp的保守元件（CTCATGAGAGGCCAA）。
