Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.
Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules.
复制标题
用线性质粒分子转化大肠杆菌期间质粒 DNA 的缺失和重排。
DOI：
10.1093/nar/14.22.8905
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发表时间：
1986
影响因子：
14.9
通讯作者：
J. Saunders
中科院分区：
文献类型：
--
作者：
E. C. Conley;V. Saunders;J. Saunders
当E.用线性化的pBR 322 DNA转化大肠杆菌，许多转化子含有带有缺失和其它重排的再环化质粒。大多数异常分子小于单体长度，并且失去了用于线性化的限制性位点，缺失区域单向延伸（Ia型）或双向延伸（Ib型）。II型缺失体大于单体但小于二聚体，并且包含在一个或两个连接处缺失的正向重复序列中的pBR 322序列（IIa型）或在两个连接处缺失序列的反向重复序列中的pBR 322序列（IIb型）。III型缺失体大于二聚体但小于三聚体，由在两个或更多个连接处缺失的正向和反向重复的pBR 322序列组成。线性DNA的转化频率大大降低，在XTH-1-细菌与IIb型缺失占主导地位的转化体。这表明核酸外切酶III对于质粒的完美纯化和I型缺失体的产生是重要的。体外连接产物的体内去甲基化解释了基因克隆过程中遇到的许多异常DNA分子。
When E. coli was transformed with linearized pBR322 DNA, many transformants contained recircularized plasmids bearing deletions and other rearrangements. Most aberrant molecules were less than monomeric length and had lost the restriction site used for linearization, with the deleted region extending mono- (type Ia) or bi-directionally (type Ib). Type II deletants were greater than monomeric but less than dimeric and contained the pBR322 sequence in direct repeat with deletion at one or both junctions (type IIa) or in inverted repeat with loss of sequence at both junctions (type IIb). Type III deletants were greater than dimeric but less than trimeric, consisting of pBR322 sequences in both direct and inverse repeat with deletions at two or more junctions. Transformation frequencies for linear DNA were drastically reduced in xth-1- bacteria with type IIb deletants predominating in transformants. This indicates that exonuclease III is important for perfect recyclization of plasmids and the generation of type I deletants. In vivo recyclization of in vitro ligation products explains many of the aberrant DNA molecules that are encountered during gene cloning.