Conley E C, Saunders V A, Saunders J R
Nucleic Acids Res. 1986 Nov 25;14(22):8905-17. doi: 10.1093/nar/14.22.8905.
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.
当用线性化的pBR322 DNA转化大肠杆菌时，许多转化体含有带有缺失和其他重排的环化质粒。大多数异常分子长度小于单体长度，并且失去了用于线性化的限制酶切位点，缺失区域单向（Ia型）或双向（Ib型）延伸。II型缺失体大于单体但小于二聚体，在一个或两个连接点处有缺失的情况下以正向重复形式包含pBR322序列（IIa型），或者在两个连接点处都有序列缺失的情况下以反向重复形式包含pBR322序列（IIb型）。III型缺失体大于二聚体但小于三聚体，由在两个或更多连接点处有缺失的正向和反向重复的pBR322序列组成。在xth-1-细菌中，线性DNA的转化频率急剧降低，转化体中IIb型缺失体占主导。这表明核酸外切酶III对于质粒的完美环化和I型缺失体的产生很重要。体外连接产物的体内环化解释了基因克隆过程中遇到的许多异常DNA分子。
