Bron S, Luxen E, Swart P
Department of Genetics, Center of Biological Sciences, Haren, The Netherlands.
Plasmid. 1988 May;19(3):231-41. doi: 10.1016/0147-619x(88)90041-8.
Two series of pUB110-derived plasmids were constructed to study segregational stability in Bacillus subtilis. pEB plasmids were based on the entire pUB110, whereas pLB plasmids lack the membrane-binding areas BA3 and BA4. Two kinds of stability defects were observed. The first was characterized by a strong size dependency and occurred with different inserts at various positions in pLB and pEB plasmids. Size-dependent reductions in plasmid copy numbers appeared to underly this phenomenon. This may render pUB110 unsuitable for the cloning of inserts larger than about 3 kb, in particular if no selective conditions can be applied. The second defect, observed with pLB plasmids, was caused by the absence of the membrane-binding areas BA3 and BA4. Deletion of BA3 resulted in the accumulation of single-stranded plasmid DNA, suggesting that BA3 contains the initiation signal for complementary strand synthesis. The BA3 region is very rich in hyphenated dyad symmetry which, in single-stranded DNA, could result in several stable alternative secondary structures. It is speculated that the activity of the BA3-associated initiation signal contributes to the segregational stability of pUB110-derived plasmids in B. subtilis. The absence of the BA3 stability function could not account for all stability defects observed. Additional stability functions seemed to be located on the BA4 fragment.
构建了两系列源自pUB110的质粒，用于研究枯草芽孢杆菌中的分离稳定性。pEB质粒基于完整的pUB110，而pLB质粒缺少膜结合区域BA3和BA4。观察到两种稳定性缺陷。第一种以强烈的大小依赖性为特征，在pLB和pEB质粒的不同位置插入不同片段时都会出现。质粒拷贝数的大小依赖性降低似乎是这种现象的基础。这可能使pUB110不适用于克隆大于约3 kb的插入片段，特别是在无法应用选择条件的情况下。观察到的第二种缺陷出现在pLB质粒中，是由膜结合区域BA3和BA4的缺失引起的。BA3的缺失导致单链质粒DNA的积累，这表明BA3包含互补链合成的起始信号。BA3区域富含连字符化的二元对称序列，在单链DNA中，这可能导致几种稳定的替代二级结构。据推测，与BA3相关的起始信号的活性有助于pUB110衍生质粒在枯草芽孢杆菌中的分离稳定性。BA3稳定性功能的缺失并不能解释观察到的所有稳定性缺陷。额外的稳定性功能似乎位于BA4片段上。
