Kahn M E, Barany F, Smith H O
Proc Natl Acad Sci U S A. 1983 Nov;80(22):6927-31. doi: 10.1073/pnas.80.22.6927.
The mechanism by which Haemophilus protects donor DNA from cellular restriction and degradative enzymes during transformation is unclear. In this report, we demonstrate that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which we have termed "transformasomes." DNA within transformasomes is in a protected state--resistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded. The ability of donor DNA to exit from transformasomes is dependent on its topological conformation. Circular DNA remains intact within transformasomes, while linear DNA rapidly exits and undergoes homologous recombination. Protected donor DNA can be preferentially removed from the surface of competent cells by extraction with organic solvents. Structurally intact transformasomes containing donor DNA could be partitioned into the organic layer and can be further purified by density centrifugation.
在转化过程中,嗜血杆菌保护供体DNA免受细胞限制酶和降解酶作用的机制尚不清楚。在本报告中,我们证明供体DNA通过特殊的膜状延伸结构进入流感嗜血杆菌,我们将其称为“转化体”。转化体内的DNA处于受保护状态——对外部DNase和细胞限制酶具有抗性,尽管仍未修饰且为双链。供体DNA从转化体中排出的能力取决于其拓扑构象。环状DNA在转化体内保持完整,而线性DNA迅速排出并进行同源重组。通过用有机溶剂提取,可以优先从感受态细胞表面去除受保护的供体DNA。含有供体DNA的结构完整的转化体可以分配到有机层中,并可通过密度离心进一步纯化。