Hogrefe C, Friedrich B
Plasmid. 1984 Nov;12(3):161-9. doi: 10.1016/0147-619x(84)90040-4.
A method is described for the preparative isolation of megaplasmids ranging in size from 340 to 700 kb. These plasmids were isolated from chemolithoautotrophic bacteria including the species Alcaligenes, Pseudomonas, and Paracoccus. The procedure was based on alkaline sodium dodecyl sulfate lysis of the cells, followed by heat treatment, salt precipitation, several phenol extractions, dialysis steps, and proteinase and RNase treatment. The various parameters were evaluated and controlled. Hydrogen-oxidizing-ability (Hox) encoding plasmids were compared by EcoRI restriction enzyme analysis. pHG plasmids from Alcaligenes eutrophus wild-type strains appeared to be closely related; plasmids derived from the type strain TF93 and from A. hydrogenophilus exhibited major differences in restriction sites. Two cryptic plasmids harbored by Pseudomonas facilis and Paracoccus denitrificans showed scarcely detectable similarity to the plasmid species of Alcaligenes.
本文描述了一种用于制备分离大小在340至700 kb之间的大质粒的方法。这些质粒是从包括产碱菌属、假单胞菌属和副球菌属在内的化能自养细菌中分离得到的。该方法基于细胞的碱性十二烷基硫酸钠裂解，随后进行热处理、盐沉淀、多次苯酚萃取、透析步骤以及蛋白酶和核糖核酸酶处理。对各种参数进行了评估和控制。通过EcoRI限制性内切酶分析比较了编码氢氧化能力（Hox）的质粒。来自嗜糖产碱菌野生型菌株的pHG质粒似乎密切相关；来自模式菌株TF93和嗜氢产碱菌的质粒在限制性酶切位点上表现出主要差异。费氏假单胞菌和反硝化副球菌携带的两个隐蔽质粒与产碱菌属的质粒种类几乎没有可检测到的相似性。
