Structure And Stabilization Of The Bacterial Nucleoid

大肠杆菌的DNA主要局限于一个或几个称为类核的致密体中。这种压缩可以引起DNA反应性的显著变化。由于缺乏合适的实验系统，影响类核压实的因素一直难以表征。在一个测试的重要性，大分子拥挤周围的细胞质对类核压实，我们裂解细胞的条件下，保留细胞边界。细胞质的释放允许DNA立即扩增以填充残留的细胞壁，这与拥挤的主导作用雅阁。然而，如果在裂解前加入多聚赖氨酸，则裂解后DNA的正常扩增被阻止;定义明确的紧凑类核持续存在。聚赖氨酸被用于一种新的类核分离方法，该方法首次释放出保持体内一般形状和压实状态的类核。该程序已被应用于指数期细胞和细胞处理氯霉素或nalidixate改变其类核的形态。利福平处理的细胞的显着不稳定的类核得到紧凑，稳定的形式与此程序。在多聚赖氨酸存在下释放的类核苷酸容易处理，并提供明确的DNA荧光和相差图像。均匀种群的类核保留特征形状后，可以分离出甲醛固定和加热与十二烷基硫酸钠。释放的类核的生化和结构表征正在进行中。

The DNA of Escherichia coli is largely confined to one or a few compact bodies known as nucleoids. Such compaction can cause dramatic changes in the reactivity of the DNA. Factors affecting nucleoid compaction have been difficult to characterize because of a lack of suitable experimental systems. In a test of the importance of macromolecular crowding by the surrounding cytoplasm on nucleoid compaction, we lysed cells under conditions that retained cell boundaries. Release of the cytoplasm allowed an immediate expansion of the DNA to fill the residual cell walls, in accord with a dominant role of crowding. However, if polylysine was added before lysis, the normal expansion of the DNA upon lysis was prevented; well defined, compact nucleoids persisted. Polylysine was used in a new procedure for nucleoid isolation that, for the first time, releases nucleoids that retain the general shape and state of compaction that was present in vivo. The procedure has been applied to exponential phase cells and cells treated with chloramphenicol or nalidixate to alter the morphology of their nucleoids. The notably unstable nucleoids of rifampicin-treated cells were obtained in compact, stable form with this procedure. Nucleoids released in the presence of polylysine were easily handled, and provided well-defined DNA fluorescence and phase contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde-fixation and heating with sodium dodecyl sulfate. Biochemical and structural characterizations of the released nucleoids are underway.