Structure And Stabilization Of The Bacterial Nucleoid

大肠杆菌的DNA主要局限于一个或几个被称为类核的致密小体。这种紧凑可能会导致DNA的反应性发生戏剧性的变化。由于缺乏合适的实验系统，影响类核压缩的因素一直很难表征。在一项关于周围细胞质的大分子拥挤对类核紧凑的重要性的测试中，我们在保持细胞边界的条件下裂解细胞。细胞质的释放允许DNA立即扩张以填充剩余的细胞壁，这与拥挤的主导作用一致。然而，如果在裂解前加入多聚赖氨酸，DNA裂解时的正常扩张就会被阻止；明确、致密的类核仍然存在。多聚赖氨酸被用在一种新的类核分离程序中，该程序首次释放出保持体内存在的一般形状和致密状态的类核。该程序已应用于指数期细胞和氯霉素或奈利地索处理的细胞，以改变其类核的形态。用这种方法获得了致密、稳定的利福平处理细胞的显著不稳定的类核。在聚赖氨酸存在下释放的类核很容易处理，并提供清晰的DNA荧光和相衬图像。在甲醛固定和十二烷基硫酸钠加热后，可以分离出具有特征形状的均匀的类核群体。释放的类核的生化和结构特征正在进行中。

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.