Structure And Stabilization Of The Bacterial Nucleoid

大肠杆菌的基因组DNA包含在一个或两个被称为类核的紧密体中。典型形状的类核的分离需要控制DNA的扩展，这里通过对聚赖氨酸-亚精胺过程的修改来完成。控制体外类核扩增的能力在类核纯化和高分辨率成像样品制备中有应用，并可能增加基因定位研究的分辨率。相对较低的平均分子量（约3 kDa）的聚赖氨酸用于生产含有类核的裂解物，这些类核相对于体内类核的大小扩大了几倍。通过进一步添加聚赖氨酸或在37℃下孵育稀释的裂解物，这些膨化形式可以转化为与细胞类核尺寸相似的致密形式。37℃的孵育伴随着大多数核糖体RNA的自溶降解。高色性和圆二色性光谱表明，在孵育过程中，聚赖氨酸- dna复合物被修饰。紧实形式的类核可以通过暴露于盐溶液中逐渐重新膨胀。在快速或缓慢生长的细胞或经过氯霉素短暂处理以减少DNA和细胞包膜之间联系的细胞的裂解物中，类核压实是相似的。

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically-shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (ca -3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 deg.C. The incubation at 37 deg. C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.