Structure And Stabilization Of The Bacterial Nucleoid

细菌核的结构和稳定性

• 批准号: 6983873
• 负责人: Steven C. Zimmerman
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6983873
• 关键词: Escherichia coli; bacterial DNA; bacterial RNA; bacterial genetics; bacterial proteins; cell component structure /function; cell nucleus; chloramphenicol; formaldehyde; method development; nucleic acid denaturation; nucleoproteins; phase contrast microscopy; polylysine; protein sequence; tissue /cell preparation

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.

大肠杆菌的基因组DNA包含在一个或两个称为类核的致密体中。典型形状的类核的分离需要控制DNA扩增，这里通过修改聚赖氨酸-亚精胺程序来完成。控制体外类核扩增的能力可应用于类核纯化和高分辨率成像样品的制备，并可提高基因定位研究的分辨率。相对低平均分子量（约3 kDa）的聚赖氨酸用于产生含有相对于体内类核苷酸的大小扩增数倍的类核苷酸的裂解物。通过进一步加入多聚赖氨酸或通过在37 ℃下孵育稀释的裂解物，可以将这些扩展形式转化为尺寸与细胞类核相似的致密形式。在37 ℃下孵育。C伴随着大多数核糖体RNA的自溶降解。多色性和圆二色性光谱表明，聚赖氨酸-DNA复合物在孵育过程中被修改。紧密形式的类核可以通过暴露于盐溶液而逐渐再膨胀。在由快速或缓慢生长的细胞或由用氯霉素简单处理以减少DNA和细胞包膜之间的连接的细胞制成的裂解物中，类核压实是相似的。