Guillen N, Le Hegaret F, Fleury A M, Hirschbein L
Nucleic Acids Res. 1978 Feb;5(2):475-89. doi: 10.1093/nar/5.2.475.
The isolation of folded DNA from Bacillus subtilis, a Gram positive bacterium is described. When the lysis was achieved with 1 M NaCl a slow sedimenting nucleoid was obtained (1600-2000 S). Conversely, when the lysis was achieved with 0.2 M NaCl a fast sedimenting nucleoid was obtained (3500-4000 S). The yield of folded DNA was between 80 to 90 % of the total lysate DNA. Both nucleoids contained the same amount of RNA, but the relative proportions of lipids and proteins were different. Folded chromosomes were prepared in the presence of spermidine: artifactual protein binding is shown to be unlikely. Electrophoresis of nucleoid proteins showed a dominant polypeptide (MW = 36,000), which remained associated with DNA after sarcosyl treatment and could be partially removed by heat mediated DNA unfolding. In vitro transcription by endogenous RNA polymerase bound to the fast sedimenting-nucleoid was rifamycin resistant; the template capacity of the fast sedimenting-nucleoid was compared with that of the completely unfolded chromosomes.
本文描述了从革兰氏阳性细菌枯草芽孢杆菌中分离折叠DNA的方法。当用1M NaCl进行裂解时，可获得沉降缓慢的类核（1600 - 2000 S）。相反，当用0.2M NaCl进行裂解时，可获得沉降快速的类核（3500 - 4000 S）。折叠DNA的产量占总裂解物DNA的80%至90%。两种类核所含RNA量相同，但脂质和蛋白质的相对比例不同。在亚精胺存在下制备折叠染色体：结果表明不太可能存在人为的蛋白质结合。类核蛋白的电泳显示有一条主要多肽（分子量 = 36,000），在肌氨酸处理后仍与DNA结合，并且可以通过热介导的DNA解折叠部分去除。与快速沉降类核结合的内源性RNA聚合酶的体外转录对利福平具有抗性；将快速沉降类核的模板能力与完全解折叠的染色体的模板能力进行了比较。
