Sheflin L G, Kowalski D
Nucleic Acids Res. 1985 Sep 11;13(17):6137-54. doi: 10.1093/nar/13.17.6137.
Mung bean nuclease was used to probe for recognizable DNA unwinding and unpairing in the plasmid pBR322. In negatively supercoiled DNA, but not relaxed DNA, cleavages occurred preferentially in non-coding regions of the genome. The types of nucleotide sequences cleaved and which non-coding regions were cleaved depended upon environmental conditions. At 37 degrees C, cleavages occurred in an 84 bp A+T-rich sequence in the terminator region of the ampicillin-resistance gene. Recognition is likely based on a novel DNA conformation which occurs in the longest, most dA+dT-rich region of pBR322. In the presence of 1 mM Mg2+, cleavages occurred in inverted repeated sequences in the promoter regions of the RNA primer for DNA replication and ampicillin- and tetracycline-resistance genes as well as the terminator of RNA-1. Potential loops of hairpin (cruciform) structures were cleaved. At 27 degrees C, cleavages occurred near a promoter activated by cAMP receptor protein in vitro and in the 3' non-coding region of the tetracycline-resistance gene. Thus, in supercoiled pBR322 DNA, recognizable DNA unwinding and unpairing occurs preferentially in regulatory regions for transcription and DNA replication.
用绿豆核酸酶探测质粒pBR322中可识别的DNA解旋和碱基对解开情况。在负超螺旋DNA中而非松弛型DNA中，切割优先发生在基因组的非编码区。被切割的核苷酸序列类型以及哪些非编码区被切割取决于环境条件。在37摄氏度时，切割发生在氨苄青霉素抗性基因终止区一个84bp富含A+T的序列中。识别可能基于一种新的DNA构象，这种构象出现在pBR322最长、富含dA+dT的区域。在1mM Mg2+存在的情况下，切割发生在DNA复制RNA引物、氨苄青霉素和四环素抗性基因启动区以及RNA-1终止区的反向重复序列中。潜在的发夹（十字形）结构环被切割。在27摄氏度时，则在体外被cAMP受体蛋白激活的启动子附近以及四环素抗性基因的3'非编码区发生切割。因此，在超螺旋pBR322 DNA中，可识别DNA解旋和碱基对解开优先发生在转录和DNA复制的调控区域。
