McKenzie T, Hoshino T, Tanaka T, Sueoka N
Plasmid. 1986 Mar;15(2):93-103. doi: 10.1016/0147-619x(86)90046-6.
For the study of DNA-membrane interaction and the regulation of replication initiation we have determined the total nucleotide sequence of pUB110. As previously reported, this plasmid replicates in B. subtilis at a copy number of 30-50 per cell, with a majority of plasmids (60-80%) bound to the membrane (type-I binding). The type-I membrane binding is apparently necessary for pUB110 initiation of replication in vivo, but the membrane binding site is not known. Furthermore, four areas of the plasmid specifically bind to Bacillus subtilis membrane in an in vitro binding reaction (type II binding). These two types of membrane binding of pUB110 are different in that the in vivo binding (type-I) requires one (dnaBI) of the host initiation genes and is high-salt resistant, whereas the in vitro binding (type-II) does not require the dnaBI gene product and is high-salt sensitive. 7-mer double-strand sequence, TCAGCAA/AGTCGTT, or one-base derivatives of this sequence are frequently (17 of 23 of the 7-mer sequences) found in or close to the type-II binding areas. One of them is found at a restriction enzyme recognition site of a binding area that destroys the type-II membrane binding. These sequences may or may not have significance in type-II membrane binding. In addition to the neomycin resistance gene, the sequence data indicate two sizable open reading frames, ORF alpha and ORF beta, and two small ORF, gamma, and delta. All of these reading frames are in the same direction, which coincides with the direction of the replication. The open reading frame alpha (ORF alpha) corresponding to 334 amino acids close to the replication origin may be essential for the initiation of replication of PUB110. The putative protein alpha corresponding to this open reading frame contains a consensus sequence of the DNA binding sites which are found in a number of known DNA-binding proteins. The consensus DNA binding site of protein alpha is flanked by two hydrophobic areas. These two observations suggest that the corresponding protein may have both an affinity to a specific site in pUB110, and an affinity to the membrane.
为了研究DNA与膜的相互作用以及复制起始的调控，我们测定了pUB110的全核苷酸序列。如先前报道，该质粒在枯草芽孢杆菌中以每个细胞30 - 50个拷贝的数量进行复制，大多数质粒（60 - 80%）与膜结合（I型结合）。I型膜结合显然是pUB110在体内复制起始所必需的，但膜结合位点尚不清楚。此外，在体外结合反应中，质粒的四个区域能特异性地与枯草芽孢杆菌膜结合（II型结合）。pUB110的这两种膜结合类型有所不同，体内结合（I型）需要一个宿主起始基因（dnaBI）且耐高盐，而体外结合（II型）不需要dnaBI基因产物且对高盐敏感。7聚体双链序列TCAGCAA/AGTCGTT或该序列的单碱基衍生物在II型结合区域或其附近经常出现（23个7聚体序列中有17个）。其中一个位于一个结合区域的限制酶识别位点，该位点会破坏II型膜结合。这些序列在II型膜结合中可能有意义，也可能没有。除了新霉素抗性基因外，序列数据还表明有两个较大的开放阅读框，即ORFα和ORFβ，以及两个小的ORF，γ和δ。所有这些阅读框方向相同，与复制方向一致。靠近复制起点的对应334个氨基酸的开放阅读框α（ORFα）可能对PUB110的复制起始至关重要。与这个开放阅读框对应的推定蛋白α包含许多已知DNA结合蛋白中发现的DNA结合位点的共有序列。蛋白α的共有DNA结合位点两侧是两个疏水区域。这两个观察结果表明，相应的蛋白可能既对pUB110中的特定位点有亲和力，又对膜有亲和力。
