Paterson E S, Iyer V N
Department of Biology, Carleton University, Ottawa, Ontario, Canada.
J Bacteriol. 1997 Sep;179(18):5768-76. doi: 10.1128/jb.179.18.5768-5776.1997.
The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.
质粒pMUR274的N型oriT克隆于一个474 bp的RsaI - SspI片段上，并测定了其核苷酸序列。对pMUR274 oriT序列与IncN质粒pCU1和IncW质粒R388的oriT序列进行比较，结果显示同源性分别为57%和28%。pCU1 oriT与pMUR274的oriT之间发生分子内位点特异性重组，形成了一个包含每个亲本序列一半的杂种oriT。杂种oriT的连接点位于两个亲本序列中均存在的一个10 bp序列GCTATACACC内，代表每个oriT的切口位点。10 bp连接序列中第一个A或第二个T残基发生突变，转移减少不到20倍，而第二个或第三个A残基发生突变，转移减少超过1000倍。野生型pCU1 oriT与这四个突变型pCU1 oriT之间的位点特异性重组表明，切口位点位于10 bp连接序列的第二个T和第二个A残基之间。野生型和突变型pCU1 oriT之间的位点特异性重组还表明，切口位点右侧的点突变会降低转移的起始和终止，而切口位点左侧的点突变会降低终止，但对起始影响很小或没有影响。切口位点右侧39个碱基处富含AT区域内的一个28 bp缺失会降低起始和终止，而最小oriT区域最右侧边界处一个6 bp反向重复序列的缺失会降低起始但不影响终止。
