Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.
Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.
复制标题
广泛宿主范围质粒 RK2 区域稳定功能的遗传表征。
DOI：
10.1128/jb.172.11.6204-6216.1990
复制
发表时间：
1990
影响因子：
3.2
通讯作者：
Helinski,DR
中科院分区：
文献类型：
--
作者：
Roberts,RC;Burioni,R;Helinski,DR
负责广宿主质粒RK2稳定遗传的区域之一包含在PstIC C片段内，位于坐标30.8至37.0kb(P.N.Saurugger，O.Hrabak，H.Schwab和R.M.Lafferty，J.Biotechnol。1986年4：333-343)。对该6.2kb区域的遗传分析表明，不存在通过选择性地杀死无质粒分离物而稳定的功能。36.0kb~37.0kb的序列使质粒拷贝数增加了一倍，但这一区域不是稳定活性所必需的。PstIC片段编码33.1-35.3kb的多聚体分辨系统。将其顺式作用位点定位到140个碱基，测序发现含有两个6和7碱基的直接重复序列和两个6和8碱基的完美反向重复序列。反式作用因子(S)被定位并在功能上被确定为编码一种能够在顺式作用位点之间以正向或反向高频催化重组的解旋酶。单靠多聚体分辨并不能解释PstIC C片段对质粒的完全稳定作用，因为移除最小MRS基因座35.3kb边界附近的区域大大降低了稳定性。完全稳定所需的最小区域，从32.8到35.9kb，能够完全稳定质粒，而不依赖于复制子或宿主的recA熟练程度。稳定活性在几种不同的革兰氏阴性菌中也得到了充分的表达，而F质粒PAR位点只在大肠杆菌中发挥作用。在这些观察的基础上，我们得出结论：在所使用的生长条件下，最小稳定轨迹既编码了MRS活性，又编码了具有PAR轨迹性质的稳定活性。
One of the regions responsible for the stable inheritance of the broad-host-range plasmid RK2 is contained within the PstI C fragment, located from coordinates 30.8 to 37.0 kb (P.N. Saurugger, O. Hrabak, H. Schwab, and R.M. Lafferty, J. Biotechnol. 4:333-343, 1986). Genetic analysis of this 6.2-kb region demonstrated that no function was present that stabilized by selectively killing plasmid-free segregants. The sequence from 36.0 to 37.0 kb mediated a twofold increase in plasmid copy number, but this region was not required for stabilization activity. The PstI C fragment was shown to encode a multimer resolution system from 33.1 to 35.3 kb. The resolution cis-acting site was mapped to 140 bp, sequenced, and observed to contain two directly repeated sequences of 6 and 7 bases and two perfect inverted repeats of 6 and 8 bases. The trans-acting factor(s) was mapped and functionally determined to encode a resolvase capable of catalyzing recombination at high frequency between cis-acting sites in either direct or inverted orientation. Multimer resolution alone did not account for complete plasmid stabilization by the PstI C fragment, since removal of regions adjacent to the 35.3-kb border of the minimal mrs locus dramatically reduced stabilization. The minimal region required for complete stabilization, from 32.8 to 35.9 kb, was capable of fully stabilizing plasmids independently of the replicon or the recA proficiency of the host. Stabilization activity was also fully expressed in several diverse gram-negative bacteria, whereas the F plasmid par locus functioned only in Escherichia coli. On the basis of these observations, we conclude that under the growth conditions used, the minimal stabilization locus encodes both an mrs activity and a stabilization activity that has the properties of a par locus.