Hampel K J, Burkholder G D, Lee J S
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Biochemistry. 1993 Feb 2;32(4):1072-7. doi: 10.1021/bi00055a012.
The ability of independent pyr.pur tracts to participate in triplex formation has been investigated in linear plasmids. The pyr.pur tract could be positioned at the ends of the plasmids or internally by a suitable choice of restriction enzyme. Dimer formation between plasmids was monitored by mobility shifts on agarose gels as well as by direct visualization in the electron microscope. Linear dimers and X and Y structures were observed. Control experiments showed that a pyr.pur tract was essential and was consistent with triplex formation in which the two pyrimidine strands were antiparallel. These structures were formed at pHs between 4 and 6, but once formed they remained stable up to pH 7. Spermine was required for formation of dimers at low ionic strength, but once formed the dimers remained stable in the absence of spermine. Additional linear plasmids were constructed with pyr.pur tracts at both ends; these formed structures at pH 4 which had mobilities identical to those of open circles. Triplex formation of this type may serve as a good model for loop formation in eukaryotic chromosomes.
已在线性质粒中研究了独立的嘧啶-嘌呤序列参与三链体形成的能力。通过适当选择限制性内切酶，嘧啶-嘌呤序列可定位在质粒末端或内部。通过琼脂糖凝胶上的迁移率变化以及电子显微镜下的直接观察来监测质粒之间的二聚体形成。观察到线性二聚体以及X和Y结构。对照实验表明，嘧啶-嘌呤序列是必不可少的，这与三链体形成一致，其中两条嘧啶链是反平行的。这些结构在pH值为4至6之间形成，但一旦形成，在pH值高达7时仍保持稳定。在低离子强度下形成二聚体需要精胺，但一旦形成，二聚体在没有精胺的情况下仍保持稳定。构建了两端都有嘧啶-嘌呤序列的额外线性质粒；这些在pH 4时形成的结构，其迁移率与开环的迁移率相同。这种类型的三链体形成可能是真核染色体中环形成的良好模型。
