Ohkawa J, Yuyama N, Takebe Y, Nishikawa S, Taira K
National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Tsukuba Science City, Japan.
Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11302-6. doi: 10.1073/pnas.90.23.11302.
The kinetic behavior of ribozymes derived from two types of multiple-ribozyme expression vector were examined. In some cases, multiple ribozymes were expressed as a single RNA molecule and all the ribozymes were simply connected in tandem (connected type). In other cases, multiple ribozymes were flanked by cis-acting ribozymes at both their 5' and 3' ends so that, upon transcription, multiple ribozymes were trimmed at both their 5' and 3' ends, with resultant liberation of multiple independent ribozymes (shotgun type). When levels of ribozyme expression were examined for the shotgun-type vector, the level of the ribozyme transcript was found to be proportional to the number of units (n) connected in tandem. Accordingly, the activities of the shotgun-type ribozymes, in terms of the cleavage of HIV-1 RNA in vitro, were also found to be proportional to the number of units connected in tandem (n). By contrast, the activities of the connected-type ribozymes reached plateau values at around n = 3. These results indicate that, when the shotgun-type expression system is used, it is theoretically possible to generate various independent ribozymes, each specific for a different target site, without sacrificing the activity of any individual ribozyme.
对源自两种类型多核糖酶表达载体的核糖酶的动力学行为进行了研究。在某些情况下，多个核糖酶作为单个RNA分子表达，所有核糖酶简单地串联连接（连接型）。在其他情况下，多个核糖酶在其5'和3'末端两侧均由顺式作用核糖酶侧翼，以便在转录时，多个核糖酶在其5'和3'末端均被修剪，从而产生多个独立的核糖酶（散弹枪型）。当检测散弹枪型载体的核糖酶表达水平时，发现核糖酶转录本的水平与串联连接的单位数（n）成正比。因此，就体外切割HIV-1 RNA而言，散弹枪型核糖酶的活性也与串联连接的单位数（n）成正比。相比之下，连接型核糖酶的活性在n = 3左右达到平台值。这些结果表明，当使用散弹枪型表达系统时，理论上有可能产生各种独立的核糖酶，每个核糖酶针对不同的靶位点具有特异性，而不会牺牲任何单个核糖酶的活性。
