Bennett M J, Cullimore J V
Department of Biological Sciences, University of Warwick, Coventry, UK.
Nucleic Acids Res. 1992 Feb 25;20(4):831-7. doi: 10.1093/nar/20.4.831.
In Phaseolus vulgaris L. (French bean) glutamine synthetase (GS) is encoded by four closely-related genes termed gln-alpha, gln-beta, gln-gamma and gln-delta. We have constructed and characterised in vitro a number of hammerhead ribozymes designed to cleave individual RNAs encoded by these genes. The three ribozymes, termed J1, J2 and J3, were targeted to cleave RNA at the start of the gamma and beta, and the middle of the gamma, GS open reading frames respectively. All three ribozymes successfully discriminated between the four (alpha, beta, gamma and delta) highly homologous sequences, even though the targeted sites of cleavage shared up to 18 out of 22 identical bases with other gene family members. The ribozyme-mediated cleavage reactions were Mg2+ dependent and enhanced at higher temperatures, although the J1 ribozyme retained considerable activity at physiological temperatures. Both J1 and J2 demonstrated a time-dependent cleavage of their targeted GS RNAs, although these two ribozymes differed markedly in their ability to cleave multiple substrate molecules. The rate of cleavage by J1 was found to be reduced in the presence of related GS RNAs and by total leaf poly(A) RNAs. The implications of these results for ribozyme activity in vivo are discussed.
在菜豆（Phaseolus vulgaris L.）中，谷氨酰胺合成酶（GS）由四个紧密相关的基因编码，分别称为gln-α、gln-β、gln-γ和gln-δ。我们构建并在体外表征了多种锤头状核酶，这些核酶旨在切割由这些基因编码的单个RNA。三种核酶，分别称为J1、J2和J3，分别靶向在γ和β亚基开放阅读框的起始位置以及γ亚基开放阅读框的中间位置切割RNA。尽管切割的靶向位点与其他基因家族成员在22个相同碱基中最多有18个相同，但所有三种核酶都成功地区分了四个（α、β、γ和δ）高度同源的序列。核酶介导的切割反应依赖于Mg2+，并且在较高温度下增强，尽管J1核酶在生理温度下仍保留相当的活性。J1和J2都表现出对其靶向的GS RNA的时间依赖性切割，尽管这两种核酶在切割多个底物分子的能力上有显著差异。发现在相关的GS RNA和总叶poly(A) RNA存在的情况下，J1的切割速率会降低。讨论了这些结果对体内核酶活性的影响。
