Perriman R, Graf L, Gerlach W L
CSIRO Division of Plant Industry, Canberra, Australia.
Antisense Res Dev. 1993 Fall;3(3):253-63. doi: 10.1089/ard.1993.3.253.
Site-directed mutagenesis has been used to produce two hammerhead ribozyme molecules targeting the chloramphenicol acetyltransferase gene (CAT). One ribozyme has a single catalytic domain between two 12-nucleotide arms that can hybridize 5' and 3' of the GUC target site of the CAT RNA transcript. The second ribozyme is a full-length antisense RNA with four catalytic domains inserted along the length, each targeting a specific GUC site within the CAT mRNA. Our results show that both ribozymes can produce almost equivalent rates of cleavage of the CAT mRNA in vitro (T1/2 of 18 or 15 min, respectively). In tobacco protoplasts we show consistently greater gene suppression in the presence of the long ribozyme molecule, compared with the equivalent antisense (22% gene reduction for antisense compared with 44% with the long ribozyme). These results suggest that hammerhead ribozymes may be developed for the inactivation of gene activity in plant cells.
定点诱变已被用于产生两个靶向氯霉素乙酰转移酶基因（CAT）的锤头状核酶分子。一种核酶在两个12个核苷酸的臂之间有一个单一的催化结构域，这两个臂可以与CAT RNA转录本的GUC靶位点的5'和3'端杂交。第二种核酶是一种全长反义RNA，沿长度方向插入了四个催化结构域，每个催化结构域靶向CAT mRNA内的一个特定GUC位点。我们的结果表明，两种核酶在体外都能产生几乎相同的CAT mRNA切割速率（T1/2分别为18分钟或15分钟）。在烟草原生质体中，我们发现，与等效的反义RNA相比，长核酶分子的存在始终能产生更强的基因抑制作用（反义RNA使基因减少22%，而长核酶使基因减少44%）。这些结果表明，锤头状核酶可能被开发用于使植物细胞中的基因活性失活。
