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首页> 外文期刊>Genetic analysis. Techniques and applications >DETECTION OF HAMMERHEAD RIBOZYME-MEDIATED CLEAVAGE AND REDUCED EXPRESSION OF LACZ' MRNA IN E-COLI
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DETECTION OF HAMMERHEAD RIBOZYME-MEDIATED CLEAVAGE AND REDUCED EXPRESSION OF LACZ' MRNA IN E-COLI

机译：锤头核酶介导的卵裂的检测和大肠杆菌中LACZ'mRNA的表达降低

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Hammerhead ribozymes have been shown to specifically suppress the expression of target genes in various cells, but their in vivo cleavage products have seldom been directly detected. A hammerhead ribozyme sequence was designed to cleave the phosphodiester bond just 3' to the GUC of SalI site of M13mp18. The ribozyme was inserted some base pairs upstream of the target region without disrupting the reading frame of the lacZ' gene and without introducing any translational stop codons. More than 90% RNAs synthesized in vitro were cleaved at the expected site after 1-h incubation in the presence of 10 mM magnesium ion at 37 and 50 degrees C. Inclusion of the designed ribozyme. sequence was also shown to suppress the expression of the fused lacZ' gene in E. coli cells. When the cells were infected by the ribozyme-containing phage, they remained colourless in the presence of X-gal, and the cellular beta-galactosidase activity was reduced by more than 90%. Insertion of the same ribozyme sequence in reverse orientation showed little effect on beta-galactosidase activity. Furthermore, a primer extension by reverse transcriptase revealed a cleavage product that resulted from cleavage of LacZ' mRNA at the targeted site as designed. Thus, our data demonstrated that the designed hammerhead ribozyme cleaves and reduces the expression of a fused LacZ' mRNA in E. coli cells.
机译：锤头状核酶已显示出特异性抑制靶基因在各种细胞中的表达，但很少能直接检测到它们的体内裂解产物。锤头状核酶序列被设计成仅在M13mp18的SalI位点的GUC上切割磷酸二酯键。将核酶插入靶区域上游的一些碱基对，而不破坏lacZ'基因的阅读框，并且不引入任何翻译终止密码子。在10 mM镁离子存在下于37和50摄氏度孵育1小时后，在预期的位点切割了超过90％的体外合成的RNA。包括了设计的核酶。还显示该序列可抑制融合的lacZ′基因在大肠杆菌细胞中的表达。当细胞被含核酶的噬菌体感染时，它们在X-gal存在下仍然是无色的，并且细胞中的β-半乳糖苷酶活性降低了90％以上。反向插入相同的核酶序列对β-半乳糖苷酶的活性影响很小。此外，通过逆转录酶进行的引物延伸揭示了裂解产物，该裂解产物是由如设计的在靶位点处的LacZ'mRNA的裂解所致。因此，我们的数据表明，设计的锤头状核酶裂解并减少了大肠杆菌细胞中融合的LacZ'mRNA的表达。

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