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机构地区:[1]中山医科大学生化教研室,广东广州510089
出 处:《中山医科大学学报》2000年第6期409-412,共4页Academic Journal of Sun Yat-sen University of Medical Sciences
基 金:国家自然科学基金资助项目(39670715)
摘 要:【目的】对Hela细胞端粒酶进行活性重组并探讨其重组特异性。【方法】以RT PCR法从纯化的人端粒酶复合体中扩增人类端粒酶RNA(hTR)基因片段 ,将其克隆至含有SP6启动子的 pGEM 3f+质粒中 ,构建hTR的体外转录体系以获得体外合成的hTR。与端粒酶蛋白组分进行活性重组。四膜虫端粒酶RNA及 16SrRNA作为重组反应对照组 ,以探测其重组特异性。【结果】经RT PCR扩增出 45 0bp片段 ;体外转录重组载体经酶切图谱和PCR扩增鉴定构建成功 ;体外转录的RNAs经聚丙烯酰胺凝胶电泳进行了鉴定 ;活性重组实验中 ,对照组的MNase处理但未作重组管为阴性 ;3组重组管中 ,经MNase处理后的端粒酶蛋白特异性地与经体外转录所得的人端粒酶RNA组分重组而重获端粒酶活性 ,与四膜虫端粒酶RNA及 16SRNA重组均不能获得酶活性 ,说明端粒酶复合体有自身不可替代的组成成分 ,而且不同生物体的端粒酶有各自特异的酶蛋白组分和RNA组分。【结论】Hela细胞端粒酶蛋白组分能特异地与体外合成的hTR重组端粒酶活性。Objective To observe reconstitution of human telomerase activity and its specificity to hTR. Method RT-PCR was used to amplify hTR from the enzyme complex, the hTR fragment was cloned into pGEM3f + and constructed phTR450, which involved SP6 promoter and transcribed in vitro RNA. RNA transcribed was used in reconstitution of telomerase. The hTR and Tetraehymena RNAs were respectively transcribed in vitro.The purified enzyme complex was treated with MNase to obtain protein components of the enzyme. The hTR and control RNA were used to reconstitute active telomerase in combination with MNase-treated purified prorein. The TRAP was used to detect telomerase activity. Result The specific hTR fragment (450 bp) was amplified by RT-PCR. The PCR product was cloned. Telomerase RNAs were transcribed in vitro. The active telomerase was reconstituted specific to hTR transcribed. Conclusion Human telomerase activity can be reconstituted using protein componats of Hela cell telomerase and synthetic RNA and the reconstitution is specific to hTR.
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