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现有知识所知道的锤头状Ribozyme结构中,剪切是在特殊位点的3′侧发生,我们将含GTΨ的酵母丙氨酸tRNA 3′半分子(40聚)作为底物,按Haseloff-Gerlach锤头结构模型设计了一个38聚的Ribozyme分子。在镁离子存在的条件下,GTΨ的3′侧发生预期的体外剪切反应。反应的米氏动力学参数为:K_m:6.7μmol/L,k_(cat):3.4×10~(-3)/min。与此同时,合成了一个17聚的底物类似物,仅以GUU代替上述的GTΨ序列。恒态动力学分析表明,两底物的K_m值大致相同,但k_(cat)值相差达294倍。实验表明,修饰核苷酸Ψ可以是Ribozyme锤头结构中的剪切位点,但具有很低的k_(cat)。 相似文献
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本文报告用T_4RNA连接酶将相应于酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))3′-半分子(36—76)的三个寡核昔酸大片段——10(36—45)(Ⅰ),12(46—57)(Ⅱ)和19p(58—76)(Ⅲ)——从3′-端向5′-端延伸逐个连接合成了这个tRNA的3′-半分子(36—76)。首先在供受体配比为1:1.5的情况下,采取三步连续反应,即19p(Ⅲ)的5′-磷酸化,然后与12(Ⅱ)的连接和连接反应产物的5′-磷酸化等反应,一次制备分离的方法,以70%的总得率合成了5′-磷酸化的三十一核苷酸(46—76)(Ⅳ)。然后,以(Ⅳ)作为下一步反应的供体和三倍量的10(Ⅰ)连接,以67%的产率合成了具有四十一核苷酸的tRNA_y~(Ala)的3′-半分子(36—76)(Ⅴ)。将这个合成的3′-半分子,5′-磷酸化以后,与天然的5′-半分子连接,人工半合成了tRNA_y~(Ala)整分子,经生物活力测定,这个人工半合成的tRNA_y~(Ala)具有接受[~3H]-丙氨酸、并能将接受的丙氨酸转移到蛋白质分子中去的生物活力。 相似文献
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本试验结果表明:1.2′-5′三腺苷酸(2′-5′P_3A_3)有激活多种来源(包括人)巨噬细胞的吞噬作用,说明2′-5′P_3A_3对巨噬细胞的激活作用具有普遍意义。2.甲胎蛋白(AFP)能抑制巨噬细胞的吞噬功能,可能对肝癌的发生有一定的作用。本工作证实2′-5′P_3A_3能抵消AFP对巨噬细胞的抑制作用。3.关于2′-5′P_3A_3生物功能的研究,过去都要用CaCl_2等方法将它引入细胞,这对临床应用是不实际的。本文不使用CaC国_2等方法,2′-5′P_3A_3即能激活巨噬细胞。 相似文献
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_(ppp)A_(2'p),'A_(2'p,)'A(2'-5'P_3A_3) activates macrophages and increases the phagocytosis of macrophages from different species including human beings. This indicates that the activation of macrophages may be a general action of 2'-5'P_3A_3. This discovery broadens the effect of 2'-5'P_3A_3 beyond the antiviral field.α-Feto-protein (AFP) inhibits the phagocytosis of macrophages and may be involved in the development of hepatoma. Data presented here show that 2'-5'P_3A_3 can antagonize this suppressive effect of AFP.Methods used so far for introducing 2'-5'P_3A_3 into the cells were made with the aid of CaCl_2, etc. under conditions which may not be the same as those used clinically. It was found that 2'-5'P_3A_3 can develop its biological effect without the aid of CaCl_2, etc. 相似文献
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反义核酸对小鼠腹水瘤病毒(反转录病毒)的抑制作用 总被引:2,自引:0,他引:2
本文研究了反义核酸(Antisense Nucleic Acids)对小鼠腹水瘤病毒(SRSV),M-MuLV反转录病毒之一种的抑制作用.设计和合成了不同序列,长度和核苷酸间磷酸根有无修饰等几种脱氧寡核苷酸片段,对经SRSV感染后的宿主细胞(NIH 3T3)的生长抑制和对无病感染的3T3正常细胞的毒性作用进行了试验.发现互补于病毒RNA基因5’端引物tRNA区序列的片段和均聚体脱氧胞嘧啶寡核苷酸片段(Homooligo-dC14)效果很好,病毒感染后宿主细胞生长抑制达到90%,并且在动物身上进行毒性试验,结果表明无毒性作用.对均聚体脱氧胞嘧啶寡核苷酸片段作了感染细胞内的反转录酶活力和细胞融合试验等,结果均与对照组有明显差别;并对它的作用机制进行了讨论. 提出上述效果很好的两种反义核酸片段是两种具有很大潜力的抑制SRSV反转录病毒的化合物. 相似文献
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The role of base modification in yeast tRNAAl(?) function in protein synthesis was examined by the use of unmodified tRNA analogues. Unmodified full length tRNAs, 5'-half tRNAs (nucleotides 1-35) and 3'-half tRNAs (nucleotides 37-75) were transcribed in vitro using T7-RNA polymerase. Unmodified tRNA half molecules were joined to normally modified half molecules (5'-half, nucleotides 1-36; 3'-half, nucleotides 36-75) by T4-RNA ligase. Using this method, we synthesized three analogues of yeast tRNAAl(?): (i) tRNAAl(?) which lacks base modifications in the 5'-half of the molecule; (ii) tRNAAl(?) which lacks base modifications in the 3'-half of the molecule; and (iii) tRNAAla completely lacking base modifications. We determined the biological activities of these analogues. In rat aminoacyl-tRNA synthetase reactions, the alanine acceptance activity decreased by 52%, 79% and 85% when modified bases were absent from the 5'-half molecule, the 3'-half molecule or the total molecule, respectively. In rabbit retic 相似文献
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A thorough study on the reaction of 32_pN_p with 14 dinucleoside monophosphates including U_pI and C_p m~1I has been reported. The 10μl reaction mixture containing 100mM Tris (pH8.3), 40mMMgCl_2, 6.6mM DIT, bovine serum albumin (20μg/ml), 1mM ATP, 0.25mM dinucleoside monophos-phate, 0.05mM 32_pN_p and T_4 RNA ligase was incubated for 24 hr at 5--10℃ (or 2 hr at 37℃).The joining yields depend on the reaction temperature, the donor species and the base compositionof acceptors,_pC_p is the best donor. At 5--10℃, it can join in all 14 dinucleoside monophosphateswith yields ranging from 20% to 80%. The next one is _pA_p, but the yields are comparatively low.Both _pG_p and p_U_p are poor donors, which react with some dinucleoside monophosphates to give ayield of only a few percent, and sometimes no product could be obtained whatsoever. 相似文献