用T7RNA聚合酶体外转录合成大鼠肝tRNA~(Ile)

采用PCR技术从rec M1 3mp1 8中扩增出 1 2 0bp的大鼠肝tRNAIle合成基因片段 ,经限制性内切酶BstNⅠ酶切后作为模板 ,利用T7RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNAIle基因 ,生成不含修饰碱基的tRNAIle,并对体外转录反应条件进行了优化 ,回收的tRNA产量可达DNA模板量的 4 0倍     

大鼠肝tRNA^Ile基因的克隆与体外转录

采用ＰＣＲ扩增出大鼠肝ｔＲＮＡ＾Ｉｌｅ基因,构建了重组质粒ｐＧＷＩＷ,并用Ｔ７ＲＮＡ聚合酶／启动子系统对其进行了体外表达。经过对转录产物片段大小及运用Ｎｏｒｔｈｅｒｍｂｌｏｔ鉴定,证明了获得了不含修饰核苷酸的大鼠肝ｔＲＮＡ＾Ｉｌｅ。生物学活性检测显示：合成基因体外转录产物氨基酰经活性是天然ｔＲＮＡ的４０％,提示修饰核苷酸在哺乳动物Ｉｌｅ－ｔＲＮＡ合成酶的识别过程中起重要作用。     

tRNA转录后修饰的功能及其与人类疾病的关系

转移核糖核酸(tRNA)的转录后修饰对tRNA正常行使生物学功能具有重要意义,这些功能包括tRNA的正确折叠和维持其稳定性、在核糖体上正确解码。虽然tRNA转录后大部分核苷酸修饰形式在20世纪70年代已被鉴定出,但最近才在大肠杆菌及酵母中鉴定出催化这些tRNA核苷酸修饰的酶的绝大部分基因。这些修饰酶基因的鉴定为研究tRNA转录后修饰的生物功能开启了新的大门。人胞质tRNA和线粒体tRNA(mt tRNA)都存在大量核苷酸修饰,这些修饰的缺陷常常与多种人类疾病相关。因此,研究tRNA核苷酸修饰有助于我们了解相关疾病的发病机理。     

用T7 RNA聚合酶体外转录合成大鼠肝tRNAIle

采用PCR技术从rec-M13mp18中扩增出120 bp的大鼠肝tRNA^Ile合成基因片段,经限制性内切酶BstNⅠ酶切后作为模板,利用T7 RNA聚合酶在体外无细胞体系转录由T7启动子带动的大鼠肝tRNA^Ile基因,生成不含修饰碱基的tRNA^Ile,并对体外转录反应条件进行了优化,回收的tRNA产量可达DNA模板量的40倍.     

大鼠肝tRNA~(Ⅱe)基因在大肠杆菌中的表达及分离纯化

利用T7RNA聚合酶／启动子表达系统在大肠杆菌JM109（DE3）中表达化学合成的大鼠肝tRNAⅡe基因．经酚抽提、DEAE-52离子交换柱层析及HPLC层析,分离纯化大鼠肝tRNAⅡe．变性聚丙烯酰胺凝胶电泳和Northern-blot鉴定表明,大鼠肝tRNAⅡe基因成功地获得表达．氨酸化活性检测表明：纯化后,每1A260单位（40μg）的tRNAⅡe可负载约650pmol的Ⅱe,纯度为54．2％．说明从大肠杆菌中分离纯化出具有一定生物学活性,一定纯度的合成基因表达产物．     

真核生物细胞质tRNA生物合成研究的进展

tRNA是蛋白质翻译机器中的必需成分,它对细胞的生长和增殖及器官的发育起着决定性作用.tRNA生物合成包括tRNA基因的转录、转录后的加工和修饰.对tRNA生物合成的研究还包括tRNA在细胞中的运输、tRNA生物合成的质量监控及其与其他重要细胞途径（如mRNA生物合成、DNA损伤应答和细胞周期）之间的相互作用,以及tRNA生物合成在生长发育和疾病中的作用.本文主要介绍了近年来真核生物细胞质tRNA生物合成研究的一些重要进展.     

大鼠肝tRNA~(He)基因化学合成及克隆

tRNA在蛋白质生物合成中的主要作用是转运氨基酸,此外,tRNA与第二套密码的研究及与多胺的作用亦受到重视。传统的专一tRNA制备方法,不仅步骤繁多且收获量极少(6.5 mg/4000g肝组织),难于满足进一步用NMR,ESR等技术研究的需要。利用基因工程的手段,在细胞中表达专一的tRNA可以克服传统制备方法的不足。为此我们设计合成了大鼠肝tRNA~(Ile)基因。合成的基因全长120bp,将其中U,Ψ,D核苷酸换成T,I换成G。为了方     

大肠杆菌等受体tRNALeu的T7 RNA聚合酶转录

用化学方法合成编码2个大肠杆菌tRNALeu(tRNALeu1和tRNALeu2)的基因和T7启动子,分别克隆到pUC19载体上,并在纯化的T7 RNA聚合酶的体外转录系统中转录出不含修饰核苷酸的tRNALeu.在T7转录体系中,亚精胺对转录有负影响.在最适转录条件下,可以得到有活力的RNA转录物的量是模板DNA的250倍左右.在大肠杆菌亮氨酰-tRNA合成酶的催化下,2种经体外转录产生的未修饰等受体tRNALeu(tRNALeu1和tRNALeu2)的亮氨酸接受能力基本相同,但只有从体内纯化对应的tRNALeu的四分之一左右,表明修饰核苷酸在tRNALeu氨酰化过程中起着较为重要但非关键的作用.     

真核基因启动子非依赖的功能转录延伸复合物的体外组装

真核生物RNA聚合酶Ⅱ的持续合成能力对基因转录过程中每一个阶段,包括启动子脱离、转录暂停、转录终止以及转录偶联DNA损伤修复过程的调节至关重要.在RNA聚合酶Ⅱ介导的转录延伸过程中,其和模板DNA及转录产物RNA紧密结合,形成一个非常稳定的延伸三维复合物(elongationcomplex,EC).此特征性“泡”状结构的形成是RNA聚合酶Ⅱ持续合成能力所必需的.在不依赖启动子及众多转录起始因子的条件下,利用人工合成的RNA与DNA寡核苷酸,在体外组装形成具有功能转录活性的延伸复合物.结果表明,长度为9个核苷酸的RNA与模板DNA形成的杂合分子对转录延伸复合物的形成是必需的,而非转录模板DNA链的加入导致最终活性转录“泡”状复合物的形成,并可转录形成与模板相关的转录产物,进一步通过在模板DNA的特定位置引入一个乙酰氧乙酰氨基芴修饰基团,可特异性地阻断转录延伸过程,从而显示该系统在研究真核基因转录及转录偶联DNA损伤修复机制中的潜在应用价值.     

大肠杆菌等受体tRNALeu的T7 RNA聚合酶转录

用化学方法合成编码 2个大肠杆菌tRNA^Leu(tRNA^Leu₁和tRNA^Leu₂ )的基因和T7启动子 ,分别克隆到pUC1 9载体上 ,并在纯化的T7RNA聚合酶的体外转录系统中转录出不含修饰核苷酸的tRNA^Leu.在T7转录体系中 ,亚精胺对转录有负影响 .在最适转录条件下 ,可以得到有活力的RNA转录物的量是模板DNA的 2 5 0倍左右 .在大肠杆菌亮氨酰 tRNA合成酶的催化下 ,2种经体外转录产生的未修饰等受体tRNA^Leu(tRNA^Leu₁和tRNA^Leu₂ )的亮氨酸接受能力基本相同 ,但只有从体内纯化对应的tRNA^Leu的四分之一左右 ,表明修饰核苷酸在tRNA^Leu氨酰化过程中起着较为重要但非关键的作用 .     

Increase in fidelity of rat liver Ile-tRNA formation by both spermine and the aminoacyl-tRNA synthetase complex

To examine the polyamine effects on the fidelity at the aminoacylation level and the physiological significance of the existence of the aminoacyl-tRNA synthetase complex (ARSC) in animal cells, a single-chain Ile-tRNA synthetase (IRSS) was isolated from the complex by treatment with trypsin. Ile-tRNA formation by IRSS was strongly stimulated by spermine, similar to the results with ARSC. Two misacylations (Val-tRNAIle and Ile-tRNAiMet formation) by IRSS were measured. The error frequency was higher in Ile-tRNAiMet formation (tRNA misacylation) than in Val-tRNAIle formation (amino acid misacylation). Spermine did not influence significantly Ile-tRNAiMet formation, but it stimulated Val-tRNAIle formation by IRSS. Accordingly, spermine decreased the error frequency of tRNA misacylation, but not amino acid misacylation. These results suggest that the conformational changes of individual tRNA by spermine differ from each other, meaning that spermine influences the interaction between individual tRNA and aminoacyl-tRNA synthetase variously. When the aminoacylations of tRNAIle from rat liver, yeast, and Escherichia coli were compared with ARSC and IRSS, the relative speed of Ile-tRNA formation with tRNAIle from other species was faster with IRSS than with ARSC. This indicates that ARSC can recognize tRNAIle from the same species more specifically than IRSS. These results show that both spermine and ARSC are involved in the increase of fidelity of rat liver Ile-tRNA formation.     

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase.

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.     

肝再生增强因子的cDNA克隆、表达及表达产物的生物活性研究

以肝部分切除后再生肝组织为起始材料,利用RT-PCR扩增出大鼠肝再生增强因子（ALR）,亚克隆于pGEM-T载体,核苷酸序列测定证实为大鼠ALR;将ALRcDNA亚克隆于pBV220质粒,构建了原核表达栽体,并获高效表达菌株,特异表达蛋白占细菌总蛋白的15％,原核表达的ALR在体外缺乏促进大鼠原代培养肝细胞及SMMC-7721肝癌细胞DNA合成的活性,但在体内1／3肝部分切除模型中可刺激肝细胞DNA合成;ALR在生物学活性方面与肝脏刺激物（HSS）存在一定差别,ALR和HSS应是两种不同的活性因子．ALR还具有促肝损伤修复的作用,对其深入研究可能为临床治疗严重肝病提供有效的药物.     

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.     

Thiaisoleucine and protein synthesis.

Thiaisoleucine is an isoleucine analogue having the gamma-methylene group of the valerianic carbon chain substituted by a sulphur atom. It has been demonstrated that thiaisoleucine is activated and transferred to tRNAIle by rat liver aminoacyl-tRNA synthetase and inhibits isoleucine incorporation into polypeptides in protein synthesizing systems from rat liver or rabbit reticulocytes, whereas it does not affect either leucine incorporation or ribosome run-off or polypeptide chain elongation rate. All tests were performed in comparison with O-methyl-threonine, an isoleucine analogue with the gamma-methylene group substituted by an oxygen atom. In all the reactions studied, both thiaisoleucine and O-methyl-threonine act as competitive inhibitors of isoleucine. With respect to O-methyl-threonine, thiaisoleucine shows higher activity as an isoleucine inhibitor.     

Cloning and amplified expression of the tyrosyl-tRNA synthetase genes of Bacillus stearothermophilus and Escherichia coli

Two fragments of DNA which carry the genes coding for the tyrosyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus have been cloned into the plasmid pBR322 and were selected by complementation of an E. coli temperature-sensitive mutant. Transformation of this strain with either of the recombinant plasmids results in a 100-fold increase in tyrosyl-tRNA synthetase activity measured in vitro and the protein products co-migrate with the corresponding purified enzymes on polyacrylamide gels.     

红细胞生成素基因在大鼠体内的表达及表达产物的生物学效应

红细胞生成素（ｅｒｙｔｈｒｏｐｏｉｅｔｉｎ,ＥＰＯ）是一种由胎儿肝脏和成人肾脏产生的多肽类生长因子,在体内的表达具有严格的组织特异性,因此,慢性肾病所引起的贫血常常难以得到有效的治疗．随着基因治疗技术的不断成熟与完善,尤其是近年一些实验室先后发现质粒...