一个新的水稻C2H2型锌指蛋白cDNA的克隆与序列分析

锌指蛋白 (ｚｉｎｃｆｉｎｇｅｒｐｒｏｔｅｉｎ)是一类具有“手指状”结构域的转录因子 ,负责调控基因的表达。锌指蛋白主要通过与核酸的相互作用 ,显示出不同的功能 ,如促进转录、抑制转录、单链ＤＮＡ结合、ＲＮＡ结合或ＲＮＡ/ＤＮＡ双向结合[1 ] 等。Ｃｙｓ2 /Ｈｉｓ2型锌指 (也称ＴＦⅢＡ型 )是真核生物的转录因子中结构描述的最为清楚的转录因子 ,其锌指区为ＣＸ2 - 4 ＣＸ3ＦＸ5ＬＸ2 ＨＸ3- 5Ｈ的结构[2 ] 。植物中目前已经克隆了一些Ｃ2Ｈ2型锌指蛋白基因 ,其中很多锌指区具有ＱＡＬＧＧＨ的保守区 ,如与拟南芥花发育相关的ＳＵ …     

堆型艾美耳球虫微线蛋白3及其结构域蛋白的免疫保护作用

[目的]本文旨在研究堆型艾美耳球虫微线蛋白3(EaMIC3)及其结构域蛋白的免疫保护作用。[方法]经原核表达和纯化获得重组蛋白EaMIC3、EaMAR3和EaMAR6,制备3种蛋白的抗血清并测其效价;分离纯化堆型艾美耳球虫卵囊。在腿部肌肉注射重组蛋白和翅静脉注射多克隆抗体血清免疫14日龄雏鸡,间隔7 d进行二免,再7 d后攻毒,评估3种蛋白抵抗堆型艾美耳球虫感染的免疫保护作用。[结果]重组蛋白免疫组和抗重组蛋白血清免疫组中,EaMIC3和EaMAR3的抗球虫指数分别为178、165和180、166,而EaMAR6的抗球虫指数分别为130、159。2种免疫方式与对照组相比,EaMIC3和EaMAR3均显示明显的免疫保护效果,且EaMIC3的免疫保护效果最好,而EaMAR6的免疫保护效果较差。[结论]结构域蛋白的免疫保护作用与其和肠上皮细胞的结合能力一致,也证明结构域蛋白在球虫入侵过程中的关键作用。     

云南烟草丛枝症病害研究 Ⅷ 验证与烟草丛枝症病害相关的稳定RNA

来自云南省２２个县市的烟草丛枝症标本均检测到与云南烟草丛枝症病害相关的稳定ＲＮＡ,大小相当于８００ｂｐ左右的ＤＮＡ;病株的根、茎、叶、花和枯叶中含有此ＲＮＡ,种子中未检测到此ＲＮＡ;此ＲＮＡ的最大吸收峰为２６９ｎｍ;能被蛇毒磷酸二酯酶Ｉ消化,不具有类病毒结构特征,推测可能是病毒的基因组分;支持莫笑晗等对此ＲＮＡ的结构推测,即可能是有广泛内部配对、具有类似发叉结构的线性长度为１６１ｋｂ的单链ＲＮＡ．与云南烟草丛枝症病害相关的低分子量ＲＮＡ目前发现的超稳定ＲＮＡ．     

免疫核糖核酸治疗羔羊痢疾的研究：II.i—RNA特异性小鼠体内保护试验

用Ｂ型魏氏梭菌免疫羊，从免疫羊肝，脾，淋巴结中胺Ｆａｉｎｂｏｉｎ等的酚－氯仿法稍加改进提取免疫核糖核酸（ｉ－ＲＮＡ）用同样的方法从正常羊的肝，脾，淋巴结中提取ＲＮＡ（ｎ－ＲＮＡ）。用ｉ－ＲＮＡ，ｎ－ＲＮＡ分别给小鼠腹腔注射，每天１ｍｇ／只，连续３ｄ，对照组用生理盐不代替，第４ｄ腹腔注射０．０１ｍｌ／只Ｂ型魏氏梭菌培养液，观察３周，结果表明：ｉ－ＲＮＡ对免疫力低下的小鼠有特异性的保护作用，保护率为６     

细菌四类胞外感觉结构域的概述

细菌通过信号转导网络系统感知外界的环境变化，并通过调节自身基因表达，控制菌体的行为、代谢以及发育等。细菌和古细菌大多数使用双组分转导系统和化学传感系统感知环境变化。基于基因组研究发现在细菌的双组份转导系统和化学传感系统中存在多种不同的胞外结构域，这些胞外结构域能感知胞外信号，对细菌的各种生理功能具有重要作用。介绍了Cache结构域、CHASE结构域、NIT结构域以及4HB结构域这4类胞外感觉结构域的结构特性以及功能，为胞外感觉结构域的分类以及识别信号的位点提供借鉴。     

苏云金芽孢杆菌杀虫晶体蛋白结构和功能研究进展

对目前解析的苏云金芽孢杆菌杀虫晶体蛋白的三维结构进行了比较分析。它们都有类似的结构,有三个结构域,结构域Ⅰ是由7个α螺旋以α5螺旋为中心形成的α螺旋束;结构域Ⅱ是由三个反平行β折叠形成的β棱柱;结构域Ⅲ是一个β"三明治"结构。对这三个结构域的Loop及氨基酸残基与杀虫活性的关系、与通道形成相关的杀虫作用机制的研究进展进行了综述。     

核酶在我国植物抗病毒领域的研究和应用现状

简要介绍了锤头型和发夹型核酶的结构、功能及催化机制,并对我国利用核酶抗植物病毒病害方面的研究和应用现状作一概述。     

长顺绿壳蛋鸡ITPR1基因RYDR-ITPR 结构域的多态性对蛋壳品质的影响

为了探究贵州特产长顺绿壳蛋鸡ITPR1基因RYDR-ITPR结构域的多态性对蛋壳品质的影响,采用在线引物设计软件Primer Premier 3.0设计1对引物,扩增ITPR1基因RYDR-ITPR结构域,利用双向直接测序和序列比对法挖掘单核苷酸多态性(SNP)位点,应用SPSS 18.0软件分析SNP位点对长顺绿壳蛋鸡蛋壳品质的影响。结果显示,在ITPR1基因第17～19内含子区共发现3个SNP位点,分别为位于18内含子上的g.18853584 GT、g.18853600 AG突变位点和19外显子上的g.18853848 CT突变位点;3个SNP位点均为中度多态,均未偏离Hardy-Weinberg平衡(P0.05),共检测到4种单倍型和7种双倍型。关联分析结果显示,位于19外显子上的g.18853848 CT突变对蛋壳厚度有显著影响,TT、CC基因型显著高于TC基因型(P0.05);双倍型H3H3的蛋壳厚度和蛋壳强度测定值最高。综上,长顺绿壳蛋鸡ITPR1基因RYDR-ITPR结构域中g.18853848 CT突变可能是影响蛋壳厚度的标记位点,双倍型H3H3可能是蛋壳厚度和蛋壳强度的有利双倍型。     

DNA催化活性的研究 Ⅲ.多聚核苷酸的催化作用

以多聚腺苷酸（ｐｏｌｙＡ），多聚尿苷酸（ｐｏｌｙＵ）及其混合复性的ｐｏｌｙＡ·ｐｏｌｙＵ与乙酸－α－萘酯及坚牢蓝ＲＲ盐混合保温，研究了多聚核苷酸催化乙酸－α－萘酯水解的作用，结果表明：由ｐｏｌｙＡ·ｐｏｌｙＵ组成的双链ＲＮＡ具有与ＤＮＡ分子相似的催化活性，ｐｏｌｙＡ这一单链多聚物也能起催化作用，ｐｏｌｙＵ则没有这一功能。据此认为，核酸分子的催化活性与核糖２＇－位脱氧与否无关，而碱基的堆积为素酯分子的疏水奈环基团提供足够的疏水空间，则是催化活性所必须的。双链ＤＮＡ及双链ＲＮＡ都能满足这一条件，ｐｏｌｙＡ中嘌呤碱基堆积则刚能容纳下萘环，所以均能催化萘酯的水解；而ｐｏｌｙＵ中嘧啶碱基的堆积不足以容纳下萘环，因而不具备这一催化作用。     

Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes

The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.     

Ribozyme-catalyzed transcription of an active ribozyme

A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of replicating a primordial RNA "genome." Here we describe the evolution and engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nucleotides in length. To overcome its sequence dependence, we recombined traits evolved separately in different ribozyme lineages. This yielded a more general polymerase ribozyme that was able to synthesize a wider spectrum of RNA sequences, as we demonstrate by the accurate synthesis of an enzymatically active RNA, a hammerhead endonuclease ribozyme. This recapitulates a central aspect of an RNA-based genetic system: the RNA-catalyzed synthesis of an active ribozyme from an RNA template.     

Capping by branching: a new ribozyme makes tiny lariats

The number of naturally occurring RNA enzymes has just been expanded by the discovery of a new branching ribozyme. But this ribozyme has unexpected relatives: group I introns.     

Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting

The intervening sequence of the ribosomal RNA precursor of Tetrahymena is a catalytic RNA molecule, or ribozyme. Acting as a sequence-specific endoribonuclease, it cleaves single-stranded RNA substrates with concomitant addition of guanosine. The chemistry of the reaction has now been studied by introduction of a single phosphorothioate in the substrate RNA at the cleavage site. Kinetic studies show no significant effect of this substitution on kcat (rate constant) or Km (Michaelis constant), providing evidence that some step other than the chemical step is rate-limiting. Product analysis reveals that the reaction proceeds with inversion of configuration at phosphorus, consistent with an in-line, SN2 (P) mechanism. Thus, the ribozyme reaction is in the same mechanistic category as the individual displacement reactions catalyzed by protein nucleotidyltransferases, phosphotransferases, and nucleases.     

A single-molecule study of RNA catalysis and folding

Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.     

Structural basis of glmS ribozyme activation by glucosamine-6-phosphate

The glmS ribozyme is the only natural catalytic RNA known to require a small-molecule activator for catalysis. This catalytic RNA functions as a riboswitch, with activator-dependent RNA cleavage regulating glmS messenger RNA expression. We report crystal structures of the glmS ribozyme in precleavage states that are unliganded or bound to the competitive inhibitor glucose-6-phosphate and in the postcleavage state. All structures superimpose closely, revealing a remarkably rigid RNA that contains a preformed active and coenzyme-binding site. Unlike other riboswitches, the glmS ribozyme binds its activator in an open, solvent-accessible pocket. Our structures suggest that the amine group of the glmS ribozyme-bound coenzyme performs general acid-base and electrostatic catalysis.     

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.