Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp)

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.     

Nucleotides in yeast tRNAPhe required for the specific recognition by its cognate synthetase

An analysis of the aminoacylation kinetics of unmodified yeast tRNAPhe mutants revealed that five single-stranded nucleotides are important for its recognition by yeast phenylalanyl-tRNA synthetase, provided they were positioned correctly in a properly folded tRNA structure. When four other tRNAs were changed to have these five nucleotides, they became near-normal substrates for the enzyme.     

Accuracy of in vivo aminoacylation requires proper balance of tRNA and aminoacyl-tRNA synthetase

The fidelity of protein biosynthesis in any cell rests on the accuracy of aminoacylation of tRNA. The exquisite specificity of this reaction is critically dependent on the correct recognition of tRNA by aminoacyl-tRNA synthetases. It is shown here that the relative concentrations of a tRNA and its cognate aminoacyl-tRNA synthetase are normally well balanced and crucial for maintenance of accurate aminoacylation. When Escherichia coli Gln-tRNA synthetase is overproduced in vivo, it incorrectly acylates the supF amber suppressor tRNA(Tyr) with Gln. This effect is abolished when the intracellular concentration of the cognate tRNA(Gln2) is also elevate. These data indicate that the presence of aminoacyl-tRNA synthetase and the cognate tRNAs in complexed form, which requires the proper balance of the two macromolecules, is critical in maintaining the fidelity of protein biosynthesis. Thus, limits exist on the relative levels of tRNAs and aminoacyl-tRNA synthetases within a cell.     

口服重组酵母(ApoB100肽段)对小鼠产生特异性抗体的诱导作用

【目的】开发一种针对低密度脂蛋白成分ApoB100的新型重组酵母疫苗,为动脉粥样硬化(Atherosclerosis,AS)的防治提供一种思路。【方法】利用PCR得到包含编码ApoB100的3 136-3 155位氨基酸残基的目的片段,将其克隆到酵母表达载体JMB88-HA-OVA-MCS上,构建JMB88-HA-OVA-hApoB载体,用硫酸铜诱导表达后获得HA-OVA-hApoB融合蛋白。采用口服的方法对昆明小白鼠免疫成功表达HA-OVA-hApoB蛋白的酵母细胞12周,每周1次,每次1×108个酵母细胞,免疫结束后4周采血,用Western blot检测小鼠是否产生特异性抗体。【结果】PCR扩增获得了859bp的人ApoB100基因。成功构建了JMB88-HA-OVA-hApoB载体。口服免疫12周后,HA-OVA-hApoB免疫组小鼠的血清中含有特异性ApoB100抗体。【结论】灌服表达人ApoB100肽段融合蛋白的重组酿酒酵母能诱导小鼠的免疫反应,使其产生特异性抗体。     

Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution

The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is     

南方鲇Hsc70 cDNA的克隆及序列分析

采用RT-PCR法和SMART RACE（rapid amplification of cDNA ends）技术从南方鲇肝脏RNA克隆获得Hsc70基因的全长cDNA（scHsc70 cDNA）.此cDNA全长2384 bp,其中,5＇＇非编码区194 bp,3＇＇非编码区249 bp,编码区域（coding sequence,CDS）长度为1941 bp.根据编码序列推导出相应的646个氨基酸,与其他脊椎动物Hsc70序列进行同源性比较,发现南方鲇Hsc70与欧洲银鲫（Carassius auratus gibelio）、鲦鱼（Pimephales promelas）、人（Homo sapien）和鸡（Gallus gallus）的Hsc70相似性分别为96.0%、95.5%、94.9%和93.8%,表现出较高的保守性.序列分析发现scHsc70 cDNA编码的氨基酸序列中含有2个HSP70家族的特征模体并具有Dnak特征性基序（DLGTY-S-V）.南方鲇Hsc70基因的克隆为进一步深入研究南方鲇抗逆机理以及指导南方鲇的遗传选育和遗传改良都具有重要的理论意义.     

蝎蝽次目线粒体基因组22个tRNA基因的比较研究(半翅目:异翅亚目)

[目的]本研究旨在对蝎蝽次目11科线粒体基因组中22个tRNA基因的序列特征、遗传距离、同源基因相同核苷酸百分比和二级结构进行比较研究,同时基于22个tRNA序列构建蝎蝽次目的系统发育树。[方法]通过GenBank获得蝎蝽次目所有12个代表种的tRNA序列,在MEGA 6.0中计算tRNA基因序列特征和遗传距离,同源基因相同核苷酸百分比在BioEdit 7.1中进行,tRNA二级结构在RNA structure 5.8中构建。在RAxML 8.2.8和Mrbayes 3.2.5中构建系统发育树。[结果]结果显示蝎蝽次目11科核苷酸碱基组成(A+T)%平均含量为76.41%,碱基A和T的含量均高于G和C,其中多数种呈现A高于T,所有种呈现G高于C。蝎蝽次目11科间的遗传距离在0.13～0.26之间。核苷酸保守性较高的tRNA-L2、tRNA-T和tRNA-M基因均位于线粒体基因组的J链,核苷酸保守性较低的tRNA-V和tRNA-F基因均位于线粒体基因组的N链。蝎蝽次目11科内tRNA二级结构中氨基酸接受茎表现为较高的保守性,而环中除了反密码子环相对保守外,其余环都有不同程度的碱基变异。系统发育结果显示固蝽总科、仰泳蝽总科和划蝽总科均为单系,其中固蝽总科和仰泳蝽总科为姐妹群关系,其余总科的单系性没有得到很好的解析,可能与tRNA基因序列能够提供的有效系统发育信号较少有关。[结论]本研究补充了tRNA在蝎蝽次目研究中的不足,揭示了tRNA基因及其二级结构在比较线粒体基因组学研究中的重要性。     

Morphiceptin (NH4-tyr-pro-phe-pro-COHN2): a potent and specific agonist for morphine (mu) receptors

The synthetic peptide NH2-Tyr-Pro-Phe-Pro-CONH2 (morphiceptin), which is the amide of a fragment of the milk protein beta-casein, has morphinelike activities and is highly specific for morphine (mu) receptors but not for enkephalin (delta) receptors. It is as active as morphine in the guinea pig ileum but much less active in the mouse and rat vas deferens. The discovery of this specific morphine receptor ligand substantiates the hypothesis of multiple opiate receptors. The ligand, which may be of physiological significance since a very similar, or identical, activity can be detected in enzymatic digests of beta-casein, may prove useful for further investigation of the functions of opiate receptor subtypes.     

药用植物猫人参有效成分研究(Ⅰ)——无机元素分析

对药用植物猫人参(大籽猕猴桃Actinidia macrosperma)中的无机元素应用电感耦合等离子体原子发射光谱法进行了半定量分析测定,结果表明,该植物中富含人体所需的各种无机元素(尤其是Ca、Mg、Mn、Fe等的含量), 具有较高的开发利用价值.     

黄鳝肌肉矿物元素的分析

本文对黄鳝的幼鳝(体重62.3-76.0g)、小成鳝(体重108.3-121.4g)和大成鳝(体重222.8-242.8g)肌肉中的矿物元素的组成进行了分析.结果表明:黄鳝肌肉中含有20种以上的矿物元素.具有重要生理功能的硒等微量元素含量较丰富.含量为0.038mg/100g.本研究在分析与比较的基础上评价了黄鳝矿物元素的营养价值.     

Assembly mechanism of the contractile ring for cytokinesis by fission yeast

Animals and fungi assemble a contractile ring of actin filaments and the motor protein myosin to separate into individual daughter cells during cytokinesis. We used fluorescence microscopy of live fission yeast cells to observe that membrane-bound nodes containing myosin were broadly distributed around the cell equator and assembled into a contractile ring through stochastic motions, after a meshwork of dynamic actin filaments appeared. Analysis of node motions and numerical simulations supported a mechanism whereby transient connections are established when myosins in one node capture and exert force on actin filaments growing from other nodes.     

响应面法优化酵母培养物的制备工艺

以秸秆、淀粉、豆粕、酒糟等农副产品为基质,制备酵母培养物微生态饲料,研究酵母培养物的最佳制备工艺条件.采用响应面Box-Benhken中心组合设计法,考察反应体系中接种量、含水量、温度和时间4个因素及其相互作用对酵母培养物中粗蛋白含量的影响.获得优化后的酵母培养物最佳制备条件为:接种量0.11％,含水量48.0％,培养温度28.6℃,培养时间50.3 h.经过发酵酵母培养物的粗蛋白含量可达32.91％.     

假松口蘑的SCAR特异性分子标记

由于假松口蘑与松口蘑外观与口感相似,常被混入松口蘑中销售,严重影响松口蘑品质。本研究基于SRAP分子标记从300对引物组合中筛选出1对假松口蘑具有特异性的引物,并将这对引物的扩增产物进行克隆、测序以及重新设计引物,成功构建了假松口蘑的SCAR特异性分子标记(502 bp)。通过进一步验证,该SCAR标记只在假松口蘑个体中出现,表明所建立的SCAR标记可在分子水平上快速、准确地鉴定出假松口蘑。     

Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP

Polyadenylate [poly(A)] polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs). The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP). Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site. The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides. The first two domains are functionally analogous to polymerase palm and fingers domains. The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer. In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1. One occupies the position of the incoming base, prior to its addition to the mRNA chain. The other is believed to occupy the position of the 3' end of the mRNA primer.     

特异性检测植物乳杆菌的多重PCR方法

【目的】建立特异性检测植物乳杆菌的方法,排除近缘菌的干扰。【方法】利用SMM系统筛选植物乳杆菌种特异序列,根据特异序列设计引物进行不同乳制品的多重PCR检测。【结果】设计的7对引物具有良好的种特异性,其中3对引物可以排除戊糖乳杆菌和副植物乳杆菌的干扰,可扩增获得植物乳杆菌的特异条带101bp（引物LP-1和LP-2）、189bp（引物LP-5和LP-6）、378bp（引物LP-9和LP-10）。该方法的检测限为2.2×10CFU/mL。采用多重PCR检测与琼脂糖凝胶电泳图谱分析对自制含有各种乳酸菌酸奶产品、自然发酵产品及市售酸乳产品中植物乳杆菌进行定性检测,可以特异性检测出植物乳杆菌,准确区分植物乳杆菌的近缘菌株。【结论】该多重PCR方法成本低、步骤简单、耗时短、灵敏度高,具有特异性,可作为快速检测植物乳杆菌种的方法在生产中使用。     

弧菌基因组中整合性接合元件 （ICEs）的预测分析

整合性接合元件(ICEs)是一种广泛存在于细菌中可以自主移动的遗传元件。ICEs通过水平移动整合到弧菌的染色体上,所携带的新基因有助于宿主在特定生存环境下获得生长优势。为考察ICEs在弧菌生长中发挥的作用,采用Prf C位点与核心基因相关的特征蛋白,进行了26株弧菌SXT/R391ICEs的预测。结果表明,在全测序的26株弧菌中,只有3株霍乱弧菌的较大染色体上存在SXT/R391ICEs,对这3株霍乱弧菌上SXT/R391ICEs的抗性基因和热点区域进行比较分析,结果表明,3株霍乱弧菌的SXT/R391ICE的抗性基因和热点区域虽然都拥有类似的骨架结构,但从获取基因的得失情况可以判断,这3株菌SXT/R391ICE的进化优先关系。     

用电感耦合等离子体发射光谱法检测北方主栽板栗品种(系)矿质元素含量

为进一步开发富矿质元素板栗新品种,研发板栗加工新产品,采用电感耦合等离子体发射光谱法(ICP-AES)测定17种板栗栗仁中12种大量元素和微量元素,以及4种重金属元素的含量。结果表明,板栗不同品种(系)栗仁中矿质元素的含量差异显著,含有许多有益的大量元素与微量元素。板栗栗仁中大量元素含量由高到低依次为KPCaMgNa,且K、P的含量远高于Ca、Mg、Na;微量元素含量由高到低依次为FeMnZnCuNiCrSe,且Fe、Mn、Zn的含量远高于Cu、Cr、Se、Ni;重金属元素As、Pb、Hg、Cd的含量极低。大量元素中Na的变异系数最大,变异系数达46.69%;K含量次之,变异系数达32.35%;P含量最低,变异系数仅为13.92%。微量元素中Cr含量的变异系数最大,变异系数达112.02%;Mn含量次之,变异系数达64.28%;Zn含量最低,变异系数仅为15.48%。     

拟南芥黑芥子酶基因根特异性表达启动子的克隆

黑芥子酶是一类催化芥子油苷水解的同工酶, pyk10 是一个在拟南芥根和下胚轴中特异表达的黑芥子酶基因。本研究用PCR方法从拟南芥Columbia生态型基因组中克隆了pyk10 基因启动子片段。序列分析表明:该片段全长1 454 bp,与已发表的pyk10 启动子序列(GenBank accession no. AJ292756)同源性达98%。该启动子片段中含有多种其他植物基因启动子中发现的通用启动子元件,并含有器官和组织特异性转录因子的结合位点ACGT-、CANNTG-、GATA-以及I盒等顺式元件。