转座子Tn917的DNA序列计算机分析

通过计算机分析转座子Ｔｎ９１７的全序列，详细阐述了其物理图谱、结构功能及其转录调节机制．Ｔｎ９１７的５个ＯＲＦｓ排列在同一条ＤＮＡ链上，且阅读方向都从左至右．ＯＲＦ１－３起始点的左侧翼排列有启动子序列和Ｓｈｉｎｅ－Ｄａｌｇａｒｎｏ序列．ＯＲＦ５（编码转座酶）和ＯＲＦ４（编码拆分酶）的转录方向是一致的，翻译也紧密偶联在一起．在ＯＲＦ３和ＯＲＦ４之间存在１个ｒｅｓ位点，与Ｔｎ３中的ｒｅｓ位点基本同源．翻译衰减的功能与ｒＲＮＡ甲基化酶（由ＯＲＦ２编码的、ｅｒｍ基因的产物）诱导有关，在这个结构基因的左侧翼有２００ｂｐ的前导区域编码一个具调控功能的３６个氨基酸组成的多肽（由ＯＲＦ１编码）．     

Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae.

An unusual protein splicing reaction joins the N-terminal segment (A) and the C-terminal segment (C) of the 119K primary translation product (ABC) of the yeast VMA1 gene to yield a 69K vacuolar H(+)-ATPase subunit (AC) and an internal 50K polypeptide (B). This 50K protein is a site-specific DNA endonuclease that shares 34% identity with the homothallic switching endonuclease. The site cleaved by the VMA1-derived endonuclease exists in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame. Cleavage at this site only occurs during meiosis and initiates 'homing', a genetic event that converts a VMA1 allele lacking the endonuclease coding sequence into one that contains it.     

万安玻璃红鲤鱼PKR的克隆鉴定与系统进化分析

本研究以万安玻璃红鲤鱼为材料,通过同源克隆的方法克隆了万安玻璃红鲤鱼双链RNA激活的蛋白激酶（PKRCcvwPKR）,并首次克隆到CcvwPKR的剪接异构体。CcvwPKR全长为2145 bp,编码714个氨基酸,CcvwPKR剪接异构体全长1431 bp,编码476个氨基酸。CcvwPKR的剪接异构体是CcvwPKR第477个密码子的G突变为T（gaa477taa）,从而使得CcvwPKR的翻译提前终止。氨基酸序列比对和空间结构SMART预测显示,CcvwPKR和CcvwPKR剪接异构体都含有三个双链RNA结合模体（dsRBM）,CcvwPKR剪接异构体缺失PKR的C端的激酶结构域。系统进化分析表明,万安玻璃红鲤鱼PKR在进化上与鲤鱼和鲫鱼密切相关。万安玻璃红鲤鱼和目前已有研究的鲤科鱼类PKR一样,分子结构都含有三个dsRBM,三个dsRBM使得其具有更强的结合dsRNA的作用,推测可能与其较强的抗病能力有关。     

Genetic insight of the H5N1 hemagglutinin cleavage site

The cleavability of the hemagglutinin (HA) plays a major role in virulence of avian influenza viruses. Detailed analyses of the cleavage sequences and their evolution would give insights into the high pathogenicity of the H5N1 virus. HA segments were visually identifiable in the cellular automata (CA) image, and a feature gene segment (FGS) was only found in H5N1 rather than any other subtype. This FGS is a 30-bp gene segment mainly consisting of ‘A’ and ‘G’. When translated into amino acids the FGS converted into a sequence of mainly basic amino acids with positive charges. This feature amino acid segment (FAAS) was located in the cleavage site loop of HA which was potentially cleavable by various proteases. The 3D structure of H5N1 HA was reconstructed using homology modelling. It was found that the cleavage site loop was well exposed to potential proteases. The molecular surfaces were reconstructed to study how mutation and deletion of some amino acids in the FAAS affected the charge distribution. It was found that some mutations had severely changed the landscape of the charge dis- tribution. Statistical analyses of FAAS were made with respect to when and where the H5N1 viruses were found. In 2005, there were less un-mutated FAAS than the other years according to temporal evolution, and more mutated FAAS appeared in China than other regions according to geographic dis- tribution. These results are helpful for exploring the evolution of virus high pathogenicity.     

麻疯树核糖体失活蛋白Curcin和Curcin C与腺苷及腺嘌呤的互作方式分析

麻疯树核糖体失活蛋白Curcin和Curcin C均具N-糖苷酶活性,然而两者的体外翻译抑制能力却具有明显差异,这暗示着两者的N-糖苷酶活性也存在差异.为了探究造成这一差异的结构基础,本研究使用trRosetta对两种蛋白进行了三级结构的预测,通过PROCHECK和Qmean对预测得到的三级结构模型进行了质量评估,利用Chem3D对小分子配体腺嘌呤和腺苷进行了结构优化,借助UCSF Chimera对Curcin及Curcin C活性位点的氨基酸组成进行了预测.最终使用分子对接软件AutoDock将预测得到的模型与小分子腺嘌呤及腺苷进行分子对接.对接结果显示,两种蛋白与腺嘌呤的相互作用模式具有较高的相似性,但Curcin的关键氨基酸Arg并未参与到与配体的相互作用.此外Curcin C与腺嘌呤和腺苷之间的结合能都低于Curcin,且其和腺苷与腺嘌呤之间结合能的差值也要高于Curcin.这一结果暗示着Curcin和Curcin C之间的活性差异与其活性位点处的结构特征有关,Curcin C中的关键氨基酸Arg与腺嘌呤及腺苷的结合位点更为靠近,从而导致Curcin C与底物之间的结合能更低,...     

武汉地区丙型肝炎病毒包膜蛋白E1的生物信息学研究

使用生物信息学的方法,分析武汉地区不同基因型、亚型的丙肝病毒的包膜E1蛋白,预测其二级结构、核苷酸变异性、糖基化位点、亲水性、跨膜区、信号肽、蛋白修饰位点、B细胞抗原.结果显示各HCV的包膜E1蛋白二级结构差距不大;序列始末段有数个氨基酸残基的差距,序列上存在高变异位点,基因型2a的型内一致性最高;序列大量糖基化,有多个糖基化基化位点;序列分布着亲水性区域,基因型1b的分值很高;基因型1b、6a、3b、3a多数亚型有1个跨膜区,基因型2a多数亚型有两个跨膜区;基因型1b、6a、3b、3a无信号肽,基因型2a都有信号肽;蛋白序列上存在多个不同修饰位点和B细胞抗原,各基因型、亚型之间有明显差距,具有较大异质性.该研究为揭示病毒感染机制和研制地区性疫苗提供一定的科学依据.     

Sequences at the somatic recombination sites of immunoglobulin light-chain genes.

The entire nucleotide sequence of a 1.7-kilobase embryonic DNA fragment containing five joining (J) DNA segments for mouse immunoglobulin kappa chain gene has been determined. Each J DNA segment can encode amino acid residues 96--108. Comparison of one of the five J DNA sequences with those of an embryonic variable (V) gene and a complete kappa chain gene permitted localisation of a precise recombination site. The 5'-flanking regions of J DNA segments could form an inverted stem structure with the 3'-non-coding region of embryonic V genes. This hypothetical structure and gel-blotting analysis of total embryo and myeloma DNA suggest that the somatic recombination may be accompanied by excision of an entire DNA segment between a V gene and a J DNA segment. Antibody diversity may in part be generated by modulation of the precise recombination sites.     

A tumour suppressor network relying on the polyamine-hypusine axis

Tumour suppressor genes encode a broad class of molecules whose mutational attenuation contributes to malignant progression. In the canonical situation, the tumour suppressor is completely inactivated through a two-hit process involving a point mutation in one allele and chromosomal deletion of the other. Here, to identify tumour suppressor genes in lymphoma, we screen a short hairpin RNA library targeting genes deleted in human lymphomas. We functionally identify those genes whose suppression promotes tumorigenesis in a mouse lymphoma model. Of the nine tumour suppressors we identified, eight correspond to genes occurring in three physically linked 'clusters', suggesting that the common occurrence of large chromosomal deletions in human tumours reflects selective pressure to attenuate multiple genes. Among the new tumour suppressors are adenosylmethionine decarboxylase 1 (AMD1) and eukaryotic translation initiation factor 5A (eIF5A), two genes associated with hypusine, a unique amino acid produced as a product of polyamine metabolism through a highly conserved pathway. Through a secondary screen surveying the impact of all polyamine enzymes on tumorigenesis, we establish the polyamine-hypusine axis as a new tumour suppressor network regulating apoptosis. Unexpectedly, heterozygous deletions encompassing AMD1 and eIF5A often occur together in human lymphomas and co-suppression of both genes promotes lymphomagenesis in mice. Thus, some tumour suppressor functions can be disabled through a two-step process targeting different genes acting in the same pathway.     

Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences

The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment.     

Ribosome-mediated incorporation of a non-standard amino acid into a peptide through expansion of the genetic code.

One serious limitation facing protein engineers is the availability of only 20 'proteinogenic' amino acids encoded by natural messenger RNA. The lack of structural diversity among these amino acids restricts the mechanistic and structural issues that can be addressed by site-directed mutagenesis. Here we describe a new technology for incorporating non-standard amino acids into polypeptides by ribosome-based translation. In this technology, the genetic code is expanded through the creation of a 65th codon-anticodon pair from unnatural nucleoside bases having non-standard hydrogen-bonding patterns. This new codon-anticodon pair efficiently supports translation in vitro to yield peptides containing a non-standard amino acid. The versatility of the ribosome as a synthetic tool offers new possibilities for protein engineering, and compares favourably with another recently described approach in which the genetic code is simply rearranged to recruit stop codons to play a coding role.     

Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance

The structure-function relationship of the nicotinic acetylcholine receptor (AChR) has been effectively studied by the combination of complementary DNA manipulation and single-channel current analysis. Previous work with chimaeras between the Torpedo californica and bovine AChR delta-subunits has shown that the region comprising the hydrophobic segment M2 and its vicinity contains an important determinant of the rate of ion transport through the AChR channel. It has also been suggested that this region is responsible for the reduction in channel conductance caused by divalent cations and that segment M2 contributes to the binding site of noncompetitive antagonists. To identify those amino acid residues that interact with permeating ions, we have introduced various point mutations into the Torpedo AChR subunit cDNAs to alter the net charge of the charged or glutamine residues around the proposed transmembrane segments. The single-channel conductance properties of these AChR mutants expressed in Xenopus laevis oocytes indicate that three clusters of negatively charged and glutamine residues neighbouring segment M2 of the alpha-, beta-, gamma- and delta-subunits, probably forming three anionic rings, are major determinants of the rate of ion transport.     

西瓜八氢番茄红素合成酶基因全长的克隆与表达分析

通过RT-PCR和RACE技术从西瓜果实中克隆八氢番茄红素合成酶（PSY）基因的cDNA全长,用生物信息学方法对其cDNA序列及推测氨基酸序列进行分析,并用实时荧光定量PCR技术研究PSY在不同瓤色西瓜果实发育过程中的表达情况.结果表明,PSY基因cDNA全长1 561 bp（GenBank登录号为KC166870）,其开放阅读框为1 266 bp,编码421个氨基酸.该基因及其推导的氨基酸序列与同为葫芦科的其他植物的PSY及氨基酸序列的同源性分别为87％和91％以上;序列分析显示,PSY氨基酸序列N末端存在转运肽信号序列.不同瓤色西瓜果实发育过程中PSY的表达存在明显差异,红瓤果实中的表达量最高,这表明PSY可能与红瓤果实中积累较多的类胡萝卜素有关;PSY的表达量均高于PSY-A,推测PSY基因主要负责果实中类胡萝卜素的合成.     

The transorientation hypothesis for codon recognition during protein synthesis

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5'-stacked and the 3'-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon-anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.     

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin （HA） cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process. Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons, and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops. Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses, that is, to inhibit the gene expression of avian influenza virus H5N1 subtypes, we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.     

钢筋混凝土梁结构中的声发射波衰减传播特征

为了解距离对弹性波传播的影响规律,制作了两种不同规格的钢筋混凝土梁,通过断铅模拟声发射信号,利用PAC-3声发射系统对弹性波在钢筋混凝土中传播的衰减规律进行试验研究。结果表明：频率、波速及振幅均随传播距离增加整体上呈衰减趋势;弹性波在钢筋混凝土传播过程中,高频信号衰减程度大于低频信号,因而低频信号在长距离传播更稳定;波速及振幅在1.0 m处都出现了较大幅度的提高,经分析可知声波在经过混凝土三相交界处时会发生多次折射、反射、吸收衰减等现象,因而离散性增大。可见实际检测过程中的探头布置间距不宜超过1.0 m。     

Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo

Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.