我国四株狂犬病毒M和P基因序列分析和所编码蛋白的分析

为了解我国狂犬病毒M、P基因序列和结构特点,用RT-PCR方法获得目的基因片段,测定核苷酸序列后,计算机分析核苷酸和氨基酸序列及其功能区位点结构。结果显示四株病毒M基因核苷酸和氨基酸序列同源性分别为83.9%～99.5%和93.1%～99%,四株狂犬病毒M蛋白上调节病毒RNA转录和复制功能的第58位氨基酸残基均为谷氨酰胺残基(E),与特异性细胞蛋白WW区域作用的PPxY结构序列均为PPEY保守序列;四株病毒P基因核苷酸和氨基酸序列同源性分别为83.6%～99.8%和87.2%～99%,P蛋白与胞浆动力蛋白轻链LC8相互作用的序列位于143～148位氨基酸残基,均为DKSTQT,四株病毒P基因与L蛋白、N蛋白作用位点序列显示未发生影响其生物学功能的变异。研究结果证实了这两种蛋白结构在病毒致病性中起重要作用的推论。     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

中国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30基质蛋白基因和融合蛋白基因的分子特征分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行基质蛋白(M)和融合蛋白(F)基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出M和F基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。China/Tib/Gej/07-30的M基因由1483个核苷酸组成,编码335个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为92.4%～97.7%和97.0%～98.2%。F基因由2411个核苷酸组成,编码546个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为85.5%～96.1%和94.3%～98.2%。China/Tib/Gej/07-30的F蛋白含有信号肽序列和跨膜结构域,序列高度变异。F蛋白第104～108位和第109～133位氨基酸位点分别是高度保守的裂解位点和融合肽结构域。F蛋白还含有序列高度保守的三个七肽重复区。China/Tib/Gej/07-30的M基因3′端的非编码区(UTR)长度为443个核苷酸,GC含量高达68.4%,与其他PPRV毒株的同源性为82.4%～93.5%。China/Tib/Gej/07-30的F基因5′UTR区长度为634个核苷酸,GC含量高达70.0%,与其他PPRV毒株序列相似性为76.2%～91.7%。     

鸡传染性支气管炎病毒纤突蛋白裂解与致病性的研究

冠状病毒的致病机理一直不清楚。对副粘病毒和正粘病毒的研究结果表明病毒的致病力与病毒纤突蛋白裂解程度有关,反过来纤突蛋白裂解程度是由纤突蛋白的连结多肽的氨基酸顺序决定的。本文试图从IBV致细胞病变作用,纤突蛋白裂解状态和连结多肽氨基酸顺序三个方面探讨IBV的致病机理。结果表明,IBV毒株在不同细胞培养(CK.CEF和Vero)中,从宿主细胞范围、致细胞病变作用和引起细胞融合几项指标表现了不同的致病力,但不同毒株从同一种细胞释放后其纤突蛋白的裂解程度无差异。连结多肽的氮基酸序列表明23株IBV的连结多肽均由5个氨基酸组成即二对碱性氨基酸Arg—Arg和Arg—Arg,中间由苯丙氨酸或丝氨酸联接。这些结果说明在致病机理方面,冠状病毒可能不同于副粘病毒和正粘病毒。     

Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis sec Y.

A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M(r) value has been calculated to be 47,100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis secY. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).     

水稻矮缩病毒最外层外壳蛋白基因（S2）cDNA克隆,序列分析及其在大？…

从水稻矮缩病毒（Ｒｉｃｅｄｗａｒｆｖｉｒｕｓ,ＲＤＶ）中国福建分离物中克隆分离了最外层外壳蛋白基因（Ｓ２）全长ｃＤＮＡ并对其进行序列分析,结果表明ＲＤＶＳ２ｃＤＮＡ全长３５１２ｂｐ,仅含一个３３４８ｂｐ的阅读框架,编码一人含有１１１６个氨基酸的蛋白（Ｐ２）。与基因库中已知基因序列比较,发现它与日本ＲＤＶＨ株系相应片段的核苷酸和氨基酸同源率分别为９４．６％和９５．４％与轮状病毒ＶＰ２氨基酸序列有一定     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.     

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).     

中国1995～2004年鸡传染性支气管炎病毒地方分离株膜蛋白基因的遗传变异分析

应用RT-PCR方法扩增到了我国1995～2004年20株IBV现地分离株的膜蛋白(Membrane,M)基因片段.序列测定表明,20株IBV分离株M基因开放阅读框由672～681bp组成,编码由223～226个氨基酸残基组成的多肽.与我国分离株LX4相比,M基因推导氨基酸序列的变异主要发生在2～17位、221～223位,其中4～6位存在氨基酸的插入和缺失,导致IBV毒株间M蛋白糖基化位点的差异.与GenBank中34株IBV参考毒株M蛋白基因推导氨基酸序列进行比较和分析,系统进化关系显示54株IBV毒株分属于5个进化群.我国IBV分离株M基因在进化关系上较为独立,主要分布在第Ⅱ群和第Ⅳ群,其中第Ⅱ群分离株和中国台湾毒株进化关系密切.此外,参考IBV国内分离株S1基因及N基因系统发育进化树的研究结果,并与M基因进行比较,表明我国IBV也存在着基因重组现象,尤其是疫苗毒和流行毒之间的重组.     

Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells

The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged β-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.     

Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor

Growth factor receptors are typically activated by the binding of soluble ligands to the extracellular domain of the receptor, but certain viral transmembrane proteins can induce growth factor receptor activation by binding to the receptor transmembrane domain. For example, homodimers of the transmembrane 44-amino acid bovine papillomavirus E5 protein bind the transmembrane region of the PDGF beta receptor tyrosine kinase, causing receptor dimerization, phosphorylation, and cell transformation. To determine whether it is possible to select novel biologically active transmembrane proteins that can activate growth factor receptors, we constructed and identified small proteins with random hydrophobic transmembrane domains that can bind and activate the PDGF beta receptor. Remarkably, cell transformation was induced by approximately 10% of the clones in a library in which 15 transmembrane amino acid residues of the E5 protein were replaced with random hydrophobic sequences. The transformation-competent transmembrane proteins formed dimers and stably bound and activated the PDGF beta receptor. Genetic studies demonstrated that the biological activity of the transformation-competent proteins depended on specific interactions with the transmembrane domain of the PDGF beta receptor. A consensus sequence distinct from the wild-type E5 sequence was identified that restored transforming activity to a non-transforming poly-leucine transmembrane sequence, indicating that divergent transmembrane sequence motifs can activate the PDGF beta receptor. Molecular modeling suggested that diverse transforming sequences shared similar protein structure, including the same homodimer interface as the wild-type E5 protein. These experiments have identified novel proteins with transmembrane sequences distinct from the E5 protein that can activate the PDGF beta receptor and transform cells. More generally, this approach may allow the creation and identification of small proteins that modulate the activity of a variety of cellular transmembrane proteins.     

Nucleus-targeting domain of the matrix protein (M1) of influenza virus.

The matrix protein M1 of influenza virus A/WSN/33 was shown by immunofluorescent staining to be transported into the nuclei of transfected cells without requiring other viral proteins. We postulated the existence of a potential signal sequence at amino acids 101 to 105 (RKLKR) that is required for nuclear localization of the M1 protein. When CV1 cells were transfected with recombinant vectors expressing the entire M1 protein (amino acids 1 to 252) or just the first 112 N-terminal amino acids, both the complete M1 protein and the truncated M1 protein were transported to the nucleus. In contrast, expression in CV1 cells of vectors coding for M1 proteins with deletions from amino acids 77 to 202 or amino acids 1 to 134 resulted only in cytoplasmic immunofluorescent staining of these truncated M1 proteins without protein being transported to the nucleus. Moreover, no nuclear membrane translocation occurred when CV1 cells were transfected with recombinant vectors expressing M1 proteins with deletions of amino acids 101 to 105 or with substitution at amino acids 101 to 105 of SNLNS for RKLKR. Furthermore, a synthetic oligopeptide corresponding to M1 protein amino acids 90 to 108 was also transported into isolated nuclei derived from CV1 cells, whereas oligopeptides corresponding to amino acid sequences 25 to 40, 67 to 81, and 135 to 164 were not transported into the isolated cell nuclei. These data suggest that the amino acid sequence 101RKLKR105 is the nuclear localization signal of the M1 protein.     

海七鳃鳗(Petromyzon marinus)anoctaimin-1蛋白的生物信息学分析

七鳃鳗是现存的最原始的无颌类脊椎动物之一,也是连接无脊椎动物与脊椎动物的重要环节,对生物的起源与进化有很高的研究价值。anoctamin-1蛋白(ANO1)是一种重要的跨膜蛋白,与细胞内阴离子的跨膜运输相关。以海七鳃鳗为例,利用不同软件对海七鳃鳗ANO1蛋白的理化性质、结构域、蛋白结构特征、物种进化保守性以及系统进化关系进行生物信息学分析表明:海七鳃鳗ANO1的开放阅读框为2 373 bp,编码791个氨基酸,属于anoctamin蛋白家族,具有7个跨膜区;二级结构含有无规则卷曲、α螺旋和β折叠。将海七鳃鳗与其他物种的ANO1氨基酸序列进行同源比对,并构建系统进化树,以确认海七鳃鳗ANO1基因的保守性和进化地位。对ANO1基因及蛋白的生物信息学分析为ANO1基因及蛋白的相关研究提供了重要的信息基础。     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.     

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum phosphofructokinase

The nucleotide sequence of a full-length cDNA encoding phosphofructokinase (PFK) enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2,653 bases comprises a single open reading frame of 2,452 bases and a noncoding region of 201 bases after the stop codon. The mature protein contains 812 amino acids and has a molecular mass of 90,900 Da. The amino acid sequences of several peptides derived from the purified protein show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the protein with those of 3 other worms as well as those of human, rabbit, and bacterial enzymes reveals highly conserved regions interrupted with stretches of lesser sequence similarity. Analyses of the subunit primary structure reveal, as in other eukaryotic PFKs, that the amino-terminal half is homologous to the carboxy-terminal half, supporting the hypothesis that the PFK gene evolved by duplication of the prokaryotic gene and that the allosteric sites arose by mutations at the catalytic site. The location of the phosphorylation site is unique and different compared with other PFKs and plays a key role in regulation of the enzyme activity. Structural motifs such as the putative substrate and effector binding domains and also the key amino acids involved therein are clearly identified by alignment of all the PFK protein sequences.     

Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii. The enzyme and its coding gene

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.     

Strong Correlation Between Statistical Transmembrane Tendency and Experimental Hydrophobicity Scales for Identification of Transmembrane Helices

Direct physical chemistry measurements of the hydrophobicity of amino acids or their derivatives have often been used to estimate the propensity of amino acids to participate in transmembrane helices. In this short note, it is found that there is a very high degree of correlation (r = 0.944–0.965) between an average physical chemistry hydrophobicity scale (an average of scales derived, e.g., from the solubility of amino acid derivatives in organic solvents versus water or their binding to hydrophobic particles) and the statistically based transmembrane tendency scale (derived from the relative abundance of residues in known transmembrane and soluble protein sequences (Zhao and London, Protein Sci 15:1987–2001, 2006)). This correlation indicates that, other than hydrophobicity, amino acid properties/interactions that promote or inhibit transmembrane helix formation in a specific membrane protein largely cancel out when averaged over all transmembrane sequences. In other words, other than hydrophobicity, there are no properties of a specific amino acid residue within a hydrophobic segment that have a strong systematic effect upon transmembrane helix formation independent of the remainder of the sequence in that hydrophobic segment. However, proline is an exception to this rule.