Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

Depositional environment and sequence stratigraphy of the Oligo-Miocene Asmari Formation in SW Iran

The Asmari Formation, a thick carbonate succession of the Oligo-Miocene in Zagros Mountains (southwest Iran), has been studied to determine its microfacies, paleoenvironments and sedimentary sequences. Detailed petrographic analysis of the deposits led to the recognition of 10 microfacies types. In addition, five major depositional environments were identified in the Asmari Formation. These include tidal flat, shelf lagoon, shoal, slope and basin environmental settings and are interpreted as a carbonate platform developed in an open shelf situation but without effective barriers separating the platform from the open ocean. The Asmari carbonate succession consists of four, thick shallowing-upward sequences (third-order cycles). No major hiatuses were recognized between these cycles. Therefore, the contacts are interpreted as SB2 sequence boundary types. The Pabdeh Formation, the deeper marine facies equivalent of the Asmari Limestone is interpreted to be deposited in an outer slope-basin environment. The microfacies of the Pabdeh Formation shows similarities to the Asmari Formation.     

利用序列保守模体和局部构象信息预测转录因子结合位点

转录因子结合位点的计算预测是研究基因转录调控的重要环节,但常用的位置特异得分矩阵方法预测特异性偏低.通过深入分析结合位点的生物特征,提出了一种综合利用序列保守模体和局部构象信息的结合位点预测方法,以极大相关得分矩阵作为保守模体的描述模型,并根据二苷参数模型计算位点序列的局部构象,将两类信息得分组合为多维特征向量,在二次判别分析的框架下进行训练和滑动预测.预测过程中还引入了位置信息量以优化似然得分和过滤备选结果.针对大肠杆菌CRP和Fis结合位点数据的留一法测试结果表明,描述模型的改进和多种信息的融合能有效地改善预测方法的性能,大幅度提高特异性.     

SHIV-XJ02170感染恒河猴后期的传代特点和env基因变异

目的研究SHIV-XJ02170在中国恒河猴感染后期传代过程中病毒和宿主的变化特点,并分析env基因的序列变异。方法将感染中国恒河猴G0401V后期（5年）的SHIV-XJ02170病毒垂直传代2只猴（G0401V→G0402V→0403V）,同时,剔除G0401V猴CD8＋T细胞使潜伏的病毒大量复制后传代1只猴（G0401V→G0404V）,应用流式细胞术、病毒载量测定、序列分析等方法研究该病毒在猴体内长期适应后的病毒和免疫学指标及序列变异特点。结果 G0401V在感染后期仍能稳定传代,且表现出毒力增强的特点。其传代猴G0402V在传代后41 d死亡,外周血CD4＋T淋巴细胞衰竭,仅为43个/mL,符合艾滋病感染猴快速进展型的特征。剔除体内CD8＋T细胞之后的传代猴G0404V的表现类似G0401V,即长期低水平的病毒血症水平。env基因序列分析发现SHIV-XJ02170在G0401V体内长期适应后发生了可遗传的序列变异,并引起糖基化位点的改变。结论 SHIV-XJ02170在猴体长期适应后的传代过程中表现出向强毒株过渡的特征,为进行SHIV-XJ02170感染性克隆的构建奠定了良好的实验基础。     

Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.     

引入CpG基序的汉滩病毒G2糖蛋白基因的克隆及表达

构建汉滩病毒G2糖蛋白的真核表达载体,并加入可增强小鼠免疫刺激作用的CpG基序,检测其可否在真核细胞中表达。参照Genebank中汉滩病毒M的全基因序列设计引物,引物两端引入可增强小鼠免疫刺激作用的CpG基序及双酶切位点,通过聚合酶链反应(PCR)获得含CpG基序的G2片段,并将其与T载体pMD18-T相连,测序后克隆至真核表达载体pcDNA3.1 上,将此真核表达载体以脂质体法转染至真核细胞Vero-E6中,利用间接免疫荧光法(IFA)检测发现转染后的Vero-E6中出现特异性的绿色荧光。结果表明本实验成功构建了汉滩病毒包膜糖蛋白G2的重组体。     

Classifier ensembles for protein structural class prediction with varying homology

Structural class characterizes the overall folding type of a protein or its domain. A number of computational methods have been proposed to predict structural class based on primary sequences; however, the accuracy of these methods is strongly affected by sequence homology. This paper proposes, an ensemble classification method and a compact feature-based sequence representation. This method improves prediction accuracy for the four main structural classes compared to competing methods, and provides highly accurate predictions for sequences of widely varying homologies. The experimental evaluation of the proposed method shows superior results across sequences that are characterized by entire homology spectrum, ranging from 25% to 90% homology. The error rates were reduced by over 20% when compared with using individual prediction methods and most commonly used composition vector representation of protein sequences. Comparisons with competing methods on three large benchmark datasets consistently show the superiority of the proposed method.     

PMWS病猪猪圆环病毒2型全基因组序列分析

根据GenBank中发表的猪圆环病毒2型(PCV2)全基因序列,设计一对特异性引物,用PCR方法直接从5省病料中分别扩增出5个PCV2毒株的全基因组,分别命名为FujianPCV、GuangxiPCV、HainanPCV、HunanPCV和ShandongPCV.将扩增片段克隆于pMD 18-T载体,进行序列测定,结果表明,除FujianPCV株核苷酸长度为1768bp,其余四株均为1767 bp.应用DNAstar序列分析软件分析表明,本实验的5个PCV2分离株与世界上其它地区的PCV2分离株密切相关,核苷酸序列同源性达93%～98.8%,与PCV1毒株的序列同源性只有68.4%～70%.其中ORF1和ORF2所编码的氨基酸序列与国内外PCV2毒株比较,同源性也很高,分别为98.1%～99.4%和88%～99.1%.