Articles Online (Volume 1, Issue 3)

Review Article

Integration of G-Protein Coupled Receptor Signaling Pathways for Activation of a Transcription Factor (EGR-3)

Xuehai Tan,Pam Sanders,Jack Bolado Jr,Mike Whitney

We recently reported the use of a gene-trapping approach to isolate cell clones in which a reporter gene had integrated into genes modulated by T-cell activation. We have now tested a panel of clones from that report and identified the one that responds to a variety of G-protein coupled receptors (GPCR). The βlactamase tagged EGR-3 Jurkat cell was used to dissect specific GPCR signaling in vivo. Three GPCRs were studied, including the chemokine receptor CXCR4 (Gicoupled) that was endogenously expressed, the platelet activation factor (PAF) receptor (Gq-coupled), andβ2 adrenergic receptor (Gs-coupled) that was both stably transfected. Agonists for each receptor activated transcription of theβ-lactamase tagged EGR-3 gene. Induction of EGR-3 through CXCR4 was blocked by pertussis toxin and PD58059, a specific inhibitor of MEK (MAPK/ERK kinase). Neither of these inhibitors blocked isoproterenol or PAF-mediated activation of EGR-3. Conversely, β2- and PAF-mediated EGR-3 activation was blocked by the p38, specific inhibitor SB580. In addition, bothβ2- and PAF-mediated EGR-3 activation could be synergistically activated by CXCR4 activation. This combined result indicates that EGR-3 can be activated through distinct signal transduction pathways by different GPCRs and that signals can be integrated and amplified to efficiently tune the level of activation.
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Page 173-179


Research Article

Complete Genome Sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04)

Shengli Bi,E'de Qin,Zuyuan Xu,Wei Li,Jing Wang,Yongwu Hu,Yong Liu,Shumin Duan,Jianfei Hu,Yujun Han,Jing Xu,Yan Li,Yao Yi,Yongdong Zhou,Wei Lin,Jie Wen,Hong Xu,Ruan Li,Zizhang Zhang,Haiyan Sun,Jingui Zhu,Man Yu,Baochang Fan,Qingfa Wu,Lin Tang,Bao'an Yang,Guoqing Li,Wenming Peng,Wenjie Li,Tao Jiang,Yajun Deng,Bohua Liu,Jianping Shi,Yongqiang Deng,Wei Wei,Hong Liu,Zongzhong Tong,Feng Zhang,Yu Zhang,Cui'e Wang,Yuquan Li,Jia Ye,Yonghua Gan,Jia Ji,Xiaoyu Li,Xiangjun Tian,Fushuang Lu,Gang Tan,Ruifu Yang,Bin Liu,Siqi Liu,Songgang Li,Jun Wang,Jian Wang,Wuchun Cao,Jun Yu,Xiaoping Dong,Huan-Ming Yang

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates B J01, B J02, B J03,and B J04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identifled from the BJ group, 32 are non-synonymous changes at the amino acid level.Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.
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Page 180-192


Research Article

The C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

Guozhen Liu,Shaohui Hu,Yongwu Hu,Peng Chen,Jianning Yin,Jie Wen,Jingqiang Wang,Liang Lin,Jinxiu Liu,Bo You,Ye Yin,Shuting Lin,Hao Wang,Yan Ren,Jia Ji,Xiaoqian Zhao,Yongqiao Sun,Xiaowei Zhang,Jianqiu Fang,Jian Wang,Siqi Liu,Jun Yu,Heng Zhu,Huan-Ming Yang

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC)gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter.Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls.The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.
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Page 193-197


Research Article

The Epitope Study on the SARS-CoV Nucleocapsid Protein

Shuting Lin,Liang Lin,Hao Wang,Jianning Yin,Yan Ren,Zhe Zhao,Jie Wen,Cuiqi Zhou,Xumin Zhang,Xiaolei Li,Jingqiang Wang,Zhengfeng Zhou,Jinxiu Liu,Jianmin Shao,Tingting Lei,Jianqiu Fang,Ningzhi Xu,Siqi Liu

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA),the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins,whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.
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Page 198-206


Research Article

A Strategy for Searching Antigenic Regions in the SARS-CoV Spike Protein

Yan Ren,Zhengfeng Zhou,Jinxiu Liu,Liang Lin,Shuting Lin,Hao Wang,Ji Xia,Zhe Zhao,Jie Wen,Cuiqi Zhou,Jingqiang Wang,Jianning Yin,Ningzhi Xu,Siqi Liu

In the face of the worldwide threat of severe acute respiratory syndrome (SARS)to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA), these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potential epitope regions were located at Codons 469-882 in the S protein, and one epitope site was located at Codons 599-620. Identification of antigenic regions in the SARS-CoV S protein may be important for the functional studies of this virus or the development of clinical diagnosis.
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Page 207-215


Research Article

Evolution and Variation of the SARS-CoV Genome

Jianfei Hu,Jing Wang,Jing Xu,Wei Li,Yujun Han,Yan Li,Jia Ji,Jia Ye,Zhao Xu,Zizhang Zhang,Wei Wei,Songgang Li,Jun Wang,Jian Wang,Jun Yu,Huan-Ming Yang

Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARSCoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.
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Page 216-225


Research Article

Genome Organization of the SARS-CoV

Jing Xu,Jianfei Hu,Jing Wang,Yujun Han,Yongwu Hu,Jie Wen,Yan Li,Jia Ji,Jia Ye,Zizhang Zhang,Wei Wei,Songgang Li,Jun Wang,Jian Wang,Jun Yu,Huan-Ming Yang

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves.Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions.The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.
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Page 226-235


Method

EST Pipeline System: Detailed and Automated EST Data Process- ing and Mining

Hao Xu,Ling He,Yuanzhong Zhu,Wei Huang,Lijun Fang,Lin Tao,Yuedong Zhu,Lin Cai,Huayong Xu,Liang Zhang,Hong Yu,Yan Zhou

Expressed sequence tags (ESTs) are widely used in gene survey research these years. The EST Pipeline System, software developed by Hangzhou Genomics In- stitute (HGI), can automatically analyze different scalar EST sequences by suitable methods. All the analysis reports, including those of vector masking, sequence as- sembly, gene annotation, Gene Ontology classification, and some other analyses, can be browsed and searched as well as downloaded in the Excel format from the web interface, saving research efforts from routine data processing for biological rules embedded in the data.
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Page 236-242