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Cell research 2001年第3期11卷 217-222页

作者： LU FANG, JIA FANG, CHANG QING CHEN shanghai research {2. of biotechnology, shanghai institutes for biological sciences the chinese academy of sciences, shanghai 2.02.3, China 1shanghai research {2. of biotechnology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200233 China. 1shanghai research {2. of biotechnology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200233 China. 1shanghai research {2. of biotechnology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200233 China.

TNF recepter-55 is the main mediator of TNF-induced apoptosis. TNF receptor-75-dependent induction or enhancement of cytotoxicity has been explained by intracellular signaling, 'ligand passing' , or induction of endogenous TNF. To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFa-induced cytotoxicity, HEp-2.cells were transfected with bicistronic expression vector of hTR75 and its deletion mutants genes. hTNFa-induced cytotoxicity was determined by crystal violet colorimetric method. The expression of hTR75 and its deletion mutants in HEp-2.cells was demonstrated by RT-PCR and indirect ELISA. We found that the overexpressed hTR75 could significantly increase the susceptibility of HEp-2.cells to hTNFa which especially required TRAF2.binding site. hTR75 could not only mediate partial hTNFa-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity

关键词： hTNFa hTNF receptors TRAF2 cytotoxcity signal transduction.

Sequence analysis of mRNA polyadenylation signals of rice genes

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chinese Science Bulletin 2006年第9期51卷 1069-1077页

作者： LU Ying1, 2. GAO Chenxi1,2.3 & HAN Bin1,3,41. National center for Gene research, {2., chinese academy of sciences, shanghai 2.02.3, China 2. Graduate School of the chinese academy of sciences, Beijing 100039, China 3. Institute of Plant Physiology and Ecology, {2., chinese academy of sciences, shanghai 2.0032. China National center for Gene research Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200233 China Graduate School of the chinese academy of sciences Beijing 100039 China Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

The formation of eukaryotic mRNAs involves the cleavage and polyadenylation of pre- mRNAs. To investigate the sequence requirement of putative polyadenylation signals (PASs), poly(A) sites and downstream elements (DUEs) in 3′-end-pro- cessing in rice, we compared expressed sequences tags (ESTs) with poly(A) extremity to full-length cDNA sequences and constructed a database of 12.69 pre- mRNA sequences in ?40―+40 nt surrounding the poly(A) sites, which were from 9953 genes. The al- ternative poly(A) sites were revealed in approxi- mately 2.% of mRNAs. Nearly 80% of pre-mRNAs showed stringent requirement of the YA (CA or UA) at poly (A) sites for polyadenylation. About 7.9% had the AAUAAA signals on ?40―?1 nt upstream of the poly(A) sites. Over 60% of mRNAs probably used the one- or two-base variants of AAUAAA hexamers as their PASs in 3′ fragments. The single-base variants of AAUGAA revealed the high frequency in 11.5% of 3′ fragments. The DUEs were detected in 90% of pre- mRNAs, especially more than half of the pre-mRNAs with multi-base variants of AAUAAA had the DUEs surrounding the poly(A) site. The location of DUE is also important for defining the cleavage site. Al- though most of the rice pre-mRNAs did not contain AAUAAA signal, the existence of downstream ele- ments ensured the efficiency of cleavage-polyade-

关键词：多聚腺苷酸序列分析 mRNA 水稻基因工程

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Recombinant scFv Antibodies against E Protein and N Protein of Severe Acute Respiratory Syndrome Virus

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 541-547页

作者： Hui LIU , Yan-Li DING, Wei HAN, Mei-Yun LIU, Rui-Yang TIAN, Sheng-Li YANG, and Yi GONGshanghai research {2. of biotechnology, shanghai institutes of biological sciences, chinese academy of sciences,Graduate School of the chinese academy of sciences, shanghai 2.02.3, China shanghai research {2. of biotechnology Shanghai Institutes of Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences Shanghai 200233 China. huiliu@***

Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C2., could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, Kd) of SARS-CoV E protein was 5.7×10–8 M for B10 and 8.9×10–8 M for C2.. The affinity of A17 for N protein was 2.1×10–6 M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

关键词： SARS-CoV antibody scFv phage display affinity chromatography

Identification and Analysis of Genes Present in Leptospira interrogans serovar lai but Absent in L. biflexa serovar monvalerio

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Acta Biochimica et Biophysica Sinica 2004年第12期36卷 832-839页

作者： Ping HE, Xiang-Yan ZHANG, Xiao-Kui GUO*, Bao-Yu HU, Xiao-Tian HUANG, Yang YANG, and Guo-Ping ZHAO1,2. Department of Microbiology and Parasitology, shanghai Second Medical University, shanghai 2.002., China 1 {2. center of biotechnology, institutes for biological sciences, chinese academy of sciences, shanghai 2.0031, China 2.10} National Human Genome center at shanghai, Zhangjiang High Tech Park, shanghai 2.12.3, China Department of Microbiology and Parasitology Shanghai Second Medical UniversityShanghai 200025 China research center of {2. Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China chinese National Human Genome center at {2.Zhangjiang High Tech Park Shanghai 201203 China

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogans serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 2. contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 2. distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12.genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridiza- tion is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.

关键词： Leptospira interrogans serovar lai suppression subtractive hybridization (SSH) specific genes

Progress in the detection of human genome structural variations

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Science China(Life sciences) 2009年第6期52卷 560-567页

作者： WU XueMei1,3 & XIAO HuaSheng1,2.1 center of Functional Genomics,Key Laboratory of System Biology,shanghai institutes for biological sciences,{2.,shanghai 2.0031,China 2.National Engineering center for Biochip at shanghai,shanghai 2.12.3,China 3 Graduate School of the {2.,shanghai 2.0031,China 1. center of Functional Genomics Key Laboratory of System Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China 3. Graduate School of the chinese academy of sciences Shanghai 200031 China 2. National Engineering center for Biochip at shanghai Shanghai 201203 China

The emerging of high-throughput and high-resolution genomic technologies led to the detection of submicroscopic variants ranging from 1 kb to 3 Mb in the human *** variants include copy number variations(CNVs),inversions,insertions,deletions and other complex rearrangements of DNA *** paper briefly reviews the commonly used technologies to discover both genomic structural variants and their potential ***,we highlight the array-based,PCR-based and sequencing-based assays,including array-based comparative genomic hybridization(aCGH),representational oligonucleotide microarray analysis(ROMA),multiplex amplifiable probe hybridization(MAPH),multiplex ligation-dependent probe amplification(MLPA),paired-end mapping(PEM),and next-generation DNA sequencing ***,we discuss the limitations and challenges of current assays and give advices on how to make the database of genomic variations more reliable.

关键词： structural variation cytogenetic microarray PCR paired end mapping(PEM) next-generation sequencing

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Direct transfer of A2. gene into pancreas protected mice from streptozotocin-induced diabetes

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Acta Pharmacologica Sinica 2004年第6期25卷 19-24页

作者： Lu-yang YU, Bo LIN, Zhen-lin ZHANG2. Li-he GUO3 Instituteof Biochemistryand Cell Biology, shanghai institutes for biological sciences, chinese {2. ofsciences, shanghai 2.0031 center for Preventing and Treating Osteoporosis, Osteoporosis research Unit, 2. shanghai JiaoTong UniversityAffiliated Sixth People’s Hospital, shanghai 2.02.3, China Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences center for Preventing and Treating Osteoporosis Osteoporosis Research Unit Shanghai JiaoTong University Affiliated Sixth People’s Hospi

AIM: To investigate the efficiency of transfer of A2. gene into pancreas against STZ-induced diabetes. METHODS: PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2. d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A2. was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A2. expression in the pancreas of pcDNA3-A2. transgenic mice was measured by RT-PCR and Western blots. Urine amylase, NO generation, and histological examination were examined. RESULTS: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen, duodenum, and islets. The development of diabetes was prevented by direct A2. gene transferring into the pancreas and A2.-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A2.-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A2. gene into the pancreas protected mice from STZ-induced diabetes.

关键词： adenoviridae streptozocin insulin-dependent diabetes mellitus gene therapy nitric oxide glucose amylases reversed transcriptase polymerase chain reaction Western blotting

miRNAs modulate the drug response of tumor cells

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Science China(Life sciences) 2009年第9期52卷 797-801页

作者： WU XueMei1,3 & XIAO HuaSheng1,2.1 center of Functional Genomics,Key Laboratory of System Biology,shanghai institutes for biological sciences,{2.,shanghai 2.0031,China 2.National Engineering center for Biochip at shanghai,shanghai 2.12.3,China 3 Graduate University of {2.,shanghai 2.0031,China center of Functional Genomics Key Laboratory of System Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China National Engineering center for Biochip at shanghai Shanghai 201203 China Graduate University of chinese academy of sciences Shanghai 200031 China

Chemotherapy is one of the major treatments of malignant carcinomas. However,its efficiency is affected by both intrinsic and acquired resistance to anticancer drugs. The cellular mechanisms of drug resistance include the overexpression of energy-dependent transporters that eject anticancer drugs from cells such as p-glycoprotein and multidrug resistance related protein (MRP),the mutation of drug targets,the activation of DNA repair pathways,the defects in cellular death pathways and so on. The genetic and epigenetic changes of these genes can lead to cancer drug resistance. Among these mechanisms,microRNAs (miRNAs) which are critical and essential for many important processes such as development,differentiation,and even carcinogenesis have been reported to regulate the chemosen-sitivity of tumor cells. In this paper we briefly review the relationship between miRNA and cancer drug resistance.

关键词： cancer drug resistance miRNAs

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Association between Alcohol, Smoking and HLA-DQA1*02.1 Genotype in Psoriasis

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Acta Biochimica et Biophysica Sinica 2004年第9期36卷 597-602页

作者： Guang-Yong ZHENG1,2., Sheng-Cai WEI3#, Tie-Liu SHI1, and Yi-Xue LI1* 1Bioinformatics center, shanghai institutes for biological sciences, {2., Graduate School of the {2., shanghai 2.0031, China research center for Proteome Analysis, Institute of Biochemistry and Cell Biology, shanghai institutes for biological sciences, {2., Graduate School of the {2., shanghai 2.0031, China 3Guangzhou Institute of Dermatology, Guangzhou 510095, China Bioinformatics center Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences

Psoriasis is a chronic skin disease triggered by genetic, environment or other risk factors such as infection, drugs, stress, moisture, alcohol, and smoking. A major psoriasis susceptibility locus at 6p2..3 has been identified. Further studies found that HLA-DQA1*02.1 allele was associated with psoriasis. However, there were few data exploring an association between the environmental factors and susceptibility genes. In this study, the samples of 189 patients with psoriasis and 333 healthy controls were collected with their consent and were carried on analysis through polymerase chain reaction sequence-specific primer (PCR- SSP) method. The proportion of male psoriasis patients engaging in the smoking and alcohol was much higher than that of the control group (P2.1 allele was present at significantly higher frequency in the patients with psoriasis (OR=4.2., P2.1 in male patients with psoriasis (OR>6.91, P<1.0×10–4).

关键词： psoriasis HLA-DQA1*02.1 allele smoking alcohol association

Conformational flexibility of β-secretase: molecular dynamics simulation and essential dynamics analysis

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Acta Pharmacologica Sinica 2004年第6期25卷 3-11页

作者： Bing XIONG, Xiao-qin HUANG, Ling-ling SHEN, Jian-hua SHEN 2.Xiao-min LUO, Xu SHEN, Hua-liang JIANG2. Kai-xian CHENDrug Discovery and Design center, State Key Laboratory of Drug research, {2. Institute of Materia Medica,shanghai institutes for biological sciences, chinese academy of sciences, {2. 2.12.3, China Drug Discovery and Design center State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 201203 China

AIM: Based on the structural analysis to reveal the mechanism of ligand binding to b-secretase and the specificityof each binding sub-site. METHODS: Molecular dynamics was used to simulate on the ligand free b-secretase andligand bound b-secretase. The trajectories were analyzed using the essential dynamics, and the significant confor-mational change was illustrated employing the DynDom program. RESULTS: The essential dynamics and DynDomanalyses clearly showed that the b-secretase experienced a large conformational change upon the substrate orinhibitor binding. The flap structure adopted a swing motion, gradually covering the active site to facilitate theligand binding process. Residues Ser86 and Ile87 served as the hinge point. Inhibitor-enzyme interaction analysisrevealed that residues at P2. P1, and P1 positions of the inhibitor were very important for the binding, and residuesat P2.and P3 positions may be modified to improve the binding specificity. S3 subsite of the enzyme still hadspace to modify the inhibitors in increasing the binding affinity. CONCLUSION: The information presented hereis valuable and could be used to identify small molecular inhibitors of b-secretase.

关键词： molecular dynamics secretase essential dynamics computer simulation Alzheimer disease

Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus *** Strain 10-2.

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 519-528页

作者： Lei QIN , Heng-An WANG1 , Zhong-Qin WU, Xiao-Feng ZHANG, Mei-Lei JIN,Zi-Xin DENG2. and Guo-Ping ZHAOshanghairesearch {2. of biotechnology, shanghai Institute for biological sciences, chinese academy of sciences,Graduate School of the chinese academy of sciences, shanghai 2.02.3, China 1School of Agriculture and Biology, shanghai Jiaotong University, shanghai 2.1101, China 2.chool of Life Science and biotechnology, shanghai Jiaotong University, shanghai 2.0030, China shanghairesearch {2. of biotechnology Shanghai Institute for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences School of Agriculture and Biology Shanghai Jiaotong University

The hmr19 gene was cloned from Streptomyces hygroscopicus subsp. yingchengensis strain 10- 2., a bacterium strain producing agricultural antibiotics. Sequence similarity comparison indicates that hmr19 gene may encode a predicted protein with 14 putative transmembrane α-helical spanners, belonging to the drug:H+ antiporter-2.family of the major facilitator superfamily. The expression of hmr19 in the mycelium of strain 10-2. was detected by Western blotting analysis. Gene replacement technology was employed to construct an hmr19 disruption mutant. The growth inhibition test against different antibiotics indicated that the mutant strain was 5–2. fold more susceptible to tetracycline, vancomycin and mitomycin C than the parental wild type strain. The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-2.. While with the addition of an energy uncoupler, carbonyl cyanide m- chlorophenylhydrazone, the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same. It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

关键词： multidrug resistance (MDR) gene replacement (knockout) Streptomyces Streptomyces hygroscopicus subsp. yingchengensis strain 10-2.