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Identification and Characterization of glnA Promoter and its Corresponding Trans-regulatory Protein GlnR in the Rifamycin SV Producing Actinomycete,Amycolatopsis mediterranei U32

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Acta Biochimica et Biophysica Sinica 2006年第12期38卷 831-843页

作者： Hao YU~(1&) Wen-Tao PENG~1 Yang LIU~1 Ting WU~2 Yu-Feng YAO~1 Ming-Xue CUI~1 Wei-Hong JIANG~1 Guo-Ping ZHAO~(1,3,4*) 1 laboratory of Molecular microbiology,Institute of Plant Physiology and Ecology,shanghai Institutes for Biological sciences,Chinese Academy of sciences,shanghai 200032,china 2 department of Medicinal Chemistry,school of Pharmacy,fudan university,shanghai 200032,china 3 state key laboratory of genetic engineering & department of microbiology,school of life science,fudan university,shanghai 200433,china 4 laboratory of Disease and Health Genomics,Chinese National Human Genome Center at shanghai,shanghai 201203,china laboratory of Molecular microbiology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China department of Medicinal Chemistry School of Pharmacy Fudan University Shanghai 200032 China state key laboratory of genetic engineering & department of microbiology School of Life Science Fudan University Shanghai 200433 China laboratory of Disease and Health Genomics Chinese National Human Genome Center at Shanghai Shanghai 201203 China

The genetic requirements for the transcription of glnA,encoding the major glutaminesynthetase in a rifamycin SV-producing Amycolatopsis mediterranei strain,U32,were *** experiments showed that the promoter of U32 glnA （pglnA） was likely to have two transcriptioninitiation sites:P1and P2,located 157 and 45 nucleotides （nt） upstream of the translational start codon,*** mobility shift and DNase I footprinting analyses revealed a 30 bp cis-element locatedat 45 to 75 nt downstream of P1,or 38 to 68 nt upstream of *** sequence of the cis-element displayedhigh similarity to the corresponding regions of pglnA from Streptomyces coelicolor and *** as a reporter gene,the expression levels of U32 pglnA and its deletion derivatives under differentnitrogen-source conditions were analyzed by detecting the catechol dioxygenase activities in ***54,*** J508 and *** FS10 （glnR mutant）.These in vivo studies showed that theactivation of U32 pglnA in *** required GlnR,and its binding to the U32 pglnA was furtherconfirmed by the gel mobility shift *** and heterologous expression of the U32 glnR allowed usto detect the in vitro interaction between the U32 GlnR and the corresponding pglnA *** shown by in vivo glnR inactivation and complementation indicated that GlnR is essential for theactive transcription of glnA in U32.

关键词： glnA promoter cis-element glnR Amycolatopsis mediterranei

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Bacterial Type Ⅰ Glutamine Synthetase of the Rifamycin SV Producing Actinomycete,Amycolatopsis mediterranei U32,is the Only Enzyme Responsible for Glutamine Synthesis under Physiological Conditions

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Acta Biochimica et Biophysica Sinica 2006年第12期 821-830页

作者： Wen-Tao PENG~(1&) Jin WANG~1 Ting WU~2 Jian-Qiang HUANG~1 Jui-Shen CHIAO~1 Guo-Ping ZHAO~(1,3,4) 1 laboratory of Molecular microbiology,Institute of Plant Physiology and Ecology,shanghai Institutes for Biological sciences,Chinese Academy of sciences,shanghai 200032,china 2 department of Medicinal Chemistry,school of Pharmacy,fudan university,shanghai 200032,china 3 state key laboratory of genetic engineering/department of microbiology,school of life science,fudan university,shanghai 200433,china 4 laboratory of Health and Disease Genomics,Chinese National Haman Genome Center at shanghai,shanghai 201203,china

The structural gene for glutamine synthetase,glnA,from Amycolatopsis mediterranei U32 wascloned via screening a genomic library using the analog gene from Streptomyces *** clone wasfunctionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutantunder the control of a lac *** analysis showed an open reading frame encoding a protein of466 amino acid *** deduced amino acid sequence bears significant homologies to other bacterialtype Ⅰ glutamine synthetases,specifically,71% and 72% identical to the enzymes of *** and Myco-bacterium tuberculosis,*** of this glnA gene in *** U32 led to glutamineauxotrophy with no detectable glutamine synthetase activity in *** contrast,the cloned glnA+gene cancomplement for both phenotypes in *** thus suggested that in *** U32,the glnA geneencoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratorydefined physiological conditions.

关键词： glnA glutamine synthetase Amycolatopsis mediterranei rifamycin SV

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cDNA Array Analysis of the Differential Expression Change in Virulence-related Genes During the Development of Resistance in Candida albicans

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Acta Biochimica et Biophysica Sinica 2005年第7期37卷 463-472页

作者： Zheng XU Yong-Bing CAO~1 Jun-Dong ZHANG~1 Ying-Ying CAO~1 Ping-Hui GAO~1 De-Jun WANG~1 Xu-Ping FU~2 Kang YING~2 Wan-Sheng CHEN~1 Yuan-Ying JIANG~1 1 department of Pharmacology,school of Pharmacy,Second Military Medical university,shanghai 200433,china 2 state key laboratory of genetic engineering,Institute of genetics.school of life science,fudan university,shanghai 200433,china department of Pharmacology School of Pharmacy Second Military Medical UniversityShanghai 200433 China state key laboratory of genetic engineering Institute of Genetics School of Life Science Fudan UniversityShanghai 200433 China

Candida albicans is the most frequently isolated fungus in immunocompromised patientsassociated with mucosal and deep-tissue *** investigate the correlation between virulence andresistance on a gene expression profile in ***,we examined the changes in virulence-related genesduring the development of resistance in *** from bone marrow transplant patients using a constructedcDNA array representing 3096 *** addition to the genes known to be associated with azole resistance,16 virulence-related genes were identified,whose differential expressions were newly found to be associatedwith the resistant *** expressions for these genes were confirmed by ***,the up-regulation of EFG1,CPH2,TEC1,CDC24,SAP10,ALS9,SNF1,SPO72and BDF1,and the down-regulation of RAD32,IPF3636 and UBI4 resulted in stronger virulence andinvasiveness in the resistant isolates compared with susceptible *** findings provide a link betweenthe expression of virulence genes and development of resistance during *** infection in bone marrowtransplant (BMT) patients,where *** induces hyphal formation and expression change in multiplevirulence factors.

关键词： Candida albicans cDNA array resistance virulence

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Reversal of hyperglycemia by protein transduction of NeuroD in vivo

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Acta Pharmacologica Sinica 2007年第8期28卷 1181-1188页

作者： Yin HUANG Jing CHEN Ge LI Tian-yue CHENG Ming-hong JIANG Shu-yu ZHANG Jun LU Shi YAN Wei-wei FAN Da-ru LU~1 state key laboratory of genetic engineering,Institute of genetics,school of life science,fudan university,shanghai 200433,china state key laboratory of genetic engineering Institute of Genetics School of Life Science Fudan University Shanghai China

Aim:To test whether the neurogenic differentiation(NeuroD)protein could alle-viate symptoms of diabetes mellitus by its transduction activity in ***:Type 1 diabetes mellitus in mice was induced by ip(intraperitoneal)injection ofstreptozotocin(150 mg/kg).One group of diabetic mice were intravenouslyinjected with the NeuroD-EGFP(Enhanced Green Fluorescent Protein)(5 mg/kg,n=6)and the other group with EGFP(5 mg/kg,n=5).After the transduction ofNeuroD-EGFP,the distribution of the protein was examined by means of frozensection under fluorescent microscope *** conducted RT-PCR andReal-time quantitative PCR to measure the transcription levels of insulin *** was utilized to detect the insulin ***-noassay was conducted to determine the serum insulin *** glucoselevels and body weights were regularly recorded after the protein ***:The NeuroD protein can be transduced into cells in vivo with a highefficiency of nearly 100%.Insulin mRNA was highly expressed in NeuroD-treateddiabetic mice,38-fold higher than that of control group(P337±39pg/mL,significantly higher than that of the control diabetic mice(n=5)which was84+23 pg/mL(P<0.01,t-test).Records of blood glucose level also displayed alle-viation of hyperglycemia after NeuroD administration(P<0.01,t-test,n=6).Conclusion:In vivo-transduced NeuroD in the small intestine remained function-ally active and could ameliorate the non-fasting glucose levels of streptozotocin-induced,diabetic mice by inducing enteric insulin expression.

关键词： neurogenic differentiation 1 type 1 diabetes mellitus intestines insulin

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Novel rAAV production system with low contamina-tion of helper virus

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Chinese science Bulletin 2003年第5期48卷 472-475页

作者： CHEN Li, CHEN Haoming, LI Ge, LU Daru & XUE Jinglun state key laboratory of genetic engineering, Institute of genetics,school of life sciences, fudan university, shanghai 200433, china Correspondence should be addressed to Xue Jinglun (e-mail: jlxue@ ***) state key laboratory of genetic engineering School of Life Sciences Fudan University Shanghai 200433 China

The Cre-loxP system was introduced in theproduction of recombinant AAV (rAAV). Defective adenovi-rus AdLC was used as the helper virus. While doing themass production of recombinant AAV carrying EGFP orhuman clotting factor Ⅸ (hFⅨ) gene, the generation ofhelper virus was significantly limited, it increased the sim-plicity of rAAV purification. The results from in vivo study demonstrated the superiority of this method. This systemprovides a novel approach for the application of rAAV sys-tem in gene therapy.

关键词：基因治疗重组腺病毒辅助病毒基因重组

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Characterization of a novel developmentally retarded mutant (drm1) associated with the autonomous flowering pathway in Arabidopsis

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Cell Research 2005年第2期15卷 133-140页

作者： Yong ZHU1,2,*, Hui Fang ZHAO1,3,*, Guo Dong REN1, Xiao Fei YU1, Shu Qing CAO1, Ben Ke KUAI1,4,**1department of Biochemistry, Ministry of Education key laboratory for Biodiversity science and Ecological engineering, school of life sciences, fudan university, 220 Handan Road, shanghai 200433, china 2school of Horticulture and Gardening, Southwest Forestry College, Kun Ming 650224, china 3department of biology, school of life and Environment science, shanghai Normal university, shanghai 200234, china4fudan Centre for genetic Diversity and designed Agriculture, fudan university, 220 Handan Road, shanghai 200433, china department of Biochemistry Ministry of Education Key laboratory for Biodiversity Science and Ecological Engineering School of Life Sciences Fudan University Shanghai China school of Horticulture and Gardening Southwest Forestry College Kun Ming China department of biology School of Life and Environment Science Shanghai Normal University Shanghai China fudan Centre for genetic Diversity and designed Agriculture Fudan University Shanghai China

A developmentally retarded mutant (drm1) was identified from ethyl methanesulfonate (EMS)-mutagenized M2 seedsin Columbia (Col-0) genetic background. The drm1 flowers 109 d after sowing, with a whole life cycle of about 160 *** also shows a pleiotropic phenotype, e.g., slow germination and lower germination rate, lower growth rate, curlingleaves and abnormal floral organs. The drm1 mutation was a single recessive nuclear mutation, which was mapped tothe bottom of chromosome 5 and located within a region of 20-30 kb around MXK3.1. There have been no mutantswith similar phenotypes reported in the literature, suggesting that DRM1 is a novel flowering promoting locus. Thefindings that the drm1 flowered lately under all photoperiod conditions and its late flowering phenotype was significantlyrestored by vernalization treatment suggest that the drm1 is a typical late flowering mutant and most likely associatedwith the autonomous flowering pathway. The conclusion was further confirmed by the revelation that the transcriptlevel of FLC was constantly upregulated in the drm1 at all the developmental phases examined, except for a very earlystage. Moreover, the transcript levels of two other important repressors, EMF and TFL1, were also upregulated in thedrm1, implying that the two repressors, along with FLC, seems to act in parallel pathways in the drm1 to regulateflowering as well as other aspects of floral development in a negatively additive way. This helps to explain why the drm1exhibits a much more severe late-flowering phenotype than most late-flowering mutants reported. It also implies that theDRM1 might act upstream of these repressors.

关键词： drm1 late-flowering autonomous flowering pathway Arabidopsis thaliana.

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Building of hFⅨ transgenic mice by spermatogenic cells

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Chinese science Bulletin 2003年第19期48卷 2061-2063页

作者： WANG Ning, CHEN Xiaoguang, CHEN Li, YAO Jihua, CHEN Haoming, SHEN Qi & XUE Jinglun state key laboratory of genetic engineering, Institute of genetics, school of life sciences, fudan university, shanghai 200433, china Correspondence should be addressed to Xue Jinglun (e-mail: jlxue @***) state key laboratory of genetic engineering Institute of Genetics School of Life Sciences Fudan University Shanghai China

Human FⅨ expression vector pCMVⅨ was packaged by EffecteneTM reagent and injected into mice seminiferous tubules with glass pipettes. The expressional frame of pCMVⅨ was examined by PCR and Southern blotting among 41 progenies. There were 2 (4%) mice being integrated with hFⅨ gene into chromosomes. 4.6 ng/mL of hFⅨ protein was expressed in plasma of one mouse, which was tested by ELISA. We demonstrated that building of transgenic animals by spermatogonial stem cells is an efficient method. Meanwhile, it has also been proved to be an alternative choice for mammary gland bioreactor.

关键词：基因转录老鼠精子生成细胞抗血友病因子 FⅨ 表达载体 pCMVⅨ 乳腺生物反应器

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Proteomic identification of potential target proteins regulated by an ASK1-mediated proteolysis pathway

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Cell Research 2006年第5期16卷 489-498页

作者： Kaiming Cao state key laboratory of genetic engineering department of Biochemistry and Molecular Biology school of life sciences fudan university shanghai 200433 china

The ASK1 (ARABIDOPSIS SKP1-LIKE) protein is a critical component of the SCF (Skpl-Cullin-F box protein)ubiquitin ligase complexes that recruit target proteins for degradation by the 26S *** investigate proteinsthat are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers,we compared the proteomes of theArabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis (2-DE).Ten protein spots withhigher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to furthermass spectrometry (MS) *** results showed that they were proteins involved in photomorphogenesis,circadianoscillation,post-translation process,stress-responses and cell expansion or elongation,suggesting that those processeswere affected in the ask1 *** transcript levels of these genes were also compared based on the Affymetrix genechip microarray *** significant difference was observed for most of the genes,suggesting that the proteins withelevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASKl-mediated *** results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes andalso demonstrate the importance and necessity of studying protein levels with respect to gene functions.

关键词： ASK1 two-dimensional polyacrylamide gel electrophoresis mass spectrometry proteolysis

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Effects of Citral on Aspergillus flavus Spores by Quasi-elastic Light Scattering and Multiplex Microanalysis Techniques

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Acta Biochimica et Biophysica Sinica 2004年第4期36卷 277-283页

作者： Man LUO1,2, Li-Ke JIANG2, Yao-Xiong HUANG3, Ming XIAO4, Bo LI4, and Guo-Lin ZOU1* 1College of life sciences, Wuhan university, Wuhan 430072, china 2College of life sciences, Anhui Agricultural university, Hefei 230036, china 3 Institute of Biomedical engineering, Ji’nan university, Guangzhou 510632, china 4Ministry of Education key laboratory for Biodiversity science and Ecological engineering, the Institute of Biodiversity science, fudan university, shanghai 200433, china College of life sciences Wuhan UniversityWuhan 430072 China College of life sciences Anhui Agricultural UniversityHefei 230036 China Institute of Biomedical engineering Ji'nan UniversityGuangzhou 510632 China Ministry of Education key laboratory for Biodiversity science and Ecological engineering the Institute of Biodiversity Science Fudan UniversityShanghai 200433 China

Citral refined from Litsea cubeba oil has been found to have a strong influence on fungi, especially Aspergillus flavus. Multiplex microanalysis and quasi-elastic light scattering techniques were applied to study the effects of citral on Aspergillus flavus spores from the levels of membrane, organelle and intracellular macromolecule. It was found that citral injured the wall and the membrane of A. flavus spore, resulting in decrease of its elasticity. After entering the cell, citral not only influenced the genetic expression of mitochondrion reduplication and its morphology, but also changed the aggregation of protein-like macromolecules. As a result, cells, organelles and macromolecules lost their normal structures and functions, eventually leading to the loss of germination ability of A. flavus spores. Since Litsea cubeba oil as food additive and antifungal agent is safe and less poisonous, it is important to elucidate the inhibitory mechanisms of Litsea cubeba oil on the germination ability of A. flavus spore.

关键词： citral Aspergillus flavus spore multiplex microanalysis techniques quasi-elastic light scattering technique

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Optical Waveguides from Organic/Inorganic Hybrid Materials

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光学学报 2003年第S1期23卷 151-152页

作者： Liying Liu Lei Xu Wencheng WangThe state key Lab for Advanced Photonic Materials and Devices, department of Optical science and engineering, fudan university, shanghai 200433, P. R. china The state key Lab for Advanced Photonic Materials and Devices Department of Optical Science and EngineeringFudan University Shanghai 200433 P. R. China

Organic/inorganic material has attracted great attentions because its importance as photonic materials. We report on our recent results on organic/inorganic hybrid sol-gel materials and optical waveguides like splitte... 详细信息

关键词： from In in as Optical Waveguides from Organic/Inorganic Hybrid Materials of mode that for be have

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