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Dose-dependent Inhibition of Gynecophoral Canal Protein Gene Expression in Vitro in the Schistosome(Schistosomajaponicum)by RNA Interference

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Acta Biochimica et Biophysica Sinica 2005年第6期37卷 386-390页

作者： Guo-Feng CHENG~1 Jiao-Jiao LIN~1 Yi SHI~2 You-Xin JIN~2 Zhi-Qiang FU~1 Ya-Mei JIN~1 Yuan-Cong ZHOU~2 You-Min CA~1 1 shanghai institute of Animal Parasitology, chinese academy of Agricultural sciences, key Lahoratory of Animal Parasitology,Ministry of Agriculture, shanghai 200232, china 2 state key laboratory, of molecular biology institute of biochemistry and cell biology shanghai institutes for biological sciences, chinese academy of sciences. shanghai 200031, china shanghai institute of Animal Parasitology Chinese Academy of Agricultural Sciences Key Laboratory of Animal Parasitology Ministry of Agriculture Shanghai 200232 China.

The gynecophoral canal protein gene SjGCP of Schistosoma japonicum that is necessary forthe pairing between the male and female worms is specifically expressed in the adult male *** is widely distributed in the adult female worm after *** transcription-polymerase chainreaction(RT-PCR)and immunofluorescence were employed to analyze the relationship between the RNAieffect and dsRNA dosage in the *** results revealed that the inhibition of SjGCP expression bysiRNA is ***-PCR analysis showed that the SjGCP transcript level was reduced by 75%when 100 nM dsRNA was applied.

关键词： RNA interference gynecophoral canal protein gene Schistosoma japonicum dosage

Studies on the Temperature Effect on Bacteriorhodopsin of Purple and Blue Membrane by Fluorescence and Absorption Spectroscopy

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 691-696页

作者： Lan-Ying CHENG~(1,2) Yue ZHANG~3 Shi-Gui LIU~1 Kun-Sheng HU~3 Kang-Cheng RUAN~2* 1 College of Life Science,Sichuan University,Chengdu 610064,china 2 state key laboratory of molecular biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china 3 institute of Biophysics,Academia Sinica,Beijing 100101,china state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of *** temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp *** binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to *** results suggest that significant structural difference existed between bRband *** was also found that both kinds of bR are highly thermal *** maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm *** transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high *** phenomenon has not been reported before.

关键词： bacteriorhodopsin purple membrane blue membrane fluorescence and absorption spectroscopy

Effect of specific nucleotide G11 on cleavage activity of hairpin ribozymes in vitro

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Progress in Natural Science:Materials International 2003年第5期 27-31页

作者： SONG Yuhu, LIN Jusheng , KONG Xinjuan, LI Bin, LIU Nanzhi and JIN Youxin (1. institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, china 2. state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, shanghai 200031, china) institute of Liver Diseases，Tongji Hospital，Tongji Medical College，Huazhong University of Science and Technology，Wuhan 430030，china state key laboratory of molecular biology，institute of biochemistry and cell biology，shanghai institutes for Biolog institute of Liver Diseases，Tongji Hospital，Tongji Medical College，Huazhong University of Science and Technology，Wuhan 430030，china state key laboratory of molecular biology，institute of biochemistry and cell biology，shanghai institutes for biological sciences，chinese academy of sciences，shanghai 200031，china

To study the effect of specific G11 nucleotide on cleavage activity of hairpin ribozyme in vitro, the 32P-labeled pKC transcript (267 nt) containing hepatitis B virus core region is used as target RNA. Synthetic hairpin ribozyme genes with G11 mutationa! site are cloned into ribozyme vector p1. 5 to create pHpRz for preparation of ribozyme. Non-labeled HpRz transcripts are incubated with 32P-labeled target-RNAs under different conditions and autoradiographed after gel electrophoresis. The results show that the cleavage activity of hairpin ribozyme is closely related to the matching of the bases between hairpin ribozyme and the substrate. The G11 site is not essential to cleavage activity of hairpin ribozyme in vitro, so the selection of target sequence matching G11 site of hairpin ribozyme is not limited to this site.

关键词： hairpin ribozyme kinetic of enzyme point mutant.

Regulation of mammalian pre-mRNA splicing

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Science china(Life sciences) 2009年第3期52卷 253-260页

作者： HUI JingYi state key laboratory of molecular biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china 1. state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

In eukaryotes,most protein-coding genes contain introns which are removed by precursor messenger RNA(pre-mRNA) *** splicing is a process by which multiple messenger RNAs(mRNAs) are generated from a single pre-mRNA,resulting in functionally distinct *** genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene ***-regulation of splicing causes a wide range of human *** review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA *** also discusses emerging directions in the field of alternative splicing.

关键词： pre-mRNA splicing alternative splicing splicing regulation signal transduction microRNA

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Cleavage of Supercoiled Circular Double-stranded DNA Induced by a Eukaryotic Cambialistic Superoxide Dismutase from Cinnamomum camphora

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Acta Biochimica et Biophysica Sinica 2004年第9期36卷 609-617页

作者： Bao-Zhong WANG, Xu-Bin WEI&, and Wang-Yi LIU* state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences,chinese academy of sciences, shanghai 200031, china state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences

A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity re- quires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA- cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

关键词： cambialistic SOD Fenton-type reaction hydroxyl radical relaxed circular duplex DNA supercoiled DNA cleavage

An Effective Method for Raising Antisera Against β-defensins: Double-copy Protein Expression of mBin1b in E. coli

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 571-576页

作者： Li-Qing XIAO, Ai-Hua LIU, and Yong-Lian ZHANG state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, Graduate School of the chinese academy of sciences, shanghai 200031, china state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences

Bin1b is a rat epididymis specific β-defensin which may have fertility related functions in addition to its antimicrobial activity. β-defensins are cysteine-rich cationic antimicrobial peptides that have their important implications in innate and adaptive immunity. Though considerable numbers of new β-defensins have been discovered, few corresponding antibodies have been reported. The small peptide with special structure and antimicrobial nature of β-defensins make them very difficult to express in prokaryotic system. Here we adopted a double-copy protein expression scheme based on which not only the mBin1b protein was successfully expressed but also the immunity of the antigen was enhanced. The validity of the antisera was verified by using Western blotting and immunohistochemical analyses. It will be a useful tool for deeply investigating the roles of Bin1b and also provide a simple but effective method in raising antisera against other members of the β-defensin gene family.

关键词： Bin1b epididymis β-defensin double-copy protein polyclonal antiserum

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 673-680页

作者： Chun-Hui HOU, Jian HUANG, and Ruo-Lan QIAN* state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, chinese academy of sciences, Graduate School of the chinese academy of sciences, shanghai 200031, china state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences

The developmental control of the human ε-globin gene expression is mediated by transcrip- tional regulatory elements in the 5’ flanking DNA of this gene. A previously identified negative regulatory element (–3028 to –2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at –3004 bp, –2975 bp and –2948 bp were characterized. Electrophoresis mobility shift assay (EMSA) showed that the putative NF-κB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (–177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at –2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-globin gene proximal promoter. However, the mutation of GATA site at –2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by ε-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at –2948 bp on SV40 promoter was not affected by the mutation of the putative NF-κB site, whereas it could be abolished by the mutation of GATA site at –2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by ε-globin gene proximal promoter. Those results suggested that ε- NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.

关键词： ε-globin gene regulatory element NF-κB GATA

Cinnamomin——a Versatile Type Ⅱ Ribosome-inactivating Protein

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Acta Biochimica et Biophysica Sinica 2004年第3期36卷 169-176页

作者： Hong XU and Wang-Yi LIU state key laboratory of molecular biology, the institute of biochemistry and cell biology, shanghai institutes for biological sciences, the chinese academy of sciences, 320 Yueyang Road, shanghai 200031, china state key laboratory of molecular biology the Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.

关键词： cinnamomin cinphorin ribosome-inactivating protein

MicroRNA in cell differentiation and development

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Science china(Life sciences) 2009年第3期52卷 205-211页

作者： SHI Yi & JIN YouXin state key laboratory of molecular biology,shanghai institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china state key laboratory of molecular biology Shanghai Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

The regulation of gene expression by microRNAs(miRNAs) is a recently discovered pattern of gene regulation in animals and *** have been implicated in various aspects of animal development and cell differentiation,such as early embryonic development,neuronal development,muscle development,and lymphocyte development,by the analysis of genetic deletions of individual miRNAs in *** studies show that miRNAs are key regulators in animal development and are potential causes of human *** we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

关键词： miRNA embryonic development neuronal development muscle development lymphocyte development

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