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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 SciencesShanghai 200031 China

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

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 SciencesShanghai 200031 China

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 SciencesShanghai 200031 China

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

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

Computational Identification of Novel Family Members of MicroRNA Genes in Arabidopsis thaliana and Oryza sativa

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Acta Biochimica et Biophysica Sinica 2005年第2期37卷 7+76-87页

作者： Yang LI Wei LI You-Xin JIN 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 SciencesShanghai 200031 China

MicroRNAs(miRNAs)are a class of endogenous small RNAs that play important regulatoryroles in both animals and plants,miRNA genes have been intensively studied in animals,but not in *** study,we adopted a homology search approach to identify homologs of previously validated plantmiRNAs in Arabidopsis thaliana and Oryza *** identified 20 potential miRNA genes in Arabidopsisand 40 in ***,providing a relatively complete enumeration of family members for these miRNAs *** addition,a greater number of Arabidopsis miRNAs(MIR168,MIR159 and MIR172)were found tobe conserved in *** the novel homologs,most of the miRNAs have closely related fellow miRNAs andthe number of paralogs varies in the different miRNA ***,a probable functional segmenthighly conserved on the elongated stem of pre-miRNA fold-backs of MIR319 and MIR159 family *** results support a model of variegated miRNA regulation in plants,in which miRNAs withdifferent functional elements on their pre-miRNA fold-backs can differ in their function or regulation,andclosely related miRNAs can be diverse in their specificity or competence to downregulate target *** that the sophisticated regulation of miRNAs can achieve complex biological effects through qualita-tive and quantitative modulation of gene expression profiles in plants.

关键词： microRNA post-transcriptional gene silencing rice Arabidopsis

Analysis of the Interaction between hPFTAIRE1 and PLZF in a Yeast Two-hybrid System

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Acta Biochimica et Biophysica Sinica 2006年第3期38卷 164-170页

作者： Yan-Kun GAO~# Mei JIANG~# Tao YANG Jiang-Ye CHEN~* 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 SciencesShanghai 200031 China

hPFTAIRE1 is a Cdc2-related kinase family *** search its substrates and regulatoryproteins,hPFTAIRE1 was fused to LexA and used as a bait to screen a human brain LexA two-hybrid *** this screening,seven hPFTAIRE1 interacting proteins,including promyelocytic leukemia zinc finger (PLZF),were *** interaction between PLZF and hPFTAIRE1 was confirmed by β-galactosidase assay andLeu growth *** encodes a transcription factor belonging to the POZ/BTB domain and Krüppelzinc finger (POK) *** highly conserved POZ/BTB domain plays a critical role in *** deleted the POZ/BTB and Krüppel zinc finger domains,respectively,and observed the inter-action between hPFTAIRE1 and truncated PLZFs by liquid β-galactosidase activity assay.A weak interactionwas detected between hPFTAIRE1 and *** also observed the interaction between PLZF and anotherCdc2-related kinase,PCTAIRE1.A similar result was *** interaction between PLZF and hPFTAIRE1or PCTAIRE1 was confirmed by co-immunoprecipitation assay in a yeast *** is a phosphoproteinand plays multiple roles during cell *** results suggest that hPFTAIRE1 and PCTAIRE1 may playimportant roles in the functional regulation of PLZF.

关键词： hPFTAIRE1 PLZF PCTIARE1 yeast two-hybrid interaction

Characterization of ATPase Activity of Recombinant Human Pif1

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Acta Biochimica et Biophysica Sinica 2006年第5期38卷 335-341页

作者： Yu HUANG Deng-Hong ZHANG Jin-Qiu ZHOU Max-Planck Junior Research Group at the state key laboratory of molecular biology,institute of biochemistry and cell biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china Max-Planck Junior Research Group at the state key laboratory of molecular biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China

Saccharomyces cerevisiae Piflp helicase is the founding member of the Pifl subfamily that isconserved from yeast to *** potential human homolog of the yeast PIFI gene has been cloned fromthe cDNA library of the Hek293 cell ***,we described a purification procedure of glutathione S-transferase（GST）-fused N terminal truncated human Pifl protein（hPif1ΔN）from yeast and characterizedthe enzymatic kinetics of its ATP hydrolysis *** ATPase activity of human Pif1 is dependent ondivalent cation,such as Mg2+,Ca2+and single-stranded *** for ATP for the ATPase activity isapproximately 200 μ*** the ATPase activity is essential for hPifl’s helicase activity,these results willfacilitate the further investigation on hPif1.

关键词： Pifl helicase telomere telomerase ATPase

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 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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Mechanisms of microRNA-mediated gene regulation

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Science china(Life sciences) 2009年第12期52卷 1111-1116页

作者： ZHAO Shuang & LIU Mo-Fang· state key laboratory of molecular biology, institute of biochemistry and cell biology, shanghai institutes for biological sciences, 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 the Chinese Academy of Sciences Shanghai China

microRNAs (miRNAs) are identified as a class of non-protein regulators and a new source for broad control of gene expression in eukaryotes. The past years have witnessed substantial progress in understanding miRNA functions and mechanisms, although a few controversies remain. Various hypotheses and models have been suggested for the mechanisms of miRNA repression, including translational inhibition at the level of initiation or elongation, rapid degradation of the nascent peptide, mRNA degradation, and mRNA sequestration into P bodies (processing bodies) and SGs (stress granules) for degradation or/and storage. Recently, some noncanonical miRNA regulation, such as miRNA activation and de-repression of miRNA inhibition, have been uncovered. This review discusses some recent advances about how miRNAs regulate their targets and various modes of miRNA function.

关键词： miRNA repression activation de-repression processing bodies stress granules

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