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A transcription assay for EWS oncoproteins in Xenopus oocytes

Protein & Cell 2010年第10期1卷 927-934页

作者： King Pan Ng Felix Cheung Kevin A.W.Lee Department of Biology The Hong Kong University of Science and TechnologyClear Water BayKowloon Hong KongChina

Aberrant chromosomal fusion of the Ewing's sarcoma oncogene(EWS)to several different cellular partners produces the Ewing's family of oncoproteins(EWSfusion-proteins,EFPs)and associated tumors(EFTs).EFPs are potent transcriptional activators,dependent on the N-terminal region of EWS(the EWS-activationdomain,EAD)and this function is thought to be central to EFT oncogenesis and maintenance.Thus EFPs are promising therapeutic targets,but detailed molecular studies will be pivotal for exploring this potential.Such studies have so far largely been restricted to intact mammalian cells while recent evidence has indicated that a mammalian cell-free transcription system may not support bona fide EAD function.Therefore,the lack of manipulatable assays for the EAD presents a significant barrier to progress.Using xenopus laevis oocytes we describe a plasmid-based micro-injection assay that supports efficient,bona fide EAD transcriptional activity and hence provides a new vehicle for molecular dissection of the EAD.

关键词： EWS/ATF1 Ewing's sarcoma microinjection Xenopus oocytes transcription EWS-activation domain

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Functional characterization of sex pheromone receptors in Spodoptera frugiperda, S. exigua, and S. litura moths

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Insect Science 2023年第2期30卷 305-320页

作者： Sai Zhang Fang Liu Bin Yang Yang Liu Gui-Rong Wang State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBejingChina College of Horticulture and Plant Protection Yangzhou UniversityYangzhouChina Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of AgricultureAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhenChina

Moths possess an extremely sensitive and diverse sex pheromone processing system,in which pheromone receptors(PRs)are essential to ensure communication between mating partners.Functional properties of some PRs are conserved among species,which is important for reproduction.However,functional differentiation has occurred in some homologous PR genes,which may drive species divergence.Here,using genome analysis,17 PR genes were identified from Spodoptera frugiperda,S.exigua,and S.litura,which belong to 6 homologous groups(odorant receptor[OR]6,11,13,16,56,and 62);of which 6 PR genes(OR6,OR11,OR13,OR16,OR56,and OR62)were identified in S.frugiperda and S.exigua,and 5 PR genes were identified in S.litura,excluding OR62.Using heterologous expression in Xenopus oocytes,we characterized the functions of PR orthologs including OR6,OR56,and OR62,which have not been clarified in previous studies.OR6 orthologs were specifically tuned to(Z,E)-9,12-tetradecadienyl acetate(Z9,E12-14:OAc),and OR62 orthologs were robustly tuned to Z7-12:OAc in S.frugiperda and S.exigua.The optimal ligand for OR56 was Z7-12:OAc in S.frugiperda,but responses were minimal in S.exigua and S.litura.In addition,SfruOR6 was male antennae-specific,whereas SfruOR56 and SfruOR62 were male antennae-biased.Our study further clarified the functional properties of PRs in 3 Spodoptera moth species,providing a comprehensive understanding of the mechanisms of intraspecific communication and interspecific isolation in Spodoptera.

关键词： pheromone pheromone receptor Spodoptera Xenopus oocytes

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Influence of Y151 F mutation in loop B on the agonist potency in insect nicotinic acetylcholine receptor

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Insect Science 2009年第5期16卷 365-369页

作者： Feng Song Yi-Xi Zhang Xiang-Mei Yao Ze-Wen Liu Key Laboratory of Monitoring and Management of Plant Disease and Insects Ministry of Agriculture Nanjing Agricultural UniversityNanjing China

Nicotinic acetylcholine receptors （nAChRs） are ligand-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in α subunits together with loops D-F present in either non-α subunits or homomer-forming α subunits. Although Y151 in loop B has been identified as important in agonist binding, various residues at the 151-site are found among vertebrate and invertebrate nAChR α subunits, such as F 151. In Xenopus oocytes expressing Nlα1 or Nlα1^Y151F plus rat β2, Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine, but not imidacloprid, including the decrease Of Imax, the increase of EC50 （the concentration causing 50% of the maximum response） and the fast time-constant of decay （τf）. By comparisons of residue structure, the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between Nlα1/β2 nAChRs and acetylcholine, and the phenyl group to be important between Nlα1/β2 nAChRs and imidacloprid.

关键词： acetylcholine agonist potency imidacloprid nicotinic acetylcholine receptor Nilaparvata lugens Xenopus oocytes

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Effects of Glutamic Acid on C-type Inactivation of Kv1.4ΔN Channel

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South China Journal of Cardiology 2008年第1期9卷 39-43页

作者：叶琤李晓艳舒周伍蒋学俊 Department of Cardiology Renmin HospitalWuhan University

Objectives Acidosis has an inhibitory effect on the inactivation of Kv1.4 ΔN channel through the position H508. So in order to show the effects of glutamic acid on the mutant Kv 1.4 channel that lacks N-type inactivation （Kv1.4 Δ2-146）, we studied in the expression system of the Xenopus oocytes. Methods The two-electrode voltage-clamp technique （TEV） was used to record the currents. Results Acidosis increased fKv1.4 Δ2-146 C-type inactivation. After application of glutamic acid （1 mmol/L） to Kv1.4 Δ2-146 increased C-type inactivation further, changed inactivation time constants from （2.02 ± 0.39 s ） to （1.71 ± 0.23 s）（P〈 0.05） at ＋50mv, and shifted the steady-state inactivation curves of Kv1.4 ΔN to positive potential, which was from （-44.30 ± 0.59 mV） to （-39.88 ± 0.29 mV）（P〈0.05）. and slowed the rate of recovery from inactivation, which was from （1.64 ± 0.19 s） to （1.91 ± 0.23 s）（P〈 0.05）. Conclusions Together, these results suggest that 1 mmol/L glutamic acid accelerates the C-type inactivation of Kv1.4 ΔN in pH 6.8.

关键词： glutamic acid Kv1.4ΔN C-type inactivation Xenopus oocytes

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绿盲蝽温度受体基因的鉴定及功能研究

绿盲蝽温度受体基因的鉴定及功能研究

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作者：付婷中国农业科学院

学位级别：硕士

昆虫的生长发育、迁飞、滞育、休眠等生命活动和重要的行为与温度变化息息相关。在长期的进化过程中,快速感知外界环境温度变化并迅速做出相应的行为反应对于昆虫的正常生存和繁衍极其重要。以前的研究表明瞬时受体电位通道（transient ... 详细信息

昆虫的生长发育、迁飞、滞育、休眠等生命活动和重要的行为与温度变化息息相关。在长期的进化过程中,快速感知外界环境温度变化并迅速做出相应的行为反应对于昆虫的正常生存和繁衍极其重要。以前的研究表明瞬时受体电位通道（transient receptor potential,TRP）参与了昆虫对温度的感知行为,被认为是哺乳动物和昆虫体内温度的受体。绿盲蝽Apolygus lucorum（Meyer-Dür）属于半翅目（Hemiptera）,盲蝽科（Miridae）,草盲蝽属（lygus Hahn）,是我国多种农作物和果树上的重要害虫。本论文通过生物信息学方法分析绿盲蝽转录组数据,获得了绿盲蝽两个温度受体基因（TRPA1和Painless）的片段,通过PCR结合RACE技术克隆了编码TRPA1和Painless的全长c DNA序列;分析了TRPA1及Painless基因在绿盲蝽不同发育阶段及成虫不同组织中的相对表达量;通过真核表达结合双电极电压钳记录揭示了绿盲蝽TRPA1及Painless基因的功能,通过行为学实验研究了绿盲蝽对温度及化合物刺激的行为反应。主要研究结果如下:1.完成了绿盲蝽TRPA1基因的鉴定及功能研究。在触角转录组测序基础上,利用生物信息学方法获得了绿盲蝽TRPA1基因的4个序列片段,通过序列比对,结合分子克隆技术得到绿盲蝽TRPA1基因的四个剪切型的全长序列,分别命名为Aluc TRPA1-A、Aluc TRPA1-B、Aluc TRPA1-C、Aluc TRPA1-D。四个剪切型均包含6个保守的跨膜结构域及16个锚蛋白重复序列,该基因在1至5龄若虫及成虫中均有表达,且1龄若虫的相对表达量最高;在成虫的不同组织中,触角中的表达量最高。四种剪切型仅在腹部及翅的相对表达量存在显著差异。体外功能研究表明,除了Aluc TRPA1-D,其他三种剪切型均可被20-40?C逐渐上升的温度激活,而且重复刺激不会引起通道的钝化。Aluc TRPA1-A及Aluc TRPA1-B的激活温度小于25?C,而Aluc TRPA1-C的激活温度在25-30?C之间。除了对温度的反应外,四种剪切型中,仅Aluc TRPA1-A和Aluc TRPA1-C对刺激性化合物异硫氰酸烯丙酯（AITC）、肉桂醛（CA）及香茅醛（citronellal）有明显反应。2.完成了绿盲蝽Painless基因的鉴定及功能研究。利用生物信息学方法得到绿盲蝽Painless基因的4个c DNA片段,通过RT-PCR结合RACE技术克隆得到绿盲蝽Painless基因的全长序列,命名为Aluc Painless。该基因全长2736 bp,编码911个氨基酸,蛋白结构包含6个保守的跨膜结构域及8个锚蛋白重复序列。时空表达谱分析结果表明,Aluc Painless基因在整个生命周期均有表达,1龄若虫的相对表达量最高;在成虫的不同组织中,足中的表达量最高,头中最低。基于爪蟾卵母细胞的体外功能研究表明,10-3 mol/L的刺激性化合物不能激活Aluc Painless通道。20-45?C逐渐上升的温度可以激活Aluc Painless通道。由于Aluc Painless通道在20-25?C、20-30?C、20-35?C及20-40?C温度区间的反应值与对照组没有显著差异,因此Aluc Painless基因的激活温度大于40?C。3.完成了绿盲蝽对温度、气味刺激的行为反应实验。绿盲蝽在温度梯度平板上的分布主要集中于25-28?C,绿盲蝽在高温区没有分布,表明绿盲蝽能够感知环境温度。双向选择实验表明1%CA、10%CA、1%citronellal及10%citronellal趋避绿盲蝽,而绿盲蝽在AITC、CA及citronellal三种气味化合物不同浓度下的轨迹追踪实验表明,绿盲蝽能够通过嗅觉感知AITC、CA及citronellal并产生不适反应。

关键词：绿盲蝽温度受体气味受体 TRPA1 Painless Xenopus oocytes

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Lipid modification and meiotic cell cycle regulation

Lipid modification and meiotic cell cycle regulation

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第三届细胞结构与功能的信号基础研讨会

作者： Huwei Wang and Jian Zhang Centre for developmental biology,Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

Maternal factors play important roles during early animal development.One of the major functions of maternal factors is to regulate meiotic cell cycle during oogenesis.We report here a maternal gene Xlacs,whose mRNA is tightly localized in the vegetal cortex of Xenopus oocytes.XLACS is a putative long chain fatty acyl-CoA synthetase,whose major products may include palmitoyl-CoA, myristoyl-CoA and oleoyl-CoA.Depletion of Xlacs mRNA by antisense oligos blocks the first cell cleavage of fertilized eggs,suggesting that Xlacs is essential for cell cycle progression.The blockage may be resulted from abnormal acceleration of G2/M transition during xenopus meiosis,which was observed by the depletion.The acceleration can be rescued by co-injection of in vitro transcribed Xlacs mRNAs, suggesting the enzyme activity of XLACS is required for controlled release G2 oocytes into M phase.We propose that XLACS may negatively regulate cell cycle progression during the meiosis in xenopus.

关键词： acyl-CoA synthetase meiotic cell cycle Xenopus oocytes maternal

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Fatty acyl-CoA synthetase and meiotic cell cycle regulation

Fatty acyl-CoA synthetase and meiotic cell cycle regulation

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“细胞活动生命活力”——中国细胞生物学学会全体会员代...

作者： Huwei Wang,Jian Zhang Centre for Developmental Biology,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing,China

Maternal factors play important roles during early animal development.One of the major functions of maternal factors is to regulate meiotic cell cycle during oogenesis.We report here a maternal gene Xacsl, whose mRNA is tightly localized in the vegetal cortex of Xenopus oocytes.Xacsl is a putative long chain fatty acyl-CoA synthetase,whose major products may include palmitoyl-CoA,myristoyl-CoA and oleoyl-CoA.Depletion of Xacsl mRNA by antisense oligos blocks the first cell cleavage of fertilized eggs, suggesting that Xacsl is essential for cell cycle progression.The blockage may be resulted from abnormal acceleration of G2/M transition during xenopus meiosis,which was observed by the depletion.The acceleration can be rescued by co-injection of in vitro transcribed Xacsl mRNAs,suggesting the enzyme activity of Xacsl is required for controlled release G2 oocytes into M phase.We propose that Xacsl may negatively regulate cell cycle progression during the meiosis in xenopus.

关键词： Fatty acyl-CoA synthetase meiotic cell cycle Xenopus oocytes maternal

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Characterization of a novel alpha4/4-conotoxin,Qc1.2,from vermivorous Conus quercinus

Characterization of a novel alpha4/4-conotoxin,Qc1.2,from ve...

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第九届中国生物毒素学术研讨会

作者： Tanya Sanders Geoffrey Chew Edward Hawrot Department of Molecular Pharmacology Physiology and BiotechnologyBrown Medical SchoolProvidenceRhode Island 02912USA

＜正＞Theα4/4-conotoxin first identified in a vermivorous Conus speices, designated Qc1.2,was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea.The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds（Ⅰ-Ⅲ,Ⅱ-Ⅳconnectivity） in a native globular configuration.The mature peptide of Qc1.2 is supposed to contain an N-terminal posttranslationally processed pyroglutamate residue and a free carboxyl C-terminus.This peptide was chemically synthesized and refolded for further characterizing its function properties.The synthetic Qc1.2 has two interconvertible conformations in aqueous solution,which may due to the cis-trans isomerization of the two successive Pro residues in its first cysteine loop.Using the xenopus oocyte heterologous expression system,Qc1.2 was shown to selectively inhibit both rat neuronalα3β2 andα3β4 subtypes of nicotinic acetylcholine receptors with low potency.A block of approximately 63%and 37%of the ACh-evoked currents was observed,respectively,and the toxin dissociated rapidly from the receptors.Compared to other characterizedα-conotoxin members,the unusual structural features in Qc1.2 that confer to its receptor recognition profile are addressed.

关键词： nicotinic acetylcholine receptor subtypes α4/4-conotoxins posttranslational modification Xenopus oocytes

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