In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI.

The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I-AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I-AniI in vitro and utilized competition assays to probe the relationship between the RNA- and DNA-binding sites. Despite functioning as an RNA maturase, I-AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA-binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I-AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I-AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I-AniI appears to contain two binding sites.     

猪肝过氧化氢酶提取条件的研究

采用水浸提法提取猪肝过氧化氢酶。选择浸提时间、温度、料水比作为单因素进行梯度实验,确定其条件范围,再通过进一步的正交实验得到猪肝过氧化氢酶提取条件的优化组合,即料水质量比1：5,浸提温度25℃,浸提时间8h。     

Residue conservation information for generating near-native structures in protein-protein docking

MOTIVATION: Protein-protein docking algorithms typically generate large numbers of possible complex structures with only a few of them resembling the native structure. Recently (Duan et al., Protein Sci, 14:316-218, 2005), it was observed that the surface density of conserved residue positions is high at the interface regions of interacting protein surfaces, except for antibody-antigen complexes, where a lesser number of conserved positions than average is observed at the interface regions. Using this observation, we identified putative interacting regions on the surface of interacting partners and significantly improved docking results by assigning top ranks to near-native complex structures. In this paper, we combine the residue conservation information with a widely used shape complementarity algorithm to generate candidate complex structures with a higher percentage of near-native structures (hits). What is new in this work is that the conservation information is used early in the generation stage and not only in the ranking stage of the docking algorithm. This results in a significantly larger number of generated hits and an improved predictive ability in identifying the native structure of protein-protein complexes. RESULTS: We report on results from 48 well-characterized protein complexes, which have enough residue conservation information from the same 59 benchmark complexes used in our previous work. We compute conservation indices of residue positions on the surfaces of interacting proteins using available homologous sequences from UNIPROT and calculate the solvent accessible surface area. We combine this information with shape-complementarity scores to generate candidate protein-protein complex structures. When compared with pure shape-complementarity algorithms, performed by FTDock, our method results in significantly more hits, with the improvement being over 100% in many instances. We demonstrate that residue conservation information is useful not only in refinement and scoring of docking solutions, but also helpful in enrichment of near-native-structures during the generation of candidate geometries of complex structures.     

PI3KγmRNA 3′非翻译区可能存在基因表达负调控区

为探索人磷脂酰肌醇 3 激酶γ(phosphoinositide 3 kinasePI3Kγ)基因 3′端非翻译区内AU富含区是否在基因表达调控中起作用 ,首先通过生物信息学分析发现在其 3′端非翻译区 (UTR)内存在0 9kb的AU富含区 ,其中包括 4个AU富含元件 ,以及 1个与众多基因非翻译区高度同源的长 130个碱基的区域 .将AU富含区插入报告基因egfp的下游构建pcDNA3 egfp AUR表达载体 .将表达载体转导NIH 3T3,74 0 2及K5 6 2细胞 ,流式细胞检测egfp的表达情况 .PI3Kγ基因 3′非翻译区AU富含区可显著降低egfp的表达 2～ 3倍 (P <0 0 1) .利用放线菌素D阻断RNA转录后 ,Northern印迹分析结果显示egfp AURmRNA较egfpmRNA不稳定 .实验结果提示 ,PI3Kγ基因 3′非翻译区AU富含区内可能存在转录后水平的基因表达负调控区 ,该负调控区可在一定程度上加速mRNA的衰变     

烟粉虱传毒相关蛋白基因的克隆及其表达量分析

【目的】烟粉虱Bemisia tabaci(Gennadius)是一种世界性的入侵性害虫,其传播的番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)给我国番茄生产造成巨大经济损失。为了阐明烟粉虱传播双生病毒的机理,本文拟研究明确B和Q烟粉虱体内传毒相关蛋白Gro EL基因及其表达量。【方法】采用特异性引物克隆了B和Q烟粉虱内共生菌编码的传毒相关蛋白Gro EL基因,并进行序列分析;并利用荧光定量PCR技术检测两种生物型及其获取双生病毒前后该基因的相对表达量。【结果】烟粉虱内共生菌Hamiltonella编码的Gro EL基因全长为1 668 bp,编码555个氨基酸;B、Q烟粉虱该基因的核苷酸序列相似性为99.94%,氨基酸同源性为99.82%;带毒烟粉虱中Gro EL基因的表达量显著高于未带毒的对应生物型烟粉虱;无论带毒与否,Q烟粉虱该基因的表达量均显著高于B(P<0.05)。【结论】烟粉虱携带TYLCV后可诱导Gro EL的表达量升高,B和Q烟粉虱中Gro EL基因及其表达量均存在差异,这可能是B和Q烟粉虱传毒效率存在显著差异的原因之一。     

白马雪山黑白仰鼻猴(Rhinopithecus bieti)垂直迁移

2000-2001年间,在白马雪山国家级自然保护区南任村附近,通过在四个山坡每隔海拔100米的样带(宽5.0米)内搜集黑白仰鼻猴的粪便来研究猴群不同季节的垂直迁移模式。同时,通过在每个地区山脊上和山沟内搜集2.5米宽垂直样带内的粪便,来研究猴群不同季节对山脊和山沟的选择性。结果表明:猴群在海拔3500-4300米的区域活动。虽然猴群全年集中利用3900-4200米的林带;但是具有季节性差异:夏季最高(4200米),依次是秋季(4100米)和冬季(4000米),夏季最低(3900米)。另外,冬季山沟中的粪便多于山脊说明猴群在沟中停留时间长,这与沟中少风且温度高有关。不同海拔带上的粪便密度和松萝量间正相关,这意味着食物资源的垂直分布是影响猴群垂直迁移的重要因素。春季,猴群会下到低海拔采食嫩芽/叶,而冬季则下到低海拔处躲避首次大雪,这很可能导致猴群集中利用第二个海拔带。     

CLA对急性缺氧大鼠肝脏线粒体呼吸链酶活性及氧化应激的影响

目的:观察共轭亚油酸(CLA)对急性缺氧大鼠肝脏线粒体呼吸链酶活性及肝脏内氧化应激(OS)的影响。方法:将60只SD大鼠随机分为正常对照组,模拟海拔5000米高原环境连续缺氧暴露3d的急性缺氧组,急性缺氧CLA干预组。生化法测定肝脏组织中丙二醛(MDA)、还原型谷胱甘肽(GSH)含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,Clark氧电极法观察大鼠肝脏线粒体呼吸链酶活性。结果:成功诱导大鼠急性缺氧模型。与对照组比,急性缺氧组大鼠肝脏MDA含量明显升高(P<0.05),同时肝脏线粒体呼吸链酶活性以及抗氧化酶活性(SOD,CAT)及GSH显著降低(P<0.05),与急性缺氧相比,CLA治疗组大鼠以上各项指标均有改善,并存在一定的剂量效应关系。结论:CLA通过抑制氧化应激增强大鼠肝脏线粒体呼吸链酶活性改善了急性缺氧大鼠肝脏的能量代谢障碍,对急性缺氧引起的氧化损伤有保护作用。     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

华东稻麦轮作生态系统的N2O排放研究

根据对华东稻麦轮作周期的N2 O排放及其影响因子的连续观测结果 ,分析了N2 O排放时间变化以及施肥、灌溉、温度、土壤湿度和土壤速效N素含量对N2 O排放的影响 ,同时还比较分析了稻田N2 O和CH4排放 .研究结果表明 ,稻麦轮作周期内 ,水稻生长季的N2 O排放量仅占 30 % ,稻田持续淹水可比常规灌溉增加CH4排放量 2 6% ,减少N2 O排放量 1 1～ 2 6% .     

受干扰的生态系统Ⅱ．柞蚕林N循环与调控

揭示了柞蚕林Ｎ循环与放蚕强度、土壤肥力、剪伐周期等因子之间的关系结果表明,柞蚕茧收获和周期性剪伐柞树枝叶是影响柞蚕场Ｎ平衡的主要因素．放蚕强度和剪伐周期必须根据柞蚕场土壤肥力水平和柞树生长势来确定．调整林下植被组成、种植固Ｎ植物能明显提高土壤供Ｎ能力,适当施用速效Ｎ肥能有效地促进保苗场幼树、幼龄柞墩和恢复老龄柞墩的生长势