cmv-3′LTR嵌合启动子对逆转录病毒载体MFG滴度及外源基因表达的影响

为减轻逆转录病毒载体介导的外源基因的沉默,进一步提高逆转录病毒载体MFG介导的外源基因的表达水平,同时探讨逆转录病毒3′端长末端重复序列(long terminalrepeat,LTR)内U3区对病毒基因表达的影响,将逆转录病毒载体3′端LTR内的U3区用cmv核心增强子、启动子序列替代,同时去除了3个与逆转录病毒载体启动子甲基化有关的序列NCR、DR,并以egfp为报告基因,构建了MFG egfp和MFG egfp cmv表达载体。结果显示:利用cmv启动子替代MFG载体3′端LTR的U3启动子序列,会显著降低MFG载体的病毒滴度及其介导的外源报告基因的表达。提示利用cmv启动子替代病毒载体的3′端LTR内的U3区,并不是提高MoMLV(moloneymurineleukemivirus)逆转录病毒载体介导外源基因的表达及其病毒滴度的理想策略。结果也同时提示:在MoMLV逆转录病毒3′端LTR的U3区,可能存在与病毒RNA加工、成熟及稳定性有关的信号序列。     

cmv-3’LTR嵌合启动子对逆转录病毒载体MFG滴度及外源基因表达的影响

为减轻逆转录病毒载体介导的外源基因的沉默,进一步提高逆转录病毒载体MFG介导的外源基因的表达水平,同时探讨逆转录病毒3’端长末端重复序列(long-terminal repeat,LTR)内U3区对病毒基因表达的影响,将逆转录病毒载体3’端LTR内的U3区用cmv核心增强子、启动子序列替代,同时去除了3个与逆转录病毒载体启动子甲基化有关的序列NCR、DR,并以egfp为报告基因,构建了MFG-egfp和MFG-egfp-cmv表达载体。结果显示：利用cmv启动子替代MFG载体3’端LTR的U3启动子序列,会显著降低MFG载体的病毒滴度及其介导的外源报告基因的表达。提示利用cmv启动子替代病毒载体的3’端LTR内的U3区,并不是提高MoMLV(moloney murineleukemi virus)逆转录病毒载体介导外源基因的表达及其病毒滴度的理想策略。结果也同时提示：在MoMLV逆转录病毒3’端LTR限的U3区,可能存在与病毒RNA加工、成熟及稳定性有关的信号序列。     

牛泡沫病毒长末端重复序列在大肠杆菌中的启动子功能

泡沫病毒具有复杂的基因组结构,包含典型反转录病毒共有的长末端重复序列(long terminal repeat,LTR)启动子,负责起始结构基因gag、pol、env的表达,并在env基因内部存在独有的内部启动子(internal promoter,IP)[13],用以起始下游反式作用因子Tas(在BFV中称Borf-1)的表达.有报道劳斯肉瘤病毒(rous sarcoma virus,RSV)和人免疫缺陷病毒(human immunodeficiency virus,HIV)LTR可在E.coli中起始基因表达[4,7].泡沫病毒至今未见报道.     

基于生物信息学的HERV研究现状与发展趋势

近年来研究证实,人内源性逆转录病毒（HERV）中长末端重复序列（LTR）和囊膜蛋白基因（env）与基因异常表达关系密切。本文阐述了HERV国内外研究现状与发展动态。从生物信息学角度对LTR中启动子、多聚腺苷化（PolyA）信号和增强子等顺式作用元件以及env中的免疫抑制区（ISD）进行了重点分析。指出上述元件和潜在功能区在基因的异常表达和囊膜蛋白的免疫抑制中起关键作用,必将成为HERV与基因异常表达关系研究的热点与趋势。通过此类研究,以期为癌症疾病的早期诊断和治疗靶位点的选择奠定理论基础。     

禽网状内皮组织增殖病病毒LTR序列的启动子功能

通过PCR方法,将禽网状内皮组织增殖病病毒(REV)的长末端重复序列(LTR)扩增并克隆进pUC-18质粒多克隆位点(MCS)的EcoR I和SacI之间,并以BGH基因的多聚腺苷酸序列作为终止子克隆到SphⅠ和Hind Ⅲ之间,构建成重组质粒pUC-LTR.将GFP基因和REV囊膜糖蛋白gp90基因分别克隆到pUC-LTR载体中,获得质粒pUC-LTR-GFP和质粒pUC-LTR-gp90.重组质粒经转染48h,能够检测到外源基因的表达.本研究提示,REV LTR能够作为启动子构建表达质粒.     

二株B亚群禽白血病病毒全基因组序列及其在细胞上的复制性比较

本研究比较了从山东地方品系鸡群分离到的二株B亚型禽白血病病毒(ALV)SDAU09E3和SDAU09C2的全基因组序列及它们在细胞培养上的复制动态。这二株ALV-B的同源性为95.4%,与GenBank中3株B亚群参考株之间的同源性也均在91.0%～94.9%间,而与其它亚群参考株的同源性均低于87.9%。与亚群无关的gag、pol基因和LTR的核苷酸序列比较表明,这二株ALV-Bgp85基因的gag和pol基因与所有比较的参考株的同源性均在93%以上。LTR与其他外源性ALV参考株的LTR间的同源性在72.6%～88.3%范围内,但与E亚群内源性ALV的LTR的同源性只有51.5%。然而,这二个ALV-B的LTR的同源性也只有74.8%,远低于其他基因组部分的同源性,特别是它们的LTR的U3区同源性只有68.8%,二者在二个CAAT分布上也显著不同。对这二株ALV-B在DF-1细胞上的复制动态比较表明,它们在细胞培养上清液中的TCID50值非常类似,但SDAU09E3株核衣壳蛋白p27抗原的含量显著高于SDAU09C2株。这表明,同一亚群的不同毒株在复制过程中,所表达的p27抗原量与所形成的具有传染性的病毒量间没有平行关系。这一差异与LTR-U3区的相关性则有待应用感染性克隆技术来做进一步深入研究。     

SPF鸡胚中内源性白血病病毒全基因序列鉴定与分析

摘要:以国内某3家SPF鸡场的SPF鸡胚成纤维细胞提取的基因组DNA为模板,参照已发表的序列,设计合成了4对检测内源性白血病病毒引物,分别检测gag基因、pol基因、env基因和LTR片段,结果显示4者检出阳性率很高(gag,29/46;pol,27/46;env,24/46;LTR,31/46).设计合成了8对引物,选取4片段检测均为阳性的样品之一,经PCR成功扩增出了8段连续的、相互部分重叠的目的DNA片段,分别连接入T载体进行克隆测序.用DNAstar软件对测序结果进行拼接,从一个鸡胚得到了内源性白血病病毒前病毒全基因组序列.比较分析发现,该序列env基因与已知的E亚群内源性病毒代表株env基因的核苷酸序列同源性在98.5%以上,全基因组序列同源性在99.1%以上,而与其他亚群代表株同源性相对较低,env基因同源性仅为56.3%～91.5%.     

马尾松毛虫CPV基因组S7的序列分析及部分序列的原核表达

马尾松毛虫质多角体病毒(湖南株)基因组S7节段(AY180908)cDNA克隆及序列分析结果表明S7由1501个碱基组成,编码由448个氨基酸组成的分子量为49.8 kDa的多肽P50.5′末端和3′末端具有5′-AGTAA-3′和5′-GTTAGCC-3′末端保守序列.该基因组与舞毒蛾质多角体病毒1型和家蚕质多角体病毒1型S7节段有很高的同源性,核苷酸序列同源性分别为97.2%和87.0%,氨基酸序列同源性分别为98.7%和92.8%.P50多肽与人型支原体的 DnaK样蛋白在C-末端有相似性.本文报道了编码P50 C259的cDNA序列的克隆并作了原核表达,当用1.0 mmol/L IPTG 诱导2h,分子量约为37.3 kDa的融合蛋白在大肠杆菌BL21中得到大量表达.     

人乙型肝炎病毒前表面抗原preS基因的克隆与序列分析

从杭州、兰州两地各一例乙型肝炎病毒(HBV)表面抗原阳性血清中提取病毒DNA,采取PCR技术扩增出前表面抗原(preS)基因片段,重组到质粒载体上,对该基因进行了全序列测定[GenBank索取号CpreS-HZAF325674;preS-LZAF325675].克隆的HBVpreS基因杭州分离物(preS-HZ)和兰州分离物(preS-LZ)全长522个核苷酸,编码174个氨基酸.preS-HZ与已发表的HBVadr亚型上海分离物[8]、北京分离物[9]、日本分离物[5]、HBVadw亚型[10]和ayw亚型[11]preS基因的核苷酸序列同源性分别为96.7%、96.2%、97.3%、88.7%和84.1%,氨基酸序列同源性分别为96.0%、94.9%、97.1%、85.1%和85.4%;preS-LZ与相应序列的核苷酸序列同源性分别为96.4%、96.2%、96.9%、88.7%、83.7%,氨基酸序列同源性分别为94.9%、94.9%、96.0%、85.1%、84.1%.分子进化分析(DNASTAR,1999)表明,克隆的两例HBVpreS基因属于adr亚型.preS-LZ与preS-HZ之间有两个核苷酸变异(对应两个氨基酸变异),相对于以上报道的序列二者含有四个特异的氨基酸突变位点.在免疫保护区内二者具有较好的保守性,可用于表达乙肝重组亚单位疫苗.     

植物活性长末端重复序列反转录转座子研究进展

长末端重复序列(Long terminal repeat,LTR)反转录转座子是真核生物基因组中普遍存在的一类可移动的DNA序列,它们以RNA为媒介,通过"复制粘贴"机制在基因组中不断自我复制。在高等植物中,许多活性的LTR反转录转座子已被详尽研究并应用于分子标记技术、基因标签、插入型突变及基因功能等分析。本文对植物活性LTR反转录转座子进行全面的调查,并对其结构、拷贝数和分布以及转座特性进行系统的归纳,分析了植物活性LTR反转录转座子的gag(种属特异抗原)和pol(聚合酶)序列特征,以及LTR序列中顺式调控元件的分布。研究发现自主有活性的LTR反转录转座子必须具备LTR区域以及编码Gag、Pr、Int、Rt和Rh蛋白的基因区。其中两端LTR区域具有高度同源性且富含顺式调控元件;Rt蛋白必备RVT结构域;Rh蛋白必备RNase_H1_RT结构域。这些结果为后续植物活性LTR反转录转座子的鉴定和功能分析奠定了重要基础。     

Land,livestock, and livelihoods: Changing dynamics of gender,caste, and ethnicity in a Nepalese Village

Over the past 10 years, Ghusel VDC, Lalitpur District has moved from primarily subsistence agriculture into the wider cash economy aided by the Small Farmers' Development Program (SFDP), which provides credit to farmers mainly for the purchase of buffalo for milk production, and by the National Dairy Corporation, which supports local dairy cooperatives. Analysis reveals that buffalo-keeping and milk sales are increasing the well-being of many households, while at the same time creating new inequalities in gender roles and responsibilities, greater inequities between Brahmin and Tamang residents in Ghusel, and placing pressures on the ecosystem for increased supplies of fodder and fuelwood. Evidence suggests that there is critical, need for attention to the social, and particularly gender-based, implications of maintaining livestock for milk sales and to the ecological underpinnings of this livelihood system.     

Tuning of electronic properties of one-dimensional cyano-bridged Cu-Ni, Cu-Pd, and Cu-Pt bimetallic assemblies by stereochemistry of ligands

Preparations, XPS and electronic spectroscopy, and magnetism of seven new one-dimensional cyano-bridged coordination polymers, chiral [Cu(RR-chxn)₂][Pd(CN)₄] · 2H₂O (1), [Cu(trans-chxn)₂][M(CN)₄] · 2H₂O (2, 4, and 6 for M = Pd, Ni, and Pt), and [Cu(cis-chxn)₂][M(CN)₄] · 2H₂O (3, 5, and 7 for M = Pd, Ni, and Pt) (RR-chxn = cyclohexane-(1R,2R)-diamine, trans-chxn = racemic trans-cyclohexane-(1,2)-diamine, and cis-chxn = racemic cis-cyclohexane-(1,2)-diamine) have been reported in view of tuning of their electronic properties by stereochemistry of chxn ligands and metal-substitution. Comparison of Cu 2p_1/2 and 2p_3/2 peaks of XPS and broad d-d bands around 18 000 cm⁻¹ of electronic spectra are described systematically for 1-7. Variable-temperature magnetic measurement shows that complexes 1-7 indicate weak antiferromagnetic interactions via cyano-bridges. Because of semi-coordination coupled with pseudo Jahn-Teller elongation and electrostatic interaction for 1, the axial Cu-N coordination bond distances of 2.330(7) and 3.092(8) Å are considerably longer than those of equatorial ones in the range from 2.016(6) to 2.030(6) Å. The former bond distances of 1 are intermediate values among the related Ni (2.324(6) and 3.120(8) Å) and Pt (2.34(1) and 3.09(1) Å) complexes.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Chiral Separation of G‐type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G‐type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical‐scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.     

Influence of Fluoride on Rat Kidney Antioxidant System: Effects of Methionine and Vitamin E

The aim of the study has been to determine and compare the influence upon the kidney antioxidative system, exercised by administration of vitamin E, and vitamin E in combination with methionine, under conditions of oxidative stress induced by sodium fluoride. The experiment was carried out on Wistar FL rats (adult males) that, for 35 days, were administered water, NaF, NaF with vitamin E, or vitamin E with methionine (doses: 10 mg NaF/kg of body mass/24 h, 3 mg vitamin E per 10 μl per rat for 24 h, 2 mg methionine per rat for 24 h). The influence of administered sodium fluoride and antioxidants upon the antioxidative system in kidney was examined by analyzing the concentration of malondialdehyde (MDA) and the activity of the most important antioxidative enzymes (SOD, total and both its isoenzymes, GPX, GST, GR, and CAT). The studies carried out confirmed the disadvantageous effect of the administered dose of NaF upon the antixodiative system in rats (increase in the concentration MDA, decrease activity of all antioxidative enzymes). The administration of vitamin E increased the activity of studied enzymes with the exception of glutathione reductase GR; it also reduced the procesess of lipid peroxidation. It has been found that combined doses of vitamin E and methionine were most effective in inhibiting lipid peroxidation processes. The results confirmed the antioxidative properties of methionine.     

Reorientation of Microfibrils and Microtubules at the Outer Epidermal Wall of Maize Coleoptiles During Auxin-Mediated Growth

Auxin-mediated elongation growth of isolated subapical coleoptile segments of maize (Zea mays L.) is controlled by the extensibility of the outer cell wall of the outer epidermis (Kutschera et al., 1987). Here we investigate the hypothesis that auxin controls the extensibility of this wall by changing the orientation of newly deposited microfibrils through a corresponding change in the orientation of cortical microtubules. On the basis of electron micrographs it is shown that cessation of growth after removal of the endogenous source of auxin is correlated with a relative increase of longitudinally orientated microfibrils and microtubules at the inner wall surface. Conversely, reinduction of growth by exogenous auxin is correlated with a relative increase of transversely orientated microfibrils and microtubules at the inner wall surface. These changes can be detected 30–60 min after the removal and addition of auxin, respectively. The functional significance of directional changes of newly desposited wall microfibrils for the control of elongation growth is discussed.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

八种脑-肠肽侧脑室内注射对大鼠基础胃酸分泌的影响

用乌拉坦麻醉大鼠作急性实验,采用连续灌流胃并收集流出液的方法,观察向侧脑室内注射微量脑-肠肽对大鼠基础胃酸分泌的影响。实验结果如下:(1)雨蛙肽、八肽胆囊收缩素、促甲状腺素释放激素及四肽胃泌素均使总酸排出量增加;(2)生长抑素、胰多肽、P 物质、胰高血糖素则使总酸排出量减少;(3)上述肽类用侧脑室注射的剂量作肌肉注射,除四肽胃泌素也产生明显的刺激胃酸分泌作用外,对胃酸分泌均无明显影响。以上结果提示,脑内的一些肽类可能以神经递质或调制物的方式,参与中枢对胃酸分泌的调节。     

A chromium-tolerant plant growing in Cr-contaminated land

In this paper, a kind of Typhaceae plant species, Typha angustifolia L, with a high tolerance to Cr is described. Experiments were carried out to examine its ability to tolerant Cr and its physiological response. The results showed that there was no difference in growth, plant height, and biomass response to external Cr (VI) between the plants exposed to 100 microM Cr (VI) and control (0 microM), while increasing Cr levels to 200-800 microM induced a significant decrease in plant height and biomass, but no significant injury was detected, even for the plants exposed to 800 microM Cr. Chromium induced significant increases in superoxide dismutase (SOD) and peroxidase (POD) activities. Meanwhile, a significantly positive correlation was found between Cr and Mn or Cu in leaves and roots, respectively. The Cr tolerance of the plant appeared to be associated with the enhancement of SOD and POD activities and the improvement in uptake and translocation of the essential microelements.