应用聚合酶链反应突变和克隆HIV-lgag基因片段

作者设计并合成了一对突变引物PGO1和PGO2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、BamHI和ATG及TAA序列,以便于HlV-1gag基因序列的定向克隆和表达。用PCO1和PGO2作引物,采用PCR方法从HIV-1基因组DNA中扩增出一个长504bp的DNA片段,用EcoRl和BamHI双酶切位点将此片段定向克隆入pUC19质粒。将克隆基因插入M13mp18进行DNA序列分析,结果表明,该基因序列及读框完全正确,且在其5′末端突变出EcoRI位点和ATG起始码,3′末端突变出TAA终业码和BamHI位点,从而为该基因的表达研究奠定了基础。     

用PCR突变技术克隆艾滋病病毒蛋白酶基因

作者设计并合成了一对用于PCR技术的突变引物HIV-1 Pr1和HIV-1Pr2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、HindⅢ和TAA序列,便于HIV-1 Pr基因的定向克隆和表达。用HIV-1 Pr1和HIV-1 Pr2作引物,采用PCR方法从HIV-1基因组DNA中扩增出了一个360bp长的DNA片段,用EcoRI和HindⅢ双酶切法将此片段定向克隆入pUC19质粒,将克隆基因插入M13mp18进行DNA序列分析。结果表明,该基因序列的读框完全正确,从而为HIV-1 Pr基因的表达及抑制剂的研究奠定了基础。     

EMS对D6-2转基因小鼠骨髓组织细胞内lacⅠ靶基因的诱变率和诱变谱

对D6 -2转基因小鼠骨髓组织细胞内lacⅠ靶基因的自发突变率以及甲基磺酸乙酯 (ethylmethanesulfonate ,EMS)诱发的突变率和突变谱进行了研究 .将带有lacⅠ靶基因的pSPORT1质粒通过酶切、环化和转化DH1 0B宿主菌的方式从经和未经诱变处理的D6- 2转基因小鼠骨髓组织基因组DNA中回收出来 ,lacⅠ靶基因突变体用M9/L选择性平板进行正向选择 .结果从经EMS诱变处理的小鼠基因组DNA中回收出的约 1 1 935个pSPORT1质粒中筛选到 6个lacⅠ-突变体 (EMS诱发的突变率为 5 0× 1 0 ^-5 ) ,而从对照组小鼠基因组DNA中回收出的约 1 1 6 4 9个质粒中没有检测到lacⅠ^-突变体 (自发突变率则小于 8.6× 1 0 ^-5 ) .对 6个突变体的 2个突变高发区进行了序列分析 ,共检测出 1 6个点突变 .其中 ,碱基置换突变为主要突变类型 ,占 87.5 % ( 1 4) ,其余 1 2 .5 % ( 2 )的突变为缺失突变 .在碱基置换突变中 ,颠换占6 4 % ;而转换仅占 36 % ,这既不同于用BigBlue 转基因小鼠所测到的lacⅠ基因在小鼠体内的自发突变谱 ,又不同于用穿梭载体 /哺乳动物细胞系统所测到的EMS在体外的诱变谱 .而突变发生的位点则与其他的研究报道相同 ,主要出现在CpG二核苷酸 ,占全部点突变的 45 % .这些结果表明 :( 1 )基于含 pSPORT1质粒的D6- 2转基因小鼠是一个能用于研究体内基因突变的分子机理和评估化学物质的遗传毒性作用的有效模型 ;( 2 )EMS在体内和体外的诱变特性似有本质的不同 ,但这有待于进一步的研究证实     

p53转录活化的p21和Gadd45在细胞DNA损伤监视中的作用

p2 1,Gadd45均是受抑癌基因p5 3转录调控的分子 ,当电离辐射引起细胞DNA损伤时 ,p2 1,Gadd45基因的转录以p5 3依赖的方式增强 .为了深入探讨p2 1,Gadd45在DNA损伤监视中的作用 ,构建了稳定表达p2 1及Gadd45反义RNA的p2 1^as/MCF 7及Gadd45 ^as/MCF 7细胞系 .对辐射引起这些细胞G₁期阻滞、DNA损伤修复和细胞凋亡的发生进行了研究 ,发现Gadd45同p2 1一样 ,当其表达受阻后辐射引起的细胞G₁期阻滞明显减弱 ,宿主细胞对辐射损伤的报告基因的修复减弱 ,还观察到辐射引起的p2 1表达受阻的细胞调亡的增加 ,结果表明p2 1,Gadd45通过参与多种辐射细胞学反应发挥DNA损伤监视作用 .     

大鼠前脂细胞质膜Na+/H+交换同时参与细胞的增殖和分化

以原代培养的大鼠前脂细胞为模型 ,以 2′ ,7′ bis ( 2 carboxyethyl) 5 ( 6 ) carboxyfluorescein (BCECF)作为检测胞内pH(pHi)的荧光探针 ,测定不同生长因子刺激下胞内pH的变化 ,证明大鼠肾周前脂细胞质膜存在Na⁺/H⁺交换活性 ,胎牛血清(FCS)能快速激活Na⁺/H⁺交换 ,导致pHi升高 (约 0 .2pH单位 ) ,并引起DNA合成 .Ethyl isopropyl amiloride (EIPA)抑制Na⁺/H⁺交换与DNA合成 .在无血清条件下 ,胰岛素不刺激DNA合成但引起细胞分化 ,表现为胞内脂滴积累和 3 磷酸 甘油脱氢酶(G₃ PDH酶 )活性增强 ,同时激活Na⁺/H⁺交换活性导致pHi升高 ;EIPA既抑制胰岛素对Na⁺/H⁺交换的激活 ,也抑制G₃ PDH酶活性增强 .结果证明 :Na⁺/H⁺交换的激活不仅与大鼠前脂细胞增殖相关 ,同时也是细胞分化的早期事件 .     

用蛋白质工程的方法改良枯草杆菌蛋白酶E(Subtilisin E)的抗氧化性

以含有蛋白酶E基因(aprE)的单链M13mp18-aprE DNA为模板,合成的寡核苷酸5′-3′为诱变引物,用缺口双链法对aprE进行Met-222-Ala点突变。经菌落印迹杂交筛选,选出阳性噬斑。用SaⅡ酶解M13mp18-aprE得到aprE,将它和pPZW103重组,转化中性、碱性蛋白酶缺失宿主菌DB104。经含卡那霉素和脱脂奶粉板筛选和比较aprE限制性内切酶NcoⅠ和SacⅡ水解电泳图谱分析,完成构建一个分泌抗氧化的枯草杆菌蛋白酶E的工程菌PW8888。     

极端嗜热古菌尿嘧啶DNA糖苷酶的研究进展

高温会加快碱基脱氨基反应形成损伤碱基的速率,进一步对脱氨基的碱基进行复制会导致突变。因此,极端嗜热古菌基因组的稳定性面临着其生存高温环境的挑战。胞嘧啶脱氨基形成尿嘧啶,是常见的脱碱基类型,复制DNA中尿嘧啶会造成GC→AT的突变。尿嘧啶DNA糖苷酶(Uracil DNA glycosylase,UDG)是修复DNA中尿嘧啶的关键酶。基于识别底物的特异性,UDG分为6个家族,广泛分布在细菌、古菌、真核生物以及一些病毒中。基因组序列显示,极端嗜热古菌至少编码一种UDG。目前,对于细菌和真核生物的UDG已进行了大量的研究,但是关于极端嗜热古菌UDG的研究相对较少,尚处于初期阶段。本文综述了极端嗜热古菌UDG的研究进展,并对今后的研究提出了展望。     

布氏菌噬菌体SA对各种布氏菌的裂解活性

布氏菌噬菌体SA是从猪种布氏菌培养物中分离到的。其裂解活性当菌液浓度为10^-4时,对光滑型猪种菌1、3型,牛种菌1、3、6型和沙林鼠种菌可产生混合性裂解,产生噬菌斑直径3—4mm。噬菌体SA原液或稀释浓度为10^-1、10^-2时,对羊种菌1、3型不裂解。对羊种菌2型、粗糙型牛种菌45/20、犬种菌和绵羊附睾菌产生混合性裂解或噬菌斑,噬菌斑为1—2mm或更小。     

Response of Saccharomyces cerevisiae to lead ion stress

The response of Saccharomyces cerevisiae to different concentrations of Pb²⁺ was investigated. The results demonstrated that the growth of S. cerevisiae in the presence of Pb²⁺ showed a lag phase much longer than that in the absence of Pb²⁺. The inhibition was dependent upon Pb²⁺ concentrations. The Pb²⁺ at a concentration of 5 μM inhibited the microbial growth by approximately 30% with regard to control, whereas Pb²⁺ at concentration of 2 μM did not have a significant effect on the microbial growth. The existence of Pb²⁺ did not perturb cell-protein synthesis and there was a good correlation between dry cell weights and total protein content (R ² = 0.98). The RNA/DNA ratio in the microbial cells varied with Pb²⁺ concentration and there was a significant positive correlation between Pb²⁺ concentration and the RNA/DNA ratio. The microbial assimilation of ammonium ion was inhibited by the presence of Pb²⁺ in the medium; when Pb²⁺ concentration was 10 μM, the microbial ammonium assimilation was inhibited about 50%, in comparison with the control experiment.     

血小板生成素改构体基因的克隆和序列分析

采用PCR技术,根据文献报道的鼠TPO成熟肽基因序列,设计并合成两对引物,以鼠TPO cDNA为模板,扩增获得mTPO N端153个氨基酸的478bp cDNA片段及鼠TPO全长1032bp cDNA片段,mTPO153片段与合成的碱性成纤维生长因子序列中Lys119-Lys135as的51bp肝素结合位点DNA片段连接,克隆到M13mp18及M13mp19载体中进行双向测序;同时将扩增的鼠TPO全长cDNA片段克隆到M13mp18及M13mp19载体中进行双向测序。证明获得鼠血小板生成素与肝素结合位点基因及鼠TPO全长基因,继之以pMAL-C2X为表达载体构建表达质粒,并经PCR及酶切鉴定。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Pathogenic Characterization and Full Length Genome Sequence of a Reassortant Infectious Bursal Disease Virus Newly Isolated in Pakistan

<正>Dear Editor,Infectious bursal disease (IBD) is one of the most important diseases of the poultry. The IBD virus (IBDV), a nonenveloped virus belonging to the Birnaviridae family with a genome consisting of two segments of double-stranded RNA (segments A and B), targets B lymphocytes of bursa of Fabricious leading to immunosuppression. In Pakistan,poultry farming is the second biggest industry and IBD is the second biggest disease threating the poultry sector.However, there is limited genome information of IBDV