马立克氏病毒DNA转录区和非转录区甲基化及脱甲基化的探讨

实验以鸡马立克氏病脾脏淋巴瘤继代细胞系MDCC MSB 1和感染马立克氏病毒的鸡胚成纤维细胞(CEF)为主要试验材料,作为参考系也使用了鸡马立克氏病卵巢淋巴瘤继代细胞系MDCC JP1、MDCC JP2、MDCC HP1及MDCC HP2细胞。用Suothern斑点分子杂交放射自显影等方法,验证了MDCC MSB 1等细胞株DNA的转录区和非转录区都存在着甲基化现象。转录区甲基化程度高于非转录区。用5 氮胞苷可以阻断DNA甲基化,阻断效果在转录区最好。     

DNA去甲基化酶对拟南芥Laccase家族基因甲基化及转录模式的影响

植物Laccase家族蛋白,是铜蓝氧化酶超家族的重要成员,具有包括抗病性在内的多种生物学功能。利用拟南芥野生型及ros1、rdd和dme突变体的全基因组甲基化测序(BS-SEQ)数据,分析了拟南芥Laccase蛋白家族基因(AtLACs)的DNA甲基化模式。结果表明:在野生型和不同DNA去甲基化酶突变体中,该家族少数成员在基因上游区和转录区存在较高水平的甲基化修饰现象。转录组测序(RNA-SEQ)数据分析结果显示,在野生型和不同突变体中,AtLAC家族基因的转录水平普遍较低。而AT5G48100基因在拟南芥胚乳细胞中剧烈表达,DME突变后,其表达水平进一步剧烈增强。综合而言,在野生型和不同DNA去甲基化酶突变体中,AtLAC基因家族大部分成员的DNA甲基化修饰与转录调控关联不强,但有些基因的DNA甲基化程度与转录水平之间有密切联系。     

小鼠胞内氯离子通道5基因启动子核心功能区域鉴定及转录调控分析

为研究胞内氯离子通道5基因(Chloride intracellular channel 5,CLIC5)广泛参与调节细胞内的各项生理活动与生化反应,并探讨该基因自身的表达调控机制,以小鼠基因组序列为模板,利用PCR技术扩增小鼠CLIC5基因5′上游调控序列,将其插入荧光素酶报告基因表达载体(pGL3-Basic)中,同时采用5′侧翼区缺失的方法构建了7个缺失不同DNA片段的荧光素酶表达载体。重组质粒与海肾荧光素酶载体(phRL-TK)共同瞬时转染HEK-293细胞,经双荧光素酶报告基因活性分析后,确定CLIC5基因的核心启动子区。利用生物信息学方法预测其中转录因子结合位点及启动子区甲基化状况。结果表明,CLIC5基因启动子缺乏TATA盒,但含有典型的GC盒及其他潜在转录因子结合位点;双荧光素酶报告基因活性分析表明,CLIC5基因-329～+1、-624～+1、-917～+1和-2 230～+1区域的启动子活性较高,其中-624～+1区域的启动子活性最强。进一步分析表明,启动子区-624～-329存在负性调控元件,预测存在转录因子结合位点RXR heterodimer binding sites与GC-Box factorsSp1/GC,-420～-283范围内存在CpG岛位点。     

萝卜-芥蓝异源四倍体F4和F10世代DNA甲基化变异的MSAP分析

以人工合成的萝卜-芥蓝异源四倍体的F4和F10世代为材料,用MSAP法检测2种材料DNA甲基化变异情况,以期为远缘杂交后不同世代间基因组DNA甲基化遗传和变异规律提供实验依据。结果表明:F4代和F10代甲基化程度分别为30.61%和33.10%,共检测12种甲基化变异模式,可以分为5种类型,甲基化变异位点占所有甲基化位点的41.71%。用甲基化抑制剂5-氮胞苷处理F4代,F10代材料,检测到甲基化程度分别为20.67%和25.75%,F代比F代受到了更大冲击,说明不稳定世代对环境的影响更加敏感。     

苹果属植物DNA去甲基化基因MdIDM1参与低温促进花色素苷的积累

[目的]为探明DNA去甲基化基因与苹果属植物低温诱导花色素苷积累的关系.[方法]以苹果'嘎啦'和观赏海棠'王族'与观赏海棠'火焰'组培苗叶片为试材,16℃低温连续处理7d,通过RNA提取和反转录cDNA,克隆得到苹果属植物DNA去甲基化关键基因MdIDM1,通过荧光定量PCR和高效液相色谱分别检测叶片中花色素苷合成基因和花色素苷含量,对去甲基化基因MdIDM1表达量进行相关性分析.[结果]低温胁迫促进苹果属植物着色,同时随着低温处理时间的增加MdIDM1的表达逐渐升高,且与花色素苷生物合成基因表达和花色素苷积累呈正相关.[结论]低温条件下,DNA去甲基化基因MdIDM1可能参与苹果属植物花色素苷积累.     

原花青素对IL-1β诱导的A549细胞环氧合酶-2启动子活性的影响

以白介素-1β(IL-1β)诱导的人肺癌细胞A549为模型,探讨原花青素(PC)对环氧合酶-2(COX-2)启动子活性的影响.用含有完整和NF-κB结合位点突变的COX-2启动子的的荧光素酶表达载体 pGL 3质粒,转染A549细胞,加原花青素(20mg/L)培养,检测COX-2基因5'调控区相对转录活性;RT-PCR检测细胞COX-2 mRNA的表达.结果发现NF-κB结合位点突变的COX-2基因转录活性极大地降低,原花青素可以显著降低完整的COX-2启动子转录活性,降低A549细胞中COX-2 mRNA的表达.     

FAM213B基因启动子在猪子宫内膜细胞的转录调控

【目的】分析猪FAM213B基因启动子转录活性区域,并检测转录因子NFκB对启动子活性及猪FAM213B基因表达影响,为深入了解FAM213B基因的转录调控机制影响前列腺素合成、母猪妊娠等繁殖活动奠定基础。【方法】采集卵泡期子宫,分离子宫内膜上皮组织,经胶原酶方法获得猪原代子宫内膜细胞,用于猪FAM213B基因启动子活性检测。参照笔者所在课题组前期研究获得的猪FAM213B基因m RNA全序列(Gen Bank登录号:KX444503)以及5￠调控区序列(Gen Bank登录号:100134955),通过PCR扩增猪FAM213B基因较长启动子序列,并进行测序鉴定;在此基础上,PCR扩增带有Mlu I和Xho I限制性酶切位点的FAM213B基因启动子7个5'端缺失片段,并通过双荧光素酶报告基因载体系统构建猪FAM213B基因启动子7个不同的5'端缺失载体。将7个载体经无内毒素处理后与p RL-TK质粒用阳离子脂质体法一起转染猪子宫内膜细胞,用双荧光素酶报告基因系统进行Luciferase活性检测,比较各启动子片段转录活性。生物信息学分析FAM213B基因启动子潜在转录因子结合位点,通过Ch IP试验验证FAM213B基因启动子与潜在转录因子NFκB的相互作用。构建NFκB1和Rel A的超表达载体,化学合成NFκB1和Rel A的干扰si RNA片段,分别转染猪子宫内膜细胞,通过双荧光素酶报告基因系统进行Luciferase活性检测和荧光定量PCR分别检测超表达和干扰表达NFκB1和Rel A对猪FAM213B基因启动子活性和m RNA表达影响。【结果】PCR和测序获得猪FAM213B基因启动子长度为2 261bp(-2178/+83)。生物信息学预测结果表明猪FAM213B基因启动子区存在潜在的CREB、CCAAT增强子结合蛋白、E-box因子的结合位点,炎性因子NFκB潜在结合位点分别位于-1143/-1132和-664/-655区间。启动子报告基因活性检测结果表明:重组载体P2(-1352/+30)荧光活性最高,极显著高于P1(-1 760/+30)(P0.01),在-1 760/-1 352区域存在负调控元件;而P2显著高于P3(-919/+30)(P0.05),表明在-1352/-919区域存在正调控元件;P3(-919/+30)活性极显著高于P4(-604/+80)(P0.01),表明在-919/-604区域存在正调控元件;P4、P5、P6和P7活性差异不显著,-1352/-919区域为核心启动子区,-1352/-604对于维持该启动子较高转录活性起着重要的作用。Ch IP结果表明启动子区-1143/-1132存在NFκB1结合位点,-664/-655存在Rel A结合位点。超表达载体pc DNA3.1-NFκB1与P2(-1352/+30)启动子片段重组载体共转染猪子宫内膜细胞后,P2启动子活性极显著高于对照组(P0.01),pc DNA3.1-NFκB1载体单独转染猪子宫内膜细胞后FAM213B基因m RNA表达量显著高于对照组(P0.05);超表达载体pc DNA3.1-Rel A与P3(-919/+30)启动子片段重组体共转染猪子宫内膜细胞,P3启动子活性极显著低于对照组(P0.05),pc DNA3.1-Rel A载体单独转染猪子宫内膜细胞后,FAM213B基因的mRNA表达量显著低于对照组。转染NFκB1和Rel A的si RNA片段干扰片段,FAM213B基因启动子活性和m RNA表达量则表现与超表达相反的结果。【结论】获得了猪FAM213B基因启动子核心启动子序列为-1352/-919区域,NFκB是FAM213B基因启动子的转录因子,NFκB成员NFκB1和Rel A在猪子宫内膜细胞中对FAM213B基因的表达起调控作用。     

马立克病病毒糖蛋白B抗原基因克隆的酶切分析

用狄可辛标记的马立克病病毒(MDV)基因组DNA的BamHⅠ-K_3片断为探针,从建于pUC18质粒中的GA株MDV感染细胞基因组DNA的基因文库中,筛选到MDV糖蛋白B抗原基因克隆,酶切分析表明,GA株MDV的B抗原基因克隆在EcoRⅠ、HindⅢ、SalⅠ及BamHⅠ等酶切位点的分布上与RBIB株MDV的B抗原基因没有区别。     

稀土钕尘肺工人血清中IL-10、IFN-γ的表达及其DNA甲基化分析

尘肺病是由于长期吸入粉尘颗粒物并沉积于肺部,引起以肺纤维病变为主的疾病。在此过程中,DNA甲基化可以调节细胞因子基因活性,影响Th1、Th2型细胞因子的转录,导致Th1/Th2细胞因子之间失衡,可能是导致尘肺形成的重要原因。为明确尘肺与DNA甲基化的关系,本研究检测了尘肺病人和健康人群的全血钕含量、血清中Th1型细胞因子IFN-γ、Th2型细胞因子IL-10的表达和IFN-γ、IL-10基因启动子区甲基化状态,了解了稀土钕尘肺工人钕暴露水平和机体免疫状况,初步探讨DNA甲基化在钕粉尘致肺纤维化中的作用。结果表明,氧化钕体内高蓄积、IL-10与IFN-γ基因启动子区的甲基化水平改变,导致IL-10表达升高、IFN-γ表达降低,在钕尘肺的病情进展中起重要作用。     

5-氮杂-2’-脱氧胞苷对胎牛成纤维细胞增殖、周期和凋亡的影响(英文)

胎牛成纤维细胞是细胞重编程和去甲基化研究的常用材料。本文以胎牛成纤维细胞为材料,用不同浓度的DNA去甲基抑制剂药物5-氮杂-2’-脱氧胞苷分别处理24、48和72 h后,研究其对胎牛成纤维细胞的增殖、活力、细胞周期分布和凋亡率的影响。结果显示,5-氮杂-2’-脱氧胞苷以浓度依赖性的方式抑制胎牛成纤维细胞的增殖、活力,并增加细胞的总凋亡率。其中,用浓度低于0.1μmol/L的5-氮杂-2’-脱氧胞苷处理24 h时,胎牛成纤维细胞受到的不利影响最小。这也说明采用相对低浓度的DNA去甲基抑制剂处理体细胞对于细胞的重编程是有益的。     

Knockdown of the Meq gene in Marek's disease tumor cell line MSB1 might induce cell apoptosis and inhibit cell proliferation and invasion

Marek's disease (MD), a highly cell-associated and contagious disease of chickens caused by Marek's disease virus (MDV) can result in neural lesions, immunosuppression and neoplasia in chicken. The Meq gene is an important oncogene in the MDV genome, and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines. An experiment was conducted to elucidate the role of Meq in MD tumor transformation. RNA interference technology was used to block its expression, and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1. A small interfering RNA with an interference efficiency of 70% (P<0.01) was transfected into MSB1 cells to knock down the expression of Meq gene. The cell proliferation, cycle and apoptosis were detected post-Meq knockdown. The results showed that MSB1 cell proliferation was downregulated remarkably at 48 h (P<0.01), 60 h (P<0.05) and 72 h (P<0.01) post-Meq knockdown. The cell cycle was unaffected (P>0.05). B-cell lymphoma 2 gene (BCL2) was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway. The activity of caspase-6 was upregulated (P<0.05) significantly and BCL2 gene expression was downregulated (P<0.05) significantly post-Meq knockdown, suggesting cell apoptosis might be induced. MSB1 cell migration did not exhibit any obvious change (P>0.05) post-Meq knockdown, but the expression of two genes (matrix metalloproteinase 2 (MMP2) and MMP9) that are correlated closely to cell invasion was downregulated (P<0.05) remarkably post-Meq knockdown. The Meq knockdown might affect the main features of tumorous cells, including proliferation, apoptosis, and invasion, suggesting that the Meq gene might play a crucial role in interfering with lymphomatous cell transformation.     

转化型人参皂苷抗MDV及诱导肿瘤细胞凋亡的作用初探

以转化型人参皂苷的8个组,按12.5μg·mL-1的剂量,在体外鸡胚成纤维细胞上进行抗MDV-RB1B强毒株空斑减数试验和诱导肝癌7402细胞株、大鼠脑胶质瘤细胞C6株细胞凋亡试验,用光学显微镜观察、琼脂糖凝胶电泳检测分析DNA损伤。结果表明:转化型人参皂苷6、7号组,在感染阻断、增殖抑制、直接杀灭3种方式的抗MDV空斑减数率都达到阿昔洛韦(ACV)抗MDV空斑减数率的80%,转化型人参皂苷4、6号组对MDV-RB1B株的直接杀灭作用与ACV的杀灭作用相同;转化型人参皂苷8号组诱导肝癌7402株细胞48h后,有60%的瘤细胞凋亡,对大鼠脑胶质瘤C6株细胞也有诱导凋亡的作用。     

鸡致病性MDV感染AGP法检测可靠性研究

常规琼脂扩散（ＡＧＰ）试验通过羽毛囊马立克氏病病毒（ＭＤＶ）特异抗原的检出来诊断鸡致病性ＭＤＶ的感染〔１,２,６〕,因特异性强、操作简便而广泛应用。但其检出率与感染鸡群在不同时期的检测〔３〕以及火鸡疱疹病毒（ＨＶＴ）疫苗免疫状态〔４〕密切相关。此现象...     

Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control

Transfer of a normal Chinese hamster X chromosome (carried in a mouse A9 donor cell line) to a nickel-transformed Chinese hamster cell line with an Xq chromosome deletion resulted in senescense of these previously immortal cells. At early passages of the A9/CX donor cells, the hamster X chromosome was highly active, inducing senescence in 100% of the colonies obtained after its transfer into the nickel-transformed cells. However, senescence was reduced to 50% when Chinese hamster X chromosomes were transferred from later passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA. These results suggest that a senescence gene or genes, which may be located on the Chinese hamster X chromosome, can be regulated by DNA methylation, and that escape from senescence and possibly loss of tumor suppressor gene activity can occur by epigenetic mechanisms.     

Adaptation of lymphadenopathy associated virus (LAV) to replication in EBV-transformed B lymphoblastoid cell lines

A strain of lymphadenopathy associated retrovirus ( LAV ) passaged in vitro was used to infect a lymphoblastoid cell line obtained by transformation with Epstein-Barr virus of B lymphocytes from a healthy donor. The virus produced from this line (B- LAV ) was also able to grow at a high rate in some other lymphoblastoid lines and in a Burkitt lymphoma line. This adapted strain retained the biochemical, ultrastructural, and antigenic characteristics of the original strain, as well as its tropism for normal T4+ lymphocytes. It is thus possible to grow LAV in large quantities that can be used for the preparation of diagnostic reagents. The interaction between such a human retrovirus and Epstein-Barr virus, a DNA virus, may have some implication for the pathology of the acquired immunodeficiency syndrome and related diseases.     

亲和层析法提纯鸡马立克病毒gB基因重组产物

应用针对鸡马立克病毒（ＭＤＶ）糖蛋白Ｂ（ｇＢ）抗原单克隆抗体ＩＡＮ８６制备亲和层析柱，从感染重组杆状病毒（ｇＢ－ＡｃＮＰＶ）的昆虫细胞中提纯鸡ＭＤＶｇＢ基因重组产物，获得较好效果。提纯的蛋白质在ＳＤＳ－ｐＡＧＥ中出现的５条条带中有４条在免疫印迹试验中出现，其分子量为１００，６０，４９，４０ｋｄ占蛋白总量的５４．７％。试验证明用单克隆抗体亲和层析法提纯ＭＤＶｇＢ基因重组产物是一个行之有效的方法。     

A histone acetyltransferase regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.     

Molecular Comparisons of Marek＇s disease virus strains of different pathotypes for their gⅠ, gE, pp38 and meq genes

Five to ten serotype I Marek‘ s disease virus (MDV1) strains of different pathotypes were compared for their DNA sequences of gⅠ, gE, pp38 and meq genes. The reference strains were vMDV GA and JM, vvMDV RB1B and Mdll(pl6), vv MDV strains 648A and 584A, vaccine strain CVI988/Rispens; Chinese strains were: v MDV strain N, vvMDV strain G2, vaccine strain 814. Only random aa changes were found in 12 positions within gE of 497 aa among 10 analyzed strains and in 10 positions within gⅠ of 355 aa among 5 strains. There was no relationship found between virus pathotypes and aa changes of both glycoproteins. The aa changes happened in 14 positions within meq of 339 aa among 5 compared strains. But a proline deletion at aa # 194 in a proline- rich domain of meq were shared by two vaccine strains CVI988/Rispens and 814, the latter was a non - pathogenic vaccine strain of serotype 1 isolated in 1983 in China. In another hand, vv MDV strains 648A demonstrated 5 unique aa changes and 4 of them also located in the proline - rich region. The pp38 was very conservative among sequenced 10 strains, there were only two positions at # 107 and # 109 with an altered in its 290 aa. All tested MDV1 strains had glufamine at # 107, instead, only vaccine CVI988 had arginine at the position and lost its epitope reactive with Mab H19, to which all ether tested MDV1 strains were positive. However, CVI988 and vMDV GA shared a Mab T65 - recognized epitope when there was glyeine at aa # 109. It was glutamie acid at aa # 109 in all other MDV1 strains which were not reactive with Mab T65. It seems like that there is some relationship between pathotypes and sequences of pp38 and meq, but more strains need to be compared.