nisZ启动子结构与功能的研究

应用βGlucuronidase基因(gusA)作为报告基因,通过定点突变方法分别缺失nisZ编码区上游两个启动子结构(promoter1和promoter2)中的一个,发现只有靠近编码区的promoter2是nisZ启动子诱导表达所必需。将promoter2中10区及其上游的一个碱基突变为乳酸菌中典型的组成型启动子的10区结构,该改变使nisZ启动子诱导功能下降;将promoter2的10区和35区的间隔区由20个碱基缺失突变为17个碱基,则nisZ启动子失去诱导功能。据此认为该间隔区的结构与nisZ启动子的诱导表达密切相关。     

马立克氏病病毒pp38基因和1.8kb转录子之间双向启动子的特性研究

从马立克氏病病毒(MDV)基因组DNA复制原点区某一点,将介于MDV pp38基因和18kb转录子之间的双向启动子分割成两个单方向的启动子。以pp38为报告基因,pUC18质粒为载体,构建了含不同方向完整启动子序列的pProfpp38和 pProrpp38质粒,以及含分割后单方向启动子序列的pdProfpp38和pdProrpp38质粒。4种质粒分别转染鸡胚成纤维细胞（Chicken embryo fibroblast, CEF）后,均能检测到pp38基因的表达。进一步以氯霉素乙酰转移酶（Chloramphenicol acetyltransferase, CAT）为报告基因,构建了含不同方向完整双向启动子的pProfCAT和 pProrCAT质粒,以及含分割后单方向启动子序列的pdProfCAT和 pdProrCAT质粒。通过转染试验,定量分析了完整启动子和分割后启动子在两个方向上的启动活性。实验结果表明,分割后的启动子在两个方向上的启动活性均比相应方向上完整启动子的活性低,其中1.8kb转录子方向上的活性下降了41倍。     

以绿荧光蛋白基因为报告基因的广宿主启动子探针载体的构建和应用

将以绿荧光蛋白基因(gfp)的 cDNA为模板,用人工合成引物经PCR扩增获得的09kbDNA片段克隆到表达载体pET11C上构建成gfp表达载体pHN115。从pHN115上切下的不含启动子,但保留了SD序列的gfp基因经克隆载体SK(+)和pIJ2925亚克隆后再克隆到广谱稳定性质粒pTR102上构建成广谱、稳定、可视的启动子探针载体pHN127。并用它从费氏中华根瘤菌HN01的总DNA中成功地钓出组成型和诱导型表达的启动子。     

利用绿色荧光蛋白基因gfp研究芽胞杆菌的启动子活性

利用绿色荧光蛋白基因gfpmut3,分别标记苏云金芽胞杆菌(Bacillus thuringiensis)的cry3A启动子Pcry3A、BtI_BtII启动子PBtI_BtII和来自蜡状芽胞杆菌特异启动子P44-12以研究其表达差异。其中,Pcry3A和PBtI_BtII分别与gfpmut3构成融合基因,以调控gfpmut3在苏云金芽胞杆菌中的表达。将重组质粒pGFP_304(含P44-12)、pGFPExpA(含Pcry3A_ gfpmut3融合基因)和pGFPExpB(含PBtI_BtII_ gfpmut3融合基因)分别导入大肠杆菌(Escherichia coli)和苏云金芽胞杆菌后发现,P44-12和PBtI_BtII在大肠杆菌与苏云金芽胞杆菌中均可表达gfpmut3,其中PBtI_BtII在大肠杆菌中具有极强的启动基因表达的能力。而Pcry3A不能启动gfpmut3在大肠杆菌中表达,在苏云金芽胞杆菌中启动的gfpmut3表达的荧光强度也较弱。进一步通过荧光显微镜和生物活性检测器对含重组质粒pGFP_304、pGFPExpA和pGFPExpB的转化子分别进行荧光检测及微量热检测。结果表明,3种启动子驱动下的gfpmut3基因均可在苏云金芽胞杆菌无晶体突变株BMB171中表达并检测得到不同的发光类型。微量热法检测发现P44_12和PBtI_BtII启动gfpmut3表达的代谢热低于Pcry3A驱动gfpmut3表达的代谢热。     

采用SM 22启动子调节荧光蛋白的策略分选和鉴定人前列腺平滑肌细胞与成纤维细胞

 平滑肌细胞的数量、表型以及在间质细胞中所占的比例在前列腺间质增生的发生和发展中占有重要的地位.获得纯的平滑肌细胞和成纤维细胞,研究它们基因表达的差异,有助于进一步揭示前列腺增生的分子病因学.构建不同长度的 SM 22启动子,测定荧光素酶活性.采用了基于启动子特异性激活红绿色荧光蛋白表达结合流式细胞分选的策略,体外分离纯的平滑肌细胞和成纤维细胞.启动子活性实验结果表明,1 396 bp的人SM22启动子具有平滑肌细胞特异性和较高的相对活性.构建了红绿荧光蛋白的表达载体pDual-color,在此载体中,RFP的表达受1 396bp的SM22启动子调控,GFP的组成型表达受CMV启动子控制.用流式细胞仪分选GFP+/RFP+和GFP+/RFP-细胞,提取总RNA,进行实时定量RT-PCR.结果显示,在分选获得的GFP+/RFP+细胞比GFP+/RFP-细胞的SM22和SMMHC表达水平高10倍以上.提示,基于启动子特异性可以在体外分离纯的平滑肌细胞和成纤维细胞.     

嗜热脂肪芽孢杆菌pgiB基因上游452bp序列的功能分析

用化学诱变剂N甲基N′硝基N亚硝基胍进行随机诱变,获得了穿梭启动子探测质粒pPGV5的温度抗性突变型pPGV5(tr65),序列分析发现质粒上卡那霉素核苷转移酶基因kan的+238位碱基发生了G→T的单点突变。以来自嗜热脂肪芽孢杆菌FDTP3菌株的耐热邻苯二酚2,3双加氧酶基因pheB作为报道基因,构建了转录融合质粒pPGVPB452,用高压电穿孔法将其转化嗜热脂肪芽孢杆菌,通过报道蛋白活性的分析,证明了嗜热脂肪芽孢杆菌T521菌株的6磷酸葡萄糖异构酶同工酶基因pgiB上游含启动子样序列的425bp片段在嗜热脂肪芽孢杆菌中不具有启动子功能。     

苏云金芽孢杆菌以色列亚种P19基因的初步研究

以含有P19和cyt1A基因的以色列亚种72MD质粒的9 7kb HindⅢ片段为模板进行PCR扩增,分别获得Ｐ19基因和cyt1A基因片段。与表达 载体pUHE24连接转化大肠杆菌XL１,获得3个克隆株。LZ19含有P19基因;pLZcyt1A含 有cyt1A基因;LZ19A含有P19和cyt1A两个基因。利用cyt1A蛋白质可使大肠杆菌细胞致 死的特性,在IPTG诱导下,测定了各克隆基因表达对大肠杆菌有致死作用;pLZ19A对大肠杆菌的起始致死作用明显快于pLZcyt1A,这种现象可能是P19基因促进cyt1A基因高表达的结果。     

K14和CMV启动子驱动裸鼠体内HPV16 E6/E7基因表达的差异

人乳头瘤病毒（human papillomavirus,HPV）是一种无包膜的环状闭合双链DNA病毒,具有严格的嗜人组织的特性．为探讨不同启动子驱动HPV E6/E7癌蛋白在裸鼠体内不同组织的表达效率,构建了带有角蛋白(K14)启动子和带有巨细胞病毒（CMV）启动子的E6/E7腺病毒载体（pAd-K14-E6/E7和pAd-CMV-E6/E7）,pAd-K14-E6/E7和pAd-CMV-6/E7、以及作为对照的重组腺病毒空载体pAdtrack- K14和pAd-CMV同源重组后,分别在293细胞中包装,收集重组病毒Ad-K14-E6/E7 、Ad-CMV-E6/E7、Adtrack-K14和Ad-CMV,通过尾缘静脉注射到随机分组的裸鼠体内,并每d向裸鼠腹腔注射0.05 mg雌激素．采用RT-PCR和Western 免疫印迹检测不同实验组E6/E7 mRNA 和蛋白表达水平,免疫组化法检测P53和Bcl-2蛋白表达．结果显示,注射病毒Ad-K14-E6/E7（实验组1）裸鼠子宫体中E6/E7 mRNA、E6蛋白质、P53和Bcl-2蛋白高表达,而其它组织中低表达;注射病毒Ad-CMV-E6/E7（实验组2）裸鼠各组织E6/E7　mRNA、E6蛋白质、P53和Bcl-2蛋白均低表达．研究表明,在裸鼠体内角蛋白K14启动子可以调控E6/E7在子宫体中表达,CMV启动子未能诱导E6/E7在子宫体中表达．     

耐辐射球菌pprI在大肠杆菌中表达增强细胞抗氧化能力的研究

将耐辐射球菌(Deinococcus radiodurans)与DNA修复有关的开关基因—pprI通过穿梭质粒pRADZ3导入大肠杆菌TG1中,使其在正常培养条件下(不需诱导剂)表达PprI蛋白,并通过Western blot证实该基因在TG1中可稳定表达。与转化了空白质粒pRADZ3 TG1对照,观察了改造后的两种大肠杆菌在有H2O2氧化压力下的存活率和大肠杆菌中两种过氧化氢酶（KatE, KatG）的活性表达差异。结果表明,无论在指数生长期还是稳定生长期,能表达PprI蛋白的大肠杆菌比对照的存活率要高出10％左右;非变性电泳结果表明,耐辐射球菌pprI 在大肠杆菌中的表达使得KatE活性在指数生长期与稳定生长期分别增加1.5～2倍和2.5～3倍。证明耐辐射球菌pprI 在大肠杆菌中的表达能够增强细胞抗氧化能力。     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.