哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

溶藻弧菌耐药基因qnr的克隆及生物信息学分析

[目的]克隆溶藻弧菌(Vibrio alginolyticus)喹诺酮类耐药基因qnr并分析其蛋白结构,为研究该蛋白的生物学功能奠定基础。[方法]根据NCBI上公布的相关序列设计qnr基因特异性引物,利用PCR方法扩增基因序列,进行DNA测序及生物信息学分析。[结果]从溶藻弧菌染色体上获得qnr基因大小为651 bp,编码216个氨基酸,进化分析可知其与副溶血弧菌有很近的亲缘关系,二级结构分析含有五肽重复序列,三维结构与大肠杆菌质粒上的qnr蛋白空间结构极为相似。[结论]通过对蛋白质序列分析和结构预测,初步确定该基因为喹诺酮耐药基因,为水产细菌的耐药机制研究奠定基础。     

大肠杆菌M增强子样序列结构和功能的研究

构建了以β-半乳精苷酶基因为报告基因,适用于原核增强序列研究的pAC载体.应用该载体进行的转录增强实验证明,一段已克隆的具有增强基因表达功能的大肠杆菌染色体M序列,对被调控基因的增强作用是发生在转录水平,其能使β-半乳糖苦酶基因特异性的mRNA转录增加6.5倍,与应用该载体测得的表达增强活性一致.通过构建一系列正、反向M序列缺失突变体并对其进行的研究表明,在靠近M序列5’端的340bp区域内存在3个相互作用、与增强活性密切相关的部位,分别位于M序列3’端上游770,610和470bp处.应用PCR技术对大肠杆菌染色体对应于M序列5’端上游207bp进行了克隆,研究显示M序列具有功能上的完整性.     

八肋游仆虫第二类释放因子基因的克隆与序列分析

分离八肋游仆虫 (Euplotesoctocarinatus)大核eRF3基因 ,为进一步研究第二类释放因子结构与功能 ,探讨低等真核生物新生肽链释放机理提供实验素材 .以八肋游仆虫基因组DNA为材料 ,根据已知的第二类释放因子eRF3保守氨基酸序列设计引物 ,扩增克隆了该游仆虫的第二类释放因子基因片段 ,并对其核苷酸序列进行了分析 .根据测得的序列设计特异性引物 ,并利用游仆虫的端粒序列 (C4 A4 C4 A4 C4 A4 C4 )为引物 ,扩增得到该基因的全序列 .序列分析表明 ,该基因位于 2 782bp长的大核染色体上 ,编码区由 2 4 0 0bp组成 ,编码 80 0个氨基酸 ,不含内含子     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。     

八肋游仆虫核糖体蛋白L11的基因克隆与序列分析

以单细胞真核生物八肋游仆虫Euplotes octocarinatus为实验材料,采用PCR、RT-PCR方法克隆了核糖体蛋白L11基因(EoRPL11).将该序列与GenBank中其他物种的RPL11基因序列进行同源性比对,采用DNAStar软件进行聚类分析.结果显示,成功克隆到游仆虫RPL11基因,大核中该基因全长709 bp,开放阅读框(ORF)为531 bp,编码176个氨基酸;cDNA序列与大核中ORF序列一致,表明该基因具有转录活性;所推导的氨基酸序列与嗜热四膜虫Tetrahymena thermophila的RPL11同源性最高,为66%.     

川芎咖啡酸-O-甲基转移酶基因的克隆及序列分析

克隆川芎(Ligusticum chuanxiong Hort)咖啡酸-O-甲基转移酶(COMT)基因并做序列分析。采用RT-PCR方法,以新鲜的川芎嫩叶c DNA为模板,克隆出川芎COMT基因的c DNA序列。此基因全长1328 bp,编码360个氨基酸。将得到的序列提交Gen Bank,序列号为KU942388。此川芎COMT具有植物O-甲基转移酶家族特征性的功能结构域,与中粒咖啡、牵牛花COMT的氨基酸序列同源性分别为70%、69%,推测其能够催化咖啡酸的氧甲基化反应。该川芎COMT基因的成功克隆为下一步活性验证及利用生物技术来生产阿魏酸奠定坚实基础。     

八肋游仆虫Rab家族新成员Eo-rab-1N基因的克隆与序列分析

Rab蛋白家族属于小分子GTP结合蛋白家族Ras超家族中最大的亚家族,主要在囊泡运输中起作用。本实验运用PCR、RT-PCR等技术,从八肋游仆虫中克隆到一种新的rab基因。序列分析结果表明：在大核中,该基因全长884bp,除去两端的端粒与非编码区,该基因在大核中由723bp组成。从小核中克隆相应的基因片段,此基因片段序列与大核中序列一致,表明该基因在小核中无内部删除序列的存在。通过RT-PCR,从mRNA获得的该基因的开放读框为663bp,表明该基因在转录过程中有内含子的删除。大核基因序列和cDNA序列比较,发现60bp的内含子序列位于大核基因的153~212bp之间,并符合一类内含子GU-AG剪切规则。在遗传密码使用上,该基因内部含有2个TGA,在游仆虫中编码半胱氨酸。同时首次发现,八肋游仆虫基因使用TAG作为终止密码子。NCBI上序列比对表明该基因翻译的蛋白与其它物种Rab1蛋白的同源性达49%~52%,因此我们将它命名为Eo-rab-1N,GenBank登录号为DQ105562。Eo-rab-1N与其他物种的Rab1蛋白构建进化树,发现该蛋白的进化与物种的进化保持一致,表明该基因在细胞中具有重要功能。     

解淀粉芽孢杆菌BamHI甲基转移酶基因克隆、功能鉴定与序列分析

从解淀粉芽孢杆菌Baillus amyloliquefaciens CICIM B2125中克隆了BamHI甲基转移酶基因(bamHIM),并在大肠杆菌JM109中得到了成功表达.该基因含有1 271 bp的开放阅读框(ORF),编码423个氨基酸,成熟蛋白分子量为49 kD.该基因在自身启动子引导下,表达了具有活性的BamHI甲基转移酶(M.BamHI).该酶可以将BamHI位点的碱基甲基化.氨基酸序列分析表明该酶存在有NADB_Rossmann结构域.     

一种改良的克隆小麦GLP3基因启动子的TAIL-PCR技术

以小麦基因组DNA为模板,用一种改良热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)技术克隆到全长为1748bp的小麦GLP3基因的上游侧翼序列。测序结果表明,TATA-box位于基因转录起始位点CAT-box上游-27bp处;CAAT-box位于CAT-box上游-163bp处;ATG位于CAT-box下游94bp处,5'UTR(非编码区)序列全长93bp,表明该序列为小麦GLP3启动子序列。     

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.