长臂同源多聚酶链反应法构建变形链球菌comD基因同源重组DNA片段

目的构建变形链球菌UAl59密度感应相关的comD基因同源重组DNA片段,为利用同源重组原理构建基因功能丧失菌株做准备。方法通过NCBI基因数据库获取变形链球菌的DNA序列,利用聚合酶链反应技术分别扩增变形链球菌UA159comD基因上、下游片段及抗红霉素基因片段,再通过长臂同源多聚酶链反应将这3个片段连接起来,形成同源重组DNA片段。结果经过PCR反应和琼脂电泳分析,得到了一个碱基数为3个单片段总和的连接片段,测序结果显示连接片段为预期的comD同源重组片段。结论成功构建了变形链球菌UA159comD基因同源重组DNA片段,可直接用于细菌转化构建comD基因缺陷菌株。     

质粒拯救法克隆细菌基因组大片段

[目的]细菌基因组大片段尤其是基因簇的克隆与操作,是细菌基因功能分析的一个难点.基因组测序工作的不断完成和序列信息的大量积累,为细菌基因组:DNA的操作提供了方便.本文报道了利用细菌的全基因组信息和质粒拯救法的原理建立的一种克隆细菌基因组大片段的方法.[方法]首先,根据基因组序列信息,在待克隆片段的一侧扩增一段DNA,并将其克隆到自杀载体上构建打靶质粒,然后,将打靶质粒整合到细菌的基因组中构建重组菌,提取重组菌的基因组DNA,酶切,自连,转化,将自杀质粒与待克隆的目的片段一起拯救出来.最后,根据需要将拯救的DNA片段亚克隆到新的载体中.[结果]我们利用该方法克隆了布鲁氏菌中长度为11kb的virB操纵子,并构建了互补质粒.将该质粒导入到virB的突变株中后使virB操纵子的转录活性得到了恢复,表明该策略切实可行.[结论]这种重组克隆策略给我们提供了一种新的对细菌基因组大片段进行操作的方法.     

细菌重组系统及其应用研究进展

利用重组酶和辅助蛋白共同作用于DNA片段上,使不同基因重新组合以完成基因重组的现象在细菌中广泛存在,基因重组对于细菌的遗传多样性、进化等具有重要意义。目前,细菌基因重组主要分为同源重组、位点特异性重组和转座重组3种类型。本文主要对细菌重组系统重组酶的种类、作用机制及其在细菌遗传操作中的应用策略进行阐述。     

与抗体形成有关的两个基因

正如26个英文字母组成千千万万单词一样,体内几百个基因通过不同组合产生无数种抗体,已经发现淋巴细胞是通过基因切粘技术来重组这些基因的,此项发现已获诺贝尔奖,但细胞究竟怎样完成这个过程却不清楚。现在科学家报道找到了与重组有关的两个基因。麻省剑桥Whitehead生物医学研究所的David等成功的克隆了一个基因,将它注入对DNA无影响的肌肉细胞可以激活基因重组。日本京都大学的 Honjo 也找到一个基因,它可编码一种蛋白,类似于病毒、细菌及酵母中的DNA内切酶,这些酶属重组酶类,可重组特定的DNA片段。虽然Whitehead研究所的科学     

嗜线虫致病杆菌Xna基因同源重组载体的构建及遗传转化体系的建立

构建嗜线虫致病杆菌Xna基因的同源重组载体和建立该细菌的遗传转化体系.扩增嗜线虫致病杆菌XNA基因的左右同源片段及质粒PEASY-T1上的卡那抗性基因Km;利用重叠延伸引物PCR法将这3个片段连接起来,然后将其克隆至自杀性载体PJQ200.采用电激法将同源重组载体DNA转化到供体细菌,再利用二亲本结合转移方法将供体细菌中的载体转化到受体细菌CB6菌株.结果成功地扩增了Xna基因的左右同源臂及卡那抗性基因的全长融合片段、构建了Xna基因的同源重组载体,并建立了嗜线虫致病杆菌北京变种的遗传转化体系.     

假单胞杆菌2-萘酸加氧酶基因的克隆与表达

采用Cosmid pLARF1构建了完整的2-萘酸代谢菌2-NAT菌株的基因文库.通过分析基因文库中产生蓝色色素的克隆验证了2-萘酸代谢基因在大肠杆菌体内得到表达,并导致重组细菌产生靛蓝.从产生靛蓝的克隆抽提重组的Cosmid DNA进行酶解分析,发现所有能产生靛蓝的克隆均含有一大小相同的DNA片段.用重组细菌进行靛蓝生物合成的试验展示了微生物法生产靛蓝的美好前景.     

人体TK-C基因片段和TK cDNA片段并发转化细胞的稳定性研究

本文报道了人体细胞质胸腺嘧啶核苷激酶(TK-C;EC2.7.1.21)基因的一个片段同TKcDNA的一个片段,一起转化小鼠TK缺陷型细胞株Ltk~-细胞时,这两个单独都不能表达TK活性的DNA片段可能通过同源重组,重新构建成一个能够表达TK的完整的功能单位。分析了TK~+并发转化细胞表达TK的稳定性,并用Southern印迹法杂交证实这两个外源DNA片段已重组成一个片段而整合在受体细胞基因组里,因而并发转化细胞全是稳定型。讨论了两个功能上可以互补的外源DNA片段,在受体细胞里重组成一个能表达基因活性的功能单位的可能机制。     

小鼠白细胞介素4基因的化学合成、克隆与表达

利用381A型DNA合成仪,分29个寡聚核苷酸片段化学合成了小鼠IL-4全基因,共442bp。以pUC12质粒作为载体,将所有合成片段分前后两组进行磷酸化、退火、连接和克隆,经过菌落原位杂交、酶切鉴定和质粒DNA序列分析,分别得到了含有小鼠IL-4前后两半基因片段的两种重组质粒,回收前半基因片段,插入到含有后半基因重组质粒的EcoRI和PstI酶切位点之间,成功地得到了含有小鼠IL-4全基因的重组质粒pFR101。将全合成基因插入到质粒pSM53中,得表达质粒pFR105,转化大肠杆菌TAP106,根据IL-4对CTLL细胞的作用,肯定了TAP106(pFR105)细菌中有小鼠IL-4活性蛋白的表达。     

假单胞杆菌2-萘酸加氧酶基因的克隆与表达

采用Cosmid pLARF1构建了完整的2-萘酸代谢菌2-NAT菌株的基因文库.通过分析基因文库中产生蓝色色素的克隆验证了2-萘酸代谢基因在大肠杆菌体内得到表达,并导致重组细菌产生靛蓝.从产生靛蓝的克隆抽提重组的Cosmid DNA进行酶解分析,发现所有能产生靛蓝的克隆均含有一大小相同的DNA片段.用重组细菌进行靛蓝生物合成的试验展示了微生物法生产靛蓝的美好前景.     

相变:一种控制因子的进化

细菌中的相变受某一特异DNA位点上同源重组的调节。这种重组事件引起一段970碱基对的倒位,这段DNA序列包括鞭毛基因转录所必要的启动子。这个可倒位的片段的两侧为倒位所必需的位点并包含有其产物能执行倒位的一个基因(hin)。hin基因与所在转座子Tn3上携带的TnpR基因显示广泛的同源性,也与在细菌噬菌体Mu上所携带的gin基因有同源性。这些关系表明相变系统可以通过转座子与一个固有的基因相结合而发生进化,随后这些因子特异化,从而调节鞭毛抗体的表达。     

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.