酵母三杂交技术的应用策略及在病毒研究中的应用

从酵母双杂交系统衍生出来的酵母三杂交系统是一种主要用于研究细胞内RNA-蛋白质相互作用强大的技术方法,也为病毒相关研究,尤其为研究病毒生活史中病毒RNA与病毒蛋白和宿主细胞蛋白的相互作用提供了新的工具。本文重点介绍了此种形式的酵母三杂交系统的原理、缺陷、应用和应用策略。另外,本文还介绍了同时发展起来的另外两种形式的酵母三杂交系统,即分别用于研究小分子配体-受体相互作用、复杂蛋白质相互作用的酵母三杂交系统。     

酵母三杂交技术的应用策略及在病毒研究中的应用

从酵母双杂交系统衍生出来的酵母三杂交系统是一种主要用于研究细胞内RNA-蛋白质相互作用强大的技术方法,也为病毒相关研究,尤其为研究病毒生活史中病毒RNA与病毒蛋白和宿主细胞蛋白的相互作用提供了新的工具。本文重点介绍了此种形式的酵母三杂交系统的原理、缺陷、应用和应用策略。另外,本文还介绍了同时发展起来的另外两种形式的酵母三杂交系统,即分别用于研究小分子配体-受体相互作用、复杂蛋白质相互作用的酵母三杂交系统。     

分离的泛素系统--一种新型的膜蛋白酵母双杂交系统

酵母双杂交系统是研究蛋白质间相互作用的重要遗传学方法之一,然而传统的酵母双杂交技术在实际应用中存在着一定的局限性.分离的泛素系统(split-ubiquitin system)作为一种新型的不依赖转录激活机制的酵母双杂交系统,原理是通过待检测蛋白质之间的相互作用将突变的、分离的泛素分子N端和C端部分融合,通过泛素专一的蛋白酶UBP的识别使报告蛋白解离入核,激活报告基因表达.该系统被研究的蛋白质间的相互作用不在局限于核内,更适合于发生在胞膜或胞质中的相互作用研究,是一种非常值得推广的遗传学方法.     

分离的泛素系统——一种新型的膜蛋白酵母双杂交系统

酵母双杂交系统是研究蛋白质问相互作用的重要遗传学方法之一，然而传统的酵母双杂交技术在实际应用中存在着一定的局限性。分离的泛素系统(split-ubiquitin system)作为一种新型的不依赖转录激活机制的酵母双杂交系统，原理是通过待检测蛋白质之间的相互作用将突变的、分离的泛素分子N端和C端部分融合，通过泛素专一的蛋白酶UBP的识别使报告蛋白解离入核，激活报告基因表达。该系统被研究的蛋白质问的相互作用不在局限于核内，更适合于发生在胞膜或胞质中的相互作用研究，是一种非常值得推广的遗传学方法。     

长链非编码RNA对神经干细胞的调控作用及其机制

正长链非编码RNA(LncRNA)作为一种重要的细胞功能调控因子引起越来越多广泛的关注。它是一种长度在200-100000 nt之间的RNA分子,位于细胞核或细胞质内,不编码蛋白质。在基因组转录产物中,lncRNA所占数量比例远远超过编码RNA的比例,与DNA、RNA、蛋白质相互作用参与细胞内多种调控过程,在生命活动调控网络中有极其重要的作用。lncRNA目前是遗传学研究的热点之一。近年来许多研究表明,lncRNA在神经干细胞的自我更新、增殖和分化中     

类mRNA RNA研究进展

类 m RNA RNA (m RNA- like RNA)是一类 3′端有 Poly A ,无典型 ORF,不编码蛋白质 ,而直接在 RNA水平上发挥作用的 RNA分子。近年来的研究表明各种不同的类 m RNA RNA广泛存在于不同的细胞内 ,并发挥着重要的调控作用。它们与细胞的生长和分化、胚胎的发育、肿瘤的形成和抑制密切相关     

直接法将质粒从酵母电转化至大肠杆菌

随着人类基因组计划的完成,蛋白质的功能研究将成为后基因组时代的主要任务１。任何蛋白质功能的发挥,离不开与其他蛋白质之间的相互作用。随着分子生物学方法的发展,出现了以基因文库为基础的蛋白探测、噬菌体呈现等技术,但这些方法反映的都只是蛋白的体外相互作用。酵母双杂交系统是一种新的直接在真核细胞内检测蛋白相互作用的新方法,灵敏度高２３。双杂交系统是基于一些转录激活因子（如酵母的Ｇａｌ４蛋白和细菌的ＬｅｘＡ蛋白）,往往由两个在结构上可以分开、功能上相互独立的结构域构成,这两个结构域在同一细胞内只…     

酵母双杂交技术筛选并回转验证在胞内与PML-C结构域相互作用的蛋白

目的 利用酵母双杂交技术在活细胞内筛选并回转验证与PML-C结构域相互作用的蛋白质.方法 通过诱饵质粒pGBKT7-PML-C,利用酵母双杂交系统从白血病细胞cDNA文库中筛选与PML-C结构域相互作用的蛋白质.结果 利用酵母双杂交技术筛选到43个能与PML-C结构域相互作用的克隆;经进一步的归类与酵母回转试验得到9个阳性克隆.结论 在细胞内PML-C结构域能与多种蛋白质有相互作用.中性粒细胞弹性蛋白酶(neutrophil elastase,NE)介导的急性早幼粒细胞白血病的发生可能与这些相互作用所致的生物学功能改变有关.     

体外翻译系统在分子生物学研究中的应用

体外翻译系统又称无细胞蛋白质合成系统 ,是分子生物学中一种常规的表达系统。该系统可用于蛋白质快速分析鉴定 ,基因转录和翻译的调控机理的研究以及分子间的相互作用的研究 ,如蛋白质和蛋白质的相互作用 ,蛋白质与 DNA的相互作用 ,蛋白质与 RNA的相互作用等     

组蛋白甲基化与lncRNAs的表达调控

LncRNAs是长度超过200 nt非编码蛋白质的RNA分子,并参与细胞内多种调控过程.LncRNA在发育和基因表达中发挥着复杂精确的调控功能,补充解释了基因组复杂性的生物意义,同时也使人们重新认识了生命过程中基因表达调控网络的复杂性.LncRNA参与了X染色体沉默、基因组印记、染色质修饰、转录激活、转录干扰以及核内运输等多种重要的调控过程.组蛋白修饰是基因表达调控的重要形式,尤其是组蛋白甲基化的调控作用.研究发现组蛋白甲基化对基因异常表达的调控参与多种肿瘤的发生.组蛋白去甲基化酶的发现又提出了一种新的理念,即组蛋白甲基化对lncRNA表达调控作用,这将是基因表达调控研究的新领域.     

Identification of a spliced leader RNA binding protein from Trypanosoma cruzi

Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.     

Prp8蛋白在前体mRNA剪接中的作用

前体mRNA的剪接是基因表达的关键一步,发生在蛋白质的转录之后与合成之前。在前体mR- NA剪接加工过程中需要将转录本中的内含子切除,因为它会干扰基因的转录。前体mRNA的剪接发生在细胞核中,是在一个大的RNA与蛋白质的复合物即剪接体的催化下完成的。Prp8(precursor mRNA process- ing)是参与前体mRNA剪接的最大的蛋白,其序列从酵母到人类是高度保守的。Prp8同时也是细胞核内一个最重要的剪接因子。在剪接过程中,Prp8组成剪接体的催化中心。有人推断Prp8是剪接体的支架蛋白,很可能在催化中心起到锚定RNA的作用,同时也调节着激活剪接体所必需的构象变化。Prp8还与色素性视网膜炎的发生密切相关。     

Beginning or end? Telomere structure, genetics and biology.

The word telomere derives from the Greek word telos meaning 'end', roughly translating as 'the thing at the end' when the end is that of a chromosome. Telomeres belie their apparent simplicity of structure by being involved in a wide range of diverse biological phenomena. Much of our understanding of telomere behaviour comes from studies in lower eukaryotes such as ciliates and yeast, the subject of many recent reviews. Here we concentrate on the mammalian telomere, recent progress in its study, and how recent evidence for an involvement of telomeres in the regulation of gene expression and DNA replication in yeast points to new aspects of mammalian telomere function yet to be explored.     

Protein-protein interactions between native Ro52 and immunoglobulin G heavy chain.

Using a yeast two-hybrid system to search for proteins interacting with Ro52 autoantigen, we identified a novel protein-protein interaction. Two different cDNA clones, which interacted with Ro52 in the yeast two-hybrid system, were identified and isolated from a human B-cell library. Surprisingly, both clones encoded the heavy chain of human IgG1. The expression of both HIS3 and beta-galactosidase reporter genes in yeast suggested that the interaction between Ro52 and IgG occurred in vivo. In vitro studies utilizing recombinant Ro52 and purified immunoglobulins indicated that the interaction was immunoglobulin class and subclass specific. Ro52 interacted with IgG1 and IgG4, but not with IgG2, IgG3, IgA or IgM. Ro52 could also precipitate IgG directly from serum. The identified cDNA clones did not include the variable region of IgG, which suggested a non-classical interaction independent of antibody specificity. We further mapped the domain of Ro52 responsible for this interaction to the C-terminus rfp-like region. In conclusion, our data support an unusual interaction between native Ro52 and IgG. The potential biological significance of this unusual protein-protein interaction is discussed.     

Computationally analyzing the possible biological function of YJL103C--an ORF potentially involved in the regulation of energy process in yeast

Although the complete genomes of a number of organisms have been sequenced, the biological functions of many genes are still not known. Because experimentally studying the functions of those genes one by one requires tremendous time, it is vital to use published resources like microarray gene expression data for computational analysis of gene functions. One example is YJL103C, a yeast gene of unknown function in the Saccharomyces Genome Database (SGD). It is possible to quickly infer its biological function by computational analysis. In this study, we present an efficient model to explore the biological function of a novel gene using microarray data. We showed that the expression pattern of YJL103C is most similar to the genes in the energy group and respiratory chain subgroup. We further found that YJL103C contains a HAP2,3,4 box in its promoter region and a cytochrome C heme-binding signature in its protein sequence. Our findings define a potential role for YJL103C in the regulation of energy metabolism, specifically in the process of oxidative phosphorylation. Similar bioinformatics methods can be applied to infer the biological functions of other novel genes in organisms for which microarray data are available. In this work, we selected a single gene of unknown function as a case study. By focusing on the power of computer analysis and bioinformatics on the available microarray data, we have determined the likely biological function of YJL103C. Our study provides a method by which to explore the potential function of other genes currently annotated as having an unknown function in any organism for which global gene expression data are available.