Applications of the yeast two-hybrid system.

In recent years, the yeast two-hybrid system has become the method of choice for detection and analysis of protein-protein interactions in an in vivo context. This system, which capitalizes on the significant genetic history and ease of protocols for manipulation of Saccharomyces cerevisiae, is accessible to most laboratories and is applicable to the pursuit of a large variety of experimental goals. To date, the two-hybrid system has seen widespread application for identification of interaction partners by screening methods using a particular protein of interest as a "bait." Large-scale ventures are also in progress, for example, a cataloging of interactions among the cellular proteins in yeast. However, this method also has tremendous potential for more focused analyses of specific proteins and should become more routine as an alternative or adjunct approach for many structure-function investigations.     

Development of a high-throughput yeast two-hybrid screening system to study protein-protein interactions in plants

We have developed a high-throughput yeast two-hybrid screening system (HTP-YTH) that incorporates yeast gap-repair cloning, multiple positive ( ADE2, HIS3, lacZ) and negative ( URA3-based) selection schemes to reduce the incidence of negative and false positive clones, and automation of laboratory procedures to increase throughput. This HTP-YTH system has been applied to the study of protein-protein interactions that are involved in rice defense signal transduction pathways. More than 100 genes involved in plant defense responses were selected from DuPont's rice expressed sequence tag (EST) databases as baits for HTP-YTH screening. Results from YTH screening of eight of these rice genes are presented in this paper. Not only have we identified known protein-protein interactions, but we have also discovered novel interactions, which may ultimately reveal the regulatory network of host defense signal transduction pathways. We have demonstrated that our HTP-YTH method can be used to map protein-protein interaction networks and signal transduction pathways in any system. In combination with other approaches, such efficient YTH screens can help us systemically to study the functions of known and unknown genes in the genomics era.     

酵母杂交系统的发展及其应用

近年来,酵母单杂交,双杂交及三杂交系统技术的出现及发展已成为分析鉴定蛋白蛋白,DNA蛋白相互作用及调控的强有力工具。本文简述了酵母杂交系统的发展及潜在的应用价值。     

基于酵母双杂交的微囊藻毒素检测系统初探

微囊藻毒素是由淡水微囊藻产生的次生代谢物,结构为环状七肽,分子量约1000Da,对生物机体具有多种毒理作用,是一类较强的肿瘤促进剂,目前没有比较简单便宜的藻毒素检测方法,本文探索建立利用酵母双杂交系统进行藻毒素检测的方法.前期实验已用噬菌体表面展示肽库技术从随机12和7肽库中筛选获得若干与藻毒素有相互作用的多肽.本文分别挑选其中亲和力较高的3个多肽,构建到酵母双杂交系统中,将不同的肽段分别融合在含有Active Domain和Binding Domain的表达载体中,共转化酵母菌株AH109,然后测定β-半乳糖苷酶活性来反映下游报告基因半乳糖苷酶的表达情况,从而通过反映培养基里面是否含有藻毒素,进而利用此方法来比较方便地检测藻毒素.     

A yeast two-hybrid smart-pool-array system for protein-interaction mapping

We present here a new two-hybrid smart pool array (SPA) system in which, instead of individual activation domain strains, well-designed activation domain pools are screened in an array format that allows built-in replication and prey-bait deconvolution. Using this method, a Saccharomyces cerevisiae genome SPA increases yeast two-hybrid screening efficiency by an order of magnitude.     

Analysing protein-protein interactions with the yeast two-hybrid system

Plant research is moving into the post-genomic era. Proteomic-based strategies are now being developed to study functional aspects of the genes predicted from the various genome-sequencing initiatives. All biological processes depend on interactions formed between proteins and the mapping of such interactions on a global scale is providing interesting functional insights. One of the techniques that has proved itself invaluable in the mapping of protein-protein interactions is the yeast two-hybrid system. This system is a sensitive molecular genetic approach for studying protein-protein interactions in vivo. In this review we will introduce the yeast two-hybrid system, discuss modifications of the system that may be of interest to the plant science community and suggest potential applications of the technology.     

神经元蛋白3.1结合蛋白酵母双杂交筛选体系的建立

神经元蛋白3.1(P311)是肺泡发育的上游调节因子。以pEGFP-P311重组质粒为模板,利用PCR方法扩增P311基因编码序列。通过Nde I和BamH I位点插入诱饵载体pGBKT7,构建重组诱饵载体pGBKT7-P311。重组体转化酵母菌AH109进行自激活和毒性检测,结果 DNA-BD-P311融合蛋白无单独激活报告基因作用,对酵母菌亦无毒性。以出生11 d小鼠肺组织为材料,提取总RNA。逆转录产生单链cDNA,通过长距离PCR进行扩增。扩增产物ds cDNA电泳后可见大小为0.2～3.0 kb间的弥散状分布条带,说明文库cDNA可满足筛选要求。诱饵载体pGBKT7-P311的构建及相应小鼠肺组织cDNA文库的建立,为进一步利用酵母双杂交技术探讨P311功能奠定了基础。     

Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system

Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein. In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis. A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein. Nine positive clones were obtained from screening 1 x 106 clones. The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product. The clone 4 translated product was named StAR-binding protein (SBP). We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector. After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased. The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells. In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones.     

应用酵母双杂交技术寻找与GGNBP相互作用的蛋白质

生殖细胞缺陷症(gcd)小鼠突变体是20个世纪90年代初发现的一种不育突变小鼠,其不育原因是由于其胚胎期原始生殖细胞的数目低于正常。FancL(也叫Pog)的缺失是引起gcd突变小鼠的原因,FancL基因缺失后可能影响了小鼠胚胎期原始生殖细胞的增殖／存活和成年期小鼠精母细胞的减数分裂。FANCL是一种含有PHD结构域的泛素E3连接酶,是Fanconi贫血复合物的组分之一。在生殖细胞中,FANCL与GGN1和GGN3相互作用,GGN1和GGN2又与一种新的蛋白质GGNBP特异作用。但GGNBP蛋白的功能还不清楚。为了研究GGNBP的功能以及揭示更多的参与该过程的蛋白质,运用Clontech公司新开发的第3套酵母双杂交系统,以GGNBP为诱饵从成年小鼠睾丸cDNA库中筛选与其相互作用的蛋白质基因,发现了一个主要在睾丸中表达的新的基因,其编码的蛋白质产物在酵母系统中与GGNBP特异作用。     

Identification of connexin-interacting proteins: application of the yeast two-hybrid screen

Protein-protein interactions are recognized as one of the fundamental mechanisms for relaying the intra- and intercellular signals that are required for normal cellular activities affecting growth, development, and maintenance of homeostasis in tissues and organs. The yeast two-hybrid screen has become a valuable tool for identifying protein-protein interactions. The gap junction protein connexin 43 (Cx43) has been implicated in a number of biological processes including development and cellular growth control. To further advance our understanding of the ways in which Cx43 may influence these cellular activities, and to extend our knowledge of the regulation of Cx43 function and/or processing, we have employed the yeast two-hybrid screen technique to identify Cx43-interacting proteins. We present detailed methods for the yeast two-hybrid screen of a mouse embryonic cDNA library using the C terminus of Cx43 as "bait." We also describe additional methods to confirm the interactions between Cx43 and the identified proteins. These methods include in vitro binding assays, coimmunoprecipitation, and subcellular localization using immunofluorescence microscopy.     

酵母双杂交在植物功能基因组研究中的应用

扼要介绍了酵母双杂交技术的原理,详细评述了该技术在植物功能基因组中的研究进展,并结合自己研究领域对该技术在植物领域发展方向作了展望。     

Automation of yeast two-hybrid screening

We have developed an automated format for screening yeast two-hybrid libraries for protein-protein interactions. The format consists of a liquid array in which pooled library subsets of yeast, expressing up to 1000 different cDNAs, are mated to a yeast strain of the opposite mating type, expressing a protein of interest. Interactors are detected by a liquid assay for beta-galacsidase following prototrophic selection. The method is demonstrated by the detection of interactions between two encoded yeast RNA polymerase subunits in simulated libraries of varied complexity. To demonstrate its utility for large scale screening of complex cDNA libraries, two nuclear receptor ligand-binding domains were screened through two cDNA libraries arrayed in pooled subsets. Screening these libraries yielded clones which had previously been identified in traditional yeast two hybrid screens, as well as several new putative interacting proteins. The formatting of the cDNA library into pooled subsets lends itself to functional subtraction of the promiscuous positive class of interactor from the library. Also, the liquid arrayed format enables electronic handling of the data derived from interaction screening, which, together with the automated handling of samples, should promote large-scale proteome analysis.