大规模酵母双杂交技术研究蛋白质相互作用的应用

简介了酵母双杂交技术原理, 总结了酵母双杂交技术大规模筛选蛋白质相互作用的基础、应用及存在的问题。因为大规模酵母双杂交技术结果有大量假阳性及假阴性问题, 因此, 有条件情况下有必要同时开展其他方法的大规模蛋白相互作用研究, 以构建规模更大可信度更高的蛋白质相互作用网络图。     

Approaches to detecting false positives in yeast two-hybrid systems

While many novel associations predicted by two-hybrid library screens reflect actual biological associations of two proteins in vivo, at times the functional co-relevance of two proteins scored as interacting in the two-hybrid system is unlikely. The reason for this positive score remains obscure, which leads to designating such clones as false positives. After investigating the effect of over-expressing a series of putative false positives in yeast, we determined that expression of some of these clones induces an array of biological effects in yeast, including altered growth rate and cell permeability, that bias perceived activity of LacZ reporters. Based on these observations, we identify four simple strategies that can assist in determining whether a protein is likely to have been selected in a two-hybrid screen because of indirect metabolic effects.     

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.     

<Emphasis Type="Italic">ScreenMill</Emphasis>: A freely available software suite for growth measurement,analysis and visualization of high-throughput screen data

Background  

Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.     

A型流感病毒NS1与突触后密度蛋白-95的相互作用

目的 A型流感病毒NS1蛋白是一种多功能的致病因子,能够与被感染细胞中的多种蛋白相互结合,影响并干扰宿主细胞内的信号转导、蛋白质合成及抗病毒反应。突触后密度蛋白(Postsynaptic density protein95,PSD-95)主要存在于神经元及SH-SY-5Y等神经来源的细胞株中。假设NS1能够与PSD-95结合,则更有利于了解A型流感病毒对神经元及相关细胞的作用机制。方法通过酵母双杂交,GST-pull down及免疫荧光技术分别从体外和体内两方面检测NS1与PSD-95的相互作用。结果酵母双杂交表明,仅转染PGAD-NS51/PGBK-PSD-95的QDO有菌落生长,且α-半乳糖苷酶活性显著高于阳性对照;而转染PGAD-NS32/PGBK-PSD-95的QDO无菌落生长;GST-pull down表明仅NS51与PSD-95孵育后,能够被Western-blot检测到;免疫荧光表明NS51与PSD-95可能存在共定位,而NS32与PSD-95则不存在共定位。结论 H5N1(A/chicken/Guangdong/1/2005)的NS1能够与PSD-95结合;反之,H3N2(A/Shantou/602/06)的NS1则不能。     

A direct comparison of protein interaction confidence assignment schemes

Background  

Recent technological advances have enabled high-throughput measurements of protein-protein interactions in the cell, producing large protein interaction networks for various species at an ever-growing pace. However, common technologies like yeast two-hybrid may experience high rates of false positive detection. To combat false positive discoveries, a number of different methods have been recently developed that associate confidence scores with protein interactions. Here, we perform a rigorous comparative analysis and performance assessment among these different methods.     

The full-length and N-terminal deletion of ORF2 protein of hepatitis E virus can dimerize

Hepatitis E virus is a human RNA virus containing three open reading frames. Of these ORF2 encodes, the major capsid protein (pORF2), may possess regulatory functions, in addition to a structural one. In this study, we have shown using the yeast two-hybrid system and in vitro immobilization experiments that full-length pORF2 is capable of self-association, thus forming a homodimer. Using mutational analysis we have studied dimerization of various truncated versions of the ORF2 capsid protein using the yeast two-hybrid system and supported our findings with in vitro immobilization experiments. Deletions of pORF2 reveal a loss of the dimerization potential for all deletions except an N-terminal 127-amino-acid deletion. Our studies suggest that the dimerization property of pORF2 may not be amino-acid sequence-dependent but instead a complex formation of a specific tertiary structure that imparts pORF2 its property to self-associate.     

A high-throughput system for two-hybrid screening based on growth curve analysis in microtiter plates

The yeast two-hybrid system is a powerful tool for identifying novel protein-protein interactions. In general, biochemical marker genes such as lacZ are exploited for indirect quantification of the interaction, and commonly involve the conduct of rather laborious beta-galactosidase assays. This paper describes a simple alternative method based on growth curve analysis of yeast cultures that is amenable to microtiter plate format, and therefore allows the quantification of large numbers of yeast two-hybrid combinations. The analyzed results of yeast cultures grown in microtiter plates were compared with those obtained from the classical beta-galactosidase assay. We conclude that the method presented here is reproducible, of equal or greater sensitivity than the beta-galactosidase assay, and can be further adapted for application to the conduct of large-scale, automated yeast two-hybrid experiments.     

SipA, a novel type of protein from Synechococcus sp. PCC 7942, binds to the kinase domain of NblS

Cyanobacteria respond to nutrient stress conditions by degrading their light-harvesting complexes for photosynthesis, a process regulated in Synechococcus sp. PCC 7942 by the sensor histidine kinase non-bleaching sensor (NblS). In yeast two-hybrid screenings for proteins interacting with NblS we have identified a novel type of protein, named SipA for NblS interacting protein A. Specific binding between NblS and SipA is observed with both yeast and bacterial two-hybrid systems. Additional yeast two-hybrid screenings with SipA as bait further confirmed the specificity of the interaction and allowed us to map their determinants to the ATP-binding domain of NblS. Strong conservation and coevolution of both NblS and SipA in cyanobacteria further suggests the importance of SipA in the context of the NblS signal transduction network.     

Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.     

Identification of proteins that interact with a protein of interest: Applications of the yeast two-hybrid system

The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.     

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.     

Protein--protein interaction maps: a lead towards cellular functions

The availability of complete genome sequences now permits the development of tools for functional biology on a proteomic scale. Several experimental approaches or in silico algorithms aim at clustering proteins into networks with biological significance. Among those, the yeast two-hybrid system is the technology of choice to detect protein-protein interactions. Recently, optimized versions were applied at a genomic scale, leading to databases on the web. However, as with any other 'genetic' assay, yeast two-hybrid assays are prone to false positives and false negatives. Here we discuss these various technologies, their general limitations and the potential advances they make possible, especially when in combination with other functional genomics or bioinformatics analyses.