关键词: Two-hybrid analysis
来源: 互联网
3471 阅读
1.Introduction and Background
There is a great need for general methods to characterize the proteins that contemporary biology makes available.The list of such proteins needing further characterization is growing and includes proteins already known to be important for specific cellular functions,mutant proteins identified in vivo or made in vitro,and very large numbers of protein being identified by genome projects.Here we describe the extension of two-hybrid approaches so that they can bear on this problem.
The recent success of two-hybrid systems is due to the fact that many cellular functions are carried out by proteins that touch one another.For example,the complex process of transcription initiation requires the ordered assembly of numerous interacting transcription factors with RNA polymerase and ancillary proteins,into a protein machine that initiates transcription (Guarente,1996; Tjian and Maniatis,1994).This machine can be viewed as a network of interacting proteins,as can the machines that control other processes,such as DNA replication,protein translation,and the cell cycle.A full understanding of these processes will require knowledge of,not only the proteins (parts)that make up each machine,but also of the topological relationships (connections)that individual parts make with one another.
Likewise,a full understanding of the function of any new protein will require knowledge of the interactions it makes with previously identified proteins.Currently,most new proteins are being identified by large scale sequencing projects.For many of these new proteins the sequence alone sheds little or no light on their function.
Two-hybrid systems have been used to probe the function of new proteins ever since they were developed (Chien et al.,1991; Fields and Song,1989).The first application of two-hybrid methods to probe protein function was to examine the interactions between proteins isolated by two hybrid methods and relatively small numbers of test proteins (see for example,Durfee et al.,1993; Gyuris et al.,1993; Harper et al.,1993; Zervos et al.,1993),but their use quickly spread to the analysis of many other proteins (Choi et al.,1994; Kranz et al.,1994; Marcus et al.,1994; Printen and Sprague,1994; Van Aelst et al.,1993; Yuan et al.,1993).In anticipation of the utility of applying these methods to larger sets,we and others began devising ways to do so.
Larger scale two hybrid approaches typically rely on interaction mating.In this method the protein fused to the DNA-binding domain (the bait)and the protein fused to the activation domain (here called the prey)are expressed in two different haploid yeast strains of opposite mating type (MATa and MATa),and the strains are mated to determine if the two proteins interact.Mating occurs when haploid yeast strains of opposite mating type come into contact,and results in fusion of the two haploids to form a diploid yeast strain.Thus,an interaction can be determined by measuring activation of a two-hybrid reporter gene in the diploid strain.
As described below,interaction mating has been used to examine interactions between small sets of tens of proteins (Finley and Brent,1994; Finley and Brent,1995; Reymond and Brent,1995),larger sets of hundreds of proteins (R.L.F.and R.B.,unpublished),to screen libraries (Bendixen et al.,1994),and to attempt to comprehensively map connections between proteins encoded by a small genome (Bartel et al.,1996).The primary advantage of this technique is that it reduces the number of yeast transformations needed to test individual interactions.For example,to test for interactions between a set of 10 bait proteins and 5 prey proteins without interaction mating would require 50 transformations to create 50 strains that carry the pair-wise combinations of baits and preys.With mating however,only 15 transformations would be needed; 10 for the different bait plasmids,and 5 for the different prey plasmids; and the resulting two sets of transformants would be mated to create the 50 combinations.The microbiology of the mating procedure (which is extremely simple)is detailed in Section 2.
Interaction mating techniques have facilitated a number of two-hybrid studies of protein protein interaction.Among its first uses was to determine the specificity of interactors isolated in library screens or interactor hunts (Harper et al.,1993).As described in the previous chapters,in the first steps of an interactor hunt,one isolates genes that encode proteins that interact with a particular bait.Before the interacting proteins are further characterized,it is necessary to determine if their interaction with the bait is specific by showing that they do not interact with other unrelated baits or with the DNA-binding domain portion of the bait.When mating is used to test specificity,the strain that contains the activation domain fused protein (prey)is mated with different yeast strains which express either the original bait protein or other,preferably unrelated baits,and the investigator verifies that the reporters are only active in diploids that contain the original bait (Finley and Brent,1994; Finley and Brent,1995; Harper et al.,1993).
For example,Harper,Elledge and colleagues used a mating assay to test the specificity of newly isolated interactors (Harper et al.,1993).The methods of these investigators also circumvented the need to isolate the prey plasmid.In their experiments,they performed two-hybrid hunts with a bait plasmid that contains a dominant marker,CYH2,that can be selected against by plating the yeast on medium containing cycloheximide,which is toxic to yeast that carry CYH2.Yeast isolated in an interactor hunt were plated on cycloheximide plates to select those that had lost the original bait plasmid but retained the library plasmid.The resulting strain was then mated with a collection of bait strains,including ones that expressed the original bait,to determine the specificity of the library-encoded prey.A mating scheme has also been used directly in an interactor hunt by mating a strain expressing a bait with a strain transformed with the library DNA; here,mating promises to bypass the need to perform separate transformations with library DNA for each new hunt (Bendixen et al.,1994).
In addition to its use in interactor hunts,mating can be used to characterize small sets of proteins as described in Section 2.1 and Protocol 1.In one example of this approach,we used interaction mating to characterize a set of seven Drosophila Cyclin-dependent kinases (Cdk)interactors,or Cdis (Finley and Brent,1994).Strains expressing versions of the Cdis fused to an activation domain were mated with 74 different strains expressing different bait proteins,including Cdks from other species and four of the Cdis themselves.The results from this study illustrate the types of information that can be derived from such a characterization.First,the experiments showed that some of the Cdis interacted with different subgroups of seven highly related Cdk baits,suggesting that the Cdis recognize structural features shared by these Cdks but absent in the non-interacting Cdks; inspection of an alignment of the Cdk protein sequences suggested residues that may be important for specific interactions with certain Cdis.Second,Cdi3,Drosophila Cyclin D,interacted much more strongly with human Cdk4 than with any of the other Cdks in the panel including the Drosophila Cdks,suggesting that there may be an as yet unidentified Drosophila Cdk4 homolog which is the true partner for Cyclin D.Third,two of the Cdis interacted with two other Cdis,indicating in each instance that each Cdi has surfaces for binding to the Cdk and to another Cdi,and suggesting that these proteins form ternary or higher order complexes.Finally,the demonstration that tw℃dis with no sequence similarity to previously identified proteins interact with each other as well as with the Cdk,but not with a panel of over 60 other proteins,provided an additional clue to their functions,strongly supporting the idea that they function along with the Cdk in the network of proteins that regulates the cell cycle.These results demonstrate that examination of the interactions between even small numbers of proteins can provide a number of functional insights.Much larger sets of proteins can be characterized by scaling up these procedures as described in Section 2.2 and discussed in Sections 6 and 7.
更有优质直播、研选好物、福利活动等你来!
本网站所有注明“来源:丁香园”的文字、图片和音视频资料,版权均属于丁香园所有,非经授权,任何媒体、网站或个人不得转载,授权转载时须注明“来源:丁香园”。本网所有转载文章系出于传递更多信息之目的,且明确注明来源和作者,不希望被转载的媒体或个人可与我们联系,我们将立即进行删除处理。
我的询价