Genetic analysis has been a very successful tool in modern biology. We have now identified the genes responsible for important cellular and developmental processes in many organisms and, in many fields, attention is being focused on determining the molecular function of the proteins these genes encode. Y2H is a mature technology to determine molecular interactions between proteins. Two major advantages of this system are its accessibility to laboratories with expertise in molecular genetics (the only starting materials required are cDNA clones of the genes of interest) and, because the protocols are largely the same for all proteins regardless of the species of origin, the ability to perform experiments in a high throughput environment. A major disadvantage of the Y2H technology is it requires a great deal of work to set up (it requires a many types of media, suitable libraries, and expertise in large-scale yeast transformation). Once established, however, it is relatively little work to perform additional assays. Therefore, the Indiana METACyt Initiative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc., has provided funding for the establishment of a yeast-two-hybrid interaction facility at Indiana University.
Yeast two hybrid analysis: In Y2H analysis bait and prey constructs are fused to the DNA-binding and activation domains of a transcription factor such as GAL4. When introduced into yeast, if the bait and prey proteins physically interact they will reconstitute a functional GAL4 transcription factor, which can then activate the transcription of one or more reporter genes. Y2H can be used to test for interactions between two known proteins or bait constructs can be used to naively screen expression libraries in which cDNA are fused the GAL4 activation domain. Practically, Y2H analysis involves a number of manipulations in E. coli (cloning) and yeast (testing for interactions). The basic steps in the screen are as follows:
1. Obtain error-free cDNA clones for genes of interest.
2. Subclone into Y2H vectors.
3. Sequence to confirm the proper reading frame between the Y2H vector and the cDNA.
4. Transform into yeast.
5. Check clones for autoactivation and determine the optimal conditions for library screening or pairwise interaction.
6. Perform library screen or check for pairwise interactions.
7. Verify interactions using selective medias and lacZ activity.
For clones identified in library screens:
1. Extract plasmids from yeast.
2. Transform recovered plasmids into E.coli.
3. Sequence to determine identity of interacting clones.
4. Retransform yeast with recovered plasmids to verify interactions.
The primary focus of the facility will be on those portions of Y2H analysis that are actually performed in yeast (i.e., steps 4-11). These steps require expertise in yeast manipulation that many laboratories lack, as well as a large number of selection medias (steps 4-11 require 10 different types of plates). Although the facility may perform cloning into the Y2H vectors (with some conditions, see “Cloning” below), it is expected that most laboratories that will make use of the facility will have sufficient expertise in basic molecular biology to create bait and prey constructs themselves.
Below is an outline of the primary services to be offered by the facility and is meant to be flexible. The facility will strive to accommodate the needs of individual researchers as much as possible.
Cloning: As mentioned above, the proposed facility will focus on the portions of Y2H analysis that are performed in yeast. It is expected that most laboratories will produce their own bait/prey constructs.
Testing clones for autoactivation: An interaction between bait and prey in the Y2H system results in the induction of HIS3, which enables yeast to grow in the absence of histidine in the media. Unfortunately, some bait clones exhibit autoactivation (i.e., they are able to turn on reporter genes in the absence of an interaction). In most cases, this autoactivation can be controlled by the addition of 3-Amino-1,2,4-Triazol (3AT), which acts as a quantitative inhibitor of the HIS3 reporter gene. Thus determining the optimal concentration of 3AT for each clone is critical. Bait and prey clones (along with control plasmids) will transformed into his- yeast and replica plated onto plates containing 0, 10, 20, 50, 75, and 100mM 3AT. The lowest concentration of 3AT that inhibits growth will be used for library screening and/or pairwise interactions. If autoactivation cannot be suppressed with the addition of 3AT, the bait clone is unsuitable for Y2H analysis and will need to be redesigned.
Library screens: It is expected that many users of the facility will be interested screening Y2H libraries to identify proteins that interact with their protein of interest. The facility will screen at least 1x107 clones for each bait clone provided. Clones are incubated at 30°C for 5-10 days, at which point clones that exhibit growth in the absence of histidine are selected for further analysis. The number of potentially interacting clones identified will vary depending on the bait used. The facility will perform further analysis on up to 91 clones (for analysis of additional clones, contact the facility).
Pairwise assays: In addition to naïve library screens, some users may already have a hypothesis that two proteins interact. In these cases the facility will perform pairwise assays between pairs of bait and prey proteins provided by the user. All clones will be tested for autoactivation as described above, then the bait and prey constructs will be co-transformed into yeast and eight independent transformants will be used to test for interaction as described below (see verifying interactions).
Verifying interactions: The Proquest vector system contains three markers that indicate an interaction between bait and prey proteins: growth in the absence of histidine, growth in the absence of uracil, and b-galactosidase (lacZ) activation. Potentially interacting proteins (from library screens or pairwise assays) will be assayed for all three markers.
Identifying interacting clones: The identity of interacting clones identified through library screening will be determined by sequencing.
Retransformation assays: In order to obtain the large number of transformants required for Y2H library screening, transformation is performed with a large quantity of library DNA (~100ug).Occasionally, yeast can take up more than one library plasmid; thus it is important to determine that the recovered and sequenced plasmid is really the interacting clone. Therefore, for clones obtained from library screens, the facility will retransform yeast with the bait and prey plasmids and verify the interaction as described above (see verifying interactions).
Sample storage: The facility will not provide long-term storage for any samples. At the conclusion of the experiments all constructs, yeast, and bacterial strains will be turned over to the user.
Because of the significant cost associated with purchasing pre-made or custom-made libraries, it is not feasible for us to obtain libraries for every organism. Our highest priorities will be to obtain suitable libraries for major model organisms, which have the potential to be used by multiple laboratories. Contact the facility about available libraries.
Please contact the facility for pricing.