Twohybridanalysisofgeneticregulatorynetworks2

2.2 Interaction mating - large scale

With a few modifications, the procedure described above can be used to test for interactions between a single prey protein and hundreds of baits (Protocol 3, see Figure 1 below). Large panels of bait strains can be collected and stored frozen indefinitely (Protocol 2) and then screened against any number of preys. One such set of bait strains contains over 700 different LexA fusion proteins from our own work and from numerous other labs that use the interaction trap (R.L.F., R.B., A. Reymond, unpublished). Screening a protein against such a panel enables one to quickly test its ability to interact with a large number of known proteins, most of which have been characterized to some extent, and have been chosen for study because of their known or suspected involvement in some biological process. Thus, the finding of an interaction between a tested protein and a member of the panel can often lead to immediate clues about the biological function of both proteins (see Section 5). While the number of proteins in the existing panel is far less than the number of proteins in a good library, this approach does offer the advantage of screening the test protein against a set of proteins enriched for those of current interest to the biological community. It is worth noting that these proteins come from many different organisms in which they are expressed in different tissues and at different developmental stages. Thus it becomes possible to identify interacting partners that have not yet been isolated from the same species, or that are not expressed in tissues from which interaction libraries have been made.

For some proteins, this approach offers additional advantages over screening a library using a traditional two-hybrid scheme. Proteins that activate transcription when fused to LexA or another DNA-binding domain can be difficult to use in conventional interactor hunts. Though methods are available to reduce the sensitivity of the reporter genes (Durfee et al., 1993; Estojak et al., 1995; Chapter 2, 3, 4) it is not always possible to reduce the reporter sensitivity below the threshold of activation for some baits. Moreover, reduction in reporter sensitivity carries with it the risk that the reporters will not detect weakly interacting proteins. Furthermore, spontaneously occurring yeast mutations, for example those that increase the copy number of the bait plasmid, can increase the activating potential of weakly activating baits (R.L.F., R.B., A. Mendelsohn, unpublished data); such mutations are typically scored as positive in the early stages of an interactor hunt, and they are not readily detected in schemes where the specificity test is performed by removing the bait plasmid from the strain containing the prey and mating the strain with other bait strains. Thus, an alternative for proteins that activate transcription as baits, is to use them as preys to screen existing panels of baits, or even libraries of baits. Interaction mating approaches also have clear advantages for proteins that are somewhat toxic to yeast; the prey vector allows conditional expression of toxic proteins in the presence of a bait, and often the interaction can be observed as the reporters are activated even if the cells are inviable. An example of the use of interaction mating together with a large panel of bait strains to characterize a protein that both activates transcription and is toxic to yeast, Drosophila Cyclin E (Finley, Zavitz, Thomas, Richardson, Zipursky, and Brent, in prep), is discussed in Section 7.

Figure 1. Mating assay for interactions between a prey and 96 baits

Figure 1.

Top. The plate on the left holds 96 different yeast strains in patches (or colonies) that each express a different bait protein. The plate on the right holds 96 patches, each of the same yeast strain (prey strain) that expresses a protein fused to an activation domain (prey). The plate of bait strains and the plate of prey strains are each pressed to the same replica velvet and the impression is lifted with a plate containing YPD medium. After one day of growth on the YPD plate, during which time the two strains mate to form diploids, the YPD plate is pressed to a new replica velvet and the impression is lifted with a plate containing diploid selection medium and an indicator like X-Gal. Blue patches (dark spots) on the X-Gal plate indicate that the lacZ reporter is transcribed, suggesting that the prey interacts with the bait at that location.

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Protocol 2. Collecting bait (and prey) strains

Materials

Freezing media: 1:1 solution of minimal glucose media lacking appropriate amino acids (e.g. -u-h Glu for bait strains) : sterile glycerol solution (65% (v/v) glycerol, 0.1 M MgSO4, 25 mM Tris-HCl pH 7.4)

1.0 to 1.5 ml cryotubes

Yeast strains freshly streaked to minimal glucose plates

Sterile wooden applicator strips

Methods

1. Streak bait strains to -u-h Glu plates, or prey strains to -w Glu plates, and incubate at 30oC for 24 to 48 hours. Yeast should be taken from the plates and frozen no more than 4 days after being streaked.

2. With a sterile wooden applicator stick, grab a dollop of yeast from the plates and inoculate 0.5 ml of freezing solution in a cryotube. Vortex lightly. This solution should have an OD600 over 3.0.

3. Alternatively, inoculate 0.5 ml of -u-h Glu liquid media to an OD600 less than 0.2, incubate at 30oC with shaking until OD600 = 1.5 to 2.0 (log phase), and add 0.25 ml of this culture to 0.25 ml of sterile glycerol solution in a cryotube.

4. Freeze by placing cryotubes in -80oC freezer. Most strains can be recovered after up to at least two years by scraping the surface of the ice and streaking to minimal glucose plates. Avoid allowing entire contents of cryotube to thaw.

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Protocol 3. Mating assay - large scale for hundreds of different bait or prey strains.

Materials

Freshly streaked bait and prey strains (see Protocol 1)

One set of the following 150 x 15 mm plates for each test of interactions between an activation domain-tagged protein (in a prey strain) and 96 baits (bait strains): -u-h- Glu; -w Glu; YPD; -u-h-w Glu X-Gal; -u-h-w Gal/Raf X-Gal

Replica plater and sterile velvets for 150 mm diameter plates. (A replica devise can be fashioned from a box of 200 µl pipet tips by stretching a velvet over the top of the box)

96-prong device (e.g. DanKar MC-96) with 3 mm diameter flat ended metal prongs in a 96-well configuration. Similar devices can be used in 48-well configurations for use with 100 mm plates.

0.5 to 4.0 ml sterilized tubes arranged in a 96-well configurations (e.g. cluster tubes such as Costar #4411). Ideally these tube can be capped and frozen at -80oC.

-u-h Glu liquid media, see Chapter 4

-w Glu liquid media, see Chapter 4

Sterile glycerol solution (65% (v/v) glycerol, 0.1 M MgSO4, 25 mM Tris-HCl pH 7.4)

Methods

1. It is most convenient to place large numbers of bait strains in a 96-well configuration (Figure 1). This can be done by inoculating 2 ml of -u-h Glu media in cluster tubes and growing to OD600 = 1.5 to 2.0. After making plates from these cultures (see step 2 below) add an equal volume of sterile glycerol solution, cap and freeze at -80oC.

2. Use the 96-prong device, sterilized in ethanol and flame, to transfer bait strains from the culture to the center of a 150 mm -u-h Glu plate. Each plate can contain 96 different bait strains. Tens of identical plates can be made from one culture. Incubate the plates at 30oC for 48 hours or until all bait strains have grown to colonies 5 mm in diameter. These plates can be stored at 4oC for up to 2 months and used to inoculate another liquid culture when more plates are needed. Several positions on each plate should contain control strains with baits that activate various levels of transcription (see Section 4 and Table 1).

3. Inoculate 50 ml of -w Glu liquid media with a prey strain and grow at 30oC with shaking to OD600 = 1.5 to 3.0. Pour the culture into a sterile 150 mm plate, or into the sterile top from a box of 200 µl pipets, and use the 96-prong device, sterilized in ethanol and flame, to transfer the culture to -w Glu plates. On these plates, all 96 positions will contain the same prey strain.

4. Follow the replica plating procedure from Protocol 1 to combine the bait and prey strains to a YPD plate, and then after growth on the YPD plate at 30oC for 24 hours, replica to X-Gal indicator, diploid selection plates (-u-h-w Glu X-Gal and -u-h-w Gal/Raf X-Gal) (see Figure 1 above).

5. Examine results after two days.

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3. Interaction mating assay with other yeast two-hybrid systems

In addition to the interaction trap, many other yeast two-hybrid systems have been developed (see Chapter 1 and Allen et al., 1995; Fields and Sternglanz, 1994; Mendelsohn and Brent, 1994, for reviews). All of these allow the analysis of individual protein-protein interactions, and permit interactor hunts to isolate new proteins that interact with a bait. In some instances plasmids or strains from one system can be used in another, but often the components are incompatible. Most often, the yeast selectable markers on the different components differ. In addition, systems that use Gal4 as the DNA binding domain cannot be used with yeast strains that have a wild-type GAL4 gene, and therefore, since the Gal4 protein is required to activate the GAL1 promoter, cannot be used with systems that use the GAL1 promoter to drive expression of the prey protein. Finally, use of interaction mating requires careful attention to the mating types of the strains and the selectable markers used to select the diploids.

4. Recording the results

Interaction between bait and prey results in the interaction phenotypes: growth of the strain on medium lacking leucine, and transcriptional activation of the lacZ reporter and production of active ß-galactosidase. On X-Gal plates the ß-galactosidase cleaves the X-Gal substrate, producing a product which turns the yeast colony blue. The amount of color provides a fast and simple method to approximate the level of lacZ expression in a strain. An interaction is scored when a the diploid colony is more blue on the X-Gal plate containing galactose than the X-Gal plate containing glucose.

Scoring these interactions benefits from inclusion of a number of controls. To control for common variations between the X-Gal plates, it is useful to include control strains that contain baits which activate transcription to varying extents. Table 1 shows some baits with known activating abilities. Inclusion of such strains on every X-Gal plate enables one to normalize the amount of blue produced by an interaction. It is also useful to include a control strain to check that the plates contain the correct carbon sources, and ensure that the GAL1 promoter which drives the expression of the prey protein is activated on the Gal/Raf plates and not the Glu plates. An ideal control of this nature consists of a diploid strain derived from a mating assay, which expresses an interacting pair of bait and prey proteins, such as any one of a number of well-characterized interacting pairs (Finley and Brent, 1994; Gyuris et al., 1993; Zervos et al., 1993). An alternative to using X-Gal plates is to perform a filter lift assay for ß-galactosidase activity in grown diploid colonies (Chapter ). Finally, every bait should be tested to see if, and how much, it activates transcription in the absence of a prey, which can be simply accomplished by mating the bait strains to a strain containing the empty prey vector. Thus, a true interaction with a prey protein is scored when the amount of galactose-dependent activation of the lacZ reporter (e.g. amount of blue) exceeds the amount produced in the absence of a prey.

Table 1. Activating and non-activating baits

5. Interpreting interaction data

5.1 Qualitative interpretation

For large amounts of information flowing from interaction mating experiments, the problem of determining whether individual interactions are meaningful is multiplied. We consider a number of these separately.

True and false positives. Any given interaction with affinity tighter than 10-6 will get detected. Although there may exist a weak positive correlation between apparent tightness and biological significance, many apparently weak interactions are real while some strong ones are not. The problem of determining which interactions have biological significance is therefore not trivial. At the moment, the most satisfying way to show biological significance is to verify the interaction by a different, biochemical technique, preferably co-precipitation from a cell in which both proteins are expressed. However, the interaction data alone can often point out probable true and false positives. For example, our experience indicates that highly specific interactions, such as between a protein that binds to one or a small set of highly related proteins and not to hundreds of unrelated proteins, are good candidates to pursue as biologically relevant. Conversely, we tend to give less weight to interactions between proteins that are sticky, or involving those proteins so ubiquitous in the life of the cell (e.g., members of the ubiquitin system or heat shock proteins) that the interactions might be meaningful but relatively uninformative.

True and false negatives. A problem less frequently considered is that of interactions that are not observed. Two observations suggest that many interactions that should be observed are not. One is that in library screens proteins that should be found occasionally are not. Although failure to recover expected proteins in this instance might be due to trivial considerations, such as the absence of the protein from the library used, another fact suggests there could be other reasons. There are now a number of examples in which known interactions are either not observed, or are subject to directionality, being observed only when one of the two proteins is a bait and the other a prey (see for example, Estojak et al., 1995). Our current doctrine for determining that individual interactions do not occur is that full length and truncated putative partners must be tested in all combinations of baits and preys, with the most sensitive reporters, before the investigator can tentatively conclude that the two proteins do not touch. Since this is impractical for mating experiments that involve a large number of baits and preys, such as the genome wide approaches discussed below, we are resigned that false negatives will arise, and we do not give the absence of interaction any weight in our data analysis. This doctrine may change as more sensitive detection methods are designed.

Multimeric complexes. Finally, it is worth noting that one can build up chains of individual binary interactions to suggest higher order complexes. This has worked well, for example with proteins in signal transduction (Choi et al., 1994; Marcus et al., 1994; Printen and Sprague, 1994), and the advent of mating techniques has made it even easier to build up such patterns (Finley and Brent, 1994; C. Kaiser and D. Shaywitz, personal communication).

5.2 Quantitative interpretation