Interacción genotipo x ambiente (GxA)

Y2H is a GAL4-based system that provides a transcriptional assay for detecting protein interactions in living yeast cells. This system can be employed to identify novel protein interactions, confirm suspected interactions, and to define interacting domains. The Y2H screen is based on the coexpression of the bait protein (CATS fused in frame to the GAL4- DBD) and a prey protein (a library of random cDNA expressing protein X fused in frame to the GAL4-AD). If there is interaction between the bait and prey proteins the GAL4-DBD and -AD are brought into proximity thus a transcriptional activation complex is formed which activates the expression of reporter genes in the yeast (Figure 2.2). The yeast strain AH109 (2.1.8), used for the screen, contains reporter constructs in which HIS3, ADE2 and MEL1/lacZ genes are under the control of distinct GAL4-UAS and TATA boxes. Expression of HIS3 and ADE2 provides nutritional selection which enable the yeast cells to grow on plates lacking certain nutrients (2.1.6.2) and MEL1 provides a catalytic color reaction on the same plates supplemented with X-∝-GAL (X-∝-GAL is converted into a blue color by MEL1 gene

product).

Fig. 2.2: The two-hybrid principle. The DBD is amino acids 1-147 of the yeast GAL4 protein, which binds to the GAL4-UAS upstream of the reporter genes. The AD is the amino acids 768-881 of the GAL4 protein and

Potential interaction between proteins identified with the screen should be confirmed by independent methods. Interaction was confirmed cotransforming the prey and bait plasmids into yeast strain AH109 (2.2.22.2) and assaying for growth on selective plates as well as by colocalization (2.2.21) and co-immunoprecipitation experiments (2.2.28).

2.2.22.1 Test of bait plasmid for autonomous activation

Some bait proteins may have intrinsic DNA-binding and/or transcriptional activating properties when fused to the GAL4-DBD activate the yeast reporter genes. Hence deletion of certain portions of the bait protein may be required to eliminate unwanted activity before the protein can be used in a two-hybrid screen. In order to test the bait protein, pGBT9-CATS construct was transformed in the yeast strain CG 1945 (2.1.8) as described in 2.2.22.2. As a control, CG 1945 cells were transformed with the empty pGBT9 plasmid (2.1.11). Since the bait expression plasmid pGBT9 harbor TRP1 gene, transformants were selected by growth on SD -Trp plates (2.1.6.2). The test of self-activation transformants were stricked onto SD -Trp, -His plates (2.1.6.2).

2.2.22.2 Transformation in yeast cell

A polyethylene/lithium acetate mediated transformation was performed to transform yeast strains AH109 or CG 1945 with plasmids expressing either/both GAL4-DBD and -AD fusion proteins (or cDNA library). Yeast cells were made competent as follow: one or more yeast colonies (≤ 4 weeks old) were resuspended in 1 ml YPD medium, mixed by vortexing and

transferred to 20 ml culture medium (2.1.6). O/N culture grown at 30°C for 16-18 hrs shaking at 250 rpm (stationary phase OD600 >1.5) was transferred to 300 ml YPD and incubated for

approx. 3 hrs to OD600 0.4-0.6. Cells were collected by centrifugation at 1 000 x g for 5 min at

RT and resuspended in 30 ml ddH2O. After a second centrifugation step, the supernatant was

discarded, and the cells were resuspended in 1.5 ml freshly prepared 1X TE/LiAc (2.1.5). For sequential transformation, 100 µl of the freshly prepared yeast competent cells were mixed with 10 µl carrier DNA (10 mg/ml), 100 ng bait or prey plasmid (or 1 µg library DNA) and 600 µl PEG/LiAc. For simultaneous cotransformation both bait and prey plasmids were added together in the reaction above at a molar ratio of 2:1 (200 ng DNA-DBD: 100 ng DNA-AD). The reaction was incubated for 30 min shaking at 30°C (200 rpm). 70 µl DMSO was added and gently mixed by inversion. Cells were heat shocked for 15 min in a 42°C water bath, chilled on ice for 2 min, collected by centrifugation at 14 000 rpm at RT for 5 sec and

resuspended in 1 ml YPD. In order to allow cells to recover from the stress, they were incubated for 1 hr at 30°C, shaking at 230 rpm. After that, cells were washed twice in 1 ml 1X TE buffer (14 000 rpm, 5 sec at RT) and finally stricked onto SD plates lacking tryptophane and/or leucine (selection for the presence of plasmid expressing the GAL4-DBD and -AD, respectively). Colonies positive for both bait and prey plasmids were then assayed for protein interaction by re-plating transformants from the SD -Trp, -Leu plate onto SD -Trp, -Leu, -His, -Ade plate supplemented with X-α-GAL. Plates were incubated at 30°C and growth

monitored for several days.

Glycerin stocks were prepared for long-term storage of the yeast clones. Single colony was inoculated into 3 ml YPD and incubated O/N shaking at 30°C. 1 ml of the yeast culture plus 300 µl glycerin were placed in a fresh 1.5 ml tube, mixed by vortexing and stored at -80°C. 2.2.22.3 Large-scale cDNA library transformation

For screening the HeLa S3-cDNA library thus obtaining a high number of cells expressing an individual prey protein, large-scale transformation of the yeast AH109 was performed. In a sequential transformation, the pGBKT7-CATS bait plasmid was firstly introduced through a small-scale transformation as described in 2.2.22.2. Colonies positive for pGBKT7-CATS were assayed by growth on SD -Trp plates. Selected transformants (pGBKT7-CATS/AH109 cells) were then grown up and made competent as described in 2.2.22.2. In order to obtain a maximal amount of transformation efficiency (4x104 cfu /µg cDNA library), the amount of the library DNA was increased to 4X and reaction was scaled up to be performed in a 11X vol. of the small-scale reaction. Thus 1.1 ml of the freshly prepared bait-containing competent cells was mixed with 44 µg cDNA library, 1.1 mg carrier DNA and 6.6 ml PEG/LiAc. Transformation reaction, divided in 11 reaction tubes (1.5 ml) proceeded as described before. Selection of cotransformants was performed using medium-stringency conditions. For that, cells transformed with the library cDNA were plated on SD -Trp, -Leu, -His plates. 200 µl vol. was used to strick each of the 150 mm selective plates (42 plates in total). Plates were grown at 30°C for 7 days. Subsequently, His positive colonies were replicated onto SD -Trp, - Leu, -His, -Ade plate supplemented with X-α-GAL to screen for ADE2 and MEL1 expression.

After 7 days incubation at 30°C, the positive colonies were identified by the blue color.

In order to assay the number of transformants screened in this experiment, 0.1 and 0.2 % of the transformation reaction (1 and 2 µl, respectively) was plated onto SD -Trp, -Leu plates and the number of cfu/ reaction vol. was determined.

2.2.22.4 Isolation of plasmid DNA from yeast cells

Prey plasmids were recovered from positive colonies identified in 2.2.22.3 by a rapid isolation of plasmid-DNA. For that, single colonies were inoculated into 2 ml YPD medium and grown at 30°C, O/N at 200 rpm. 1.5 ml of the O/N culture was transferred to a 1.5 ml tube and spin down for 5 sec at high speed. Supernatant was removed and the pellet was briefly vortexed. Cells were lysed in 200 µl Lysis solution for yeast cells (2.1.5) plus 0.3 g glass beads and 200 µl of phenol/chloroform/isoamyl (25:24:1), vortexed for 2 min and centrifuged for 5 min at 14 000 rpm. Approx. 250 µl of supernatant was transferred to a fresh 1.5 ml tube and ethanol precipitated as described in 2.2.3. DNA was resuspended in 20 µl ddH2O and 5 µl was used

for electroporation (2.2.5.4).