Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Suppressor screens identify genetic interactions that would shed light on the functions of the gene of interest as well as the pathways and biological processes in which they may be involved in vivo. This technique can identify a genetic relationship among two gene products that may not have been demonstrated using other approaches. The results of such screens in yeast can be extended to high eukaryotic organisms, since most fundamental biological pathways and processes are conserved across evolution.

The success of the scheme depends upon the availability, of high-quality and the high efficiency of its transformation into yeast cells. Thus, transformation protocol with the plasmid before Grow the Schizosaccharomyces pombe ell1 deletion strain at 32 degrees Celsius on YE medium plate. Take a loop full of ell1 mutant strain from the plate, inoculate five milliliters of supplemented YE medium, and then incubate the vial while shaking at 32 degrees Celsius overnight.

After incubation, dilute the overnight growth culture in 100 milliliters of fresh YE medium containing the desired supplements to attain 0.3 optical density at 600 nanometers. Incubate the medium at 32 degrees Celsius while shaking at 200 to 550 RPM until the optical density at 600 nanometers reaches the mid-log phase. After dividing the 100 milliliters of culture into two 50-milliliter centrifuge tubes, pellet the cells at 4, 000 times G for 10 minutes at room temperature.

Once the supernatant is discarded, resuspend the cell pellet in one milliliter of sterile water. Centrifuge and discard the supernatant once again and then resuspend the cell pellet in one milliliter of lithium acetate TE.Pellet the cell by centrifugation and resuspend each cell pellet in 250 microliters of lithium acetate TE.Next, transfer 125 microliters of the competent cells into four different sterile microcentrifuge tubes for subsequent transformation steps. Boil carrier DNA from salmon testes or herring sperm for one minute and immediately place the tube on ice.

For each transformation, add 10 microliters of single-stranded carrier DNA to the microcentrifuge tube containing 125 microliters of the competent cells and mix gently by pipetting. Subsequently, add 50 micrograms of the Schizosaccharomyces pombe cDNA library to the tube containing competent cells and carrier DNA mixture. After incubating the tube at room temperature for 10 minutes, add 260 microliters of polyethylene glycol-lithium acetate solution and mix by pipetting.

Incubate the tube containing the transformation mixture at 32 degrees Celsius for two hours without shaking. Add 43 microliters of pre-warmed dimethyl sulfoxide to the tube and mix gently. Now, expose the tube to heat shock at 42 degrees Celsius for five minutes.

Pellet the cells and discard the supernatant as demonstrated previously and then pipette out the residual polyethylene glycol lithium acetate TE solution. Resuspend the cells in 100 microliters of sterile water and plate them on one EMM2 plate containing the required supplements and 0.2 micrograms per milliliter of 4-NQO. Then, allow colonies to appear on the plates by incubating the plates at 32 degrees Celsius for five to six days.

For further screening, streak the obtained colonies on the same medium plate in the presence of 0.2 micrograms per milliliter of 4-NQO. For one library transformation experiment, use 500 microliters of competent cells for transformation and plate 1-by-10th of the transformation mixture on EMM2 plate with required supplements lacking 4-NQO to calculate the total number of library clones obtained after transformation and screen for the suppressor clones. Add 100 to 200 microliters of sterile water to each well of a sterile 96-well microtiter plate.

Using a sterile toothpick or a 20-to 200-microliter micropipette tip, pick a small amount of inoculum from each colony obtained after cDNA library transformation on the plate containing 4-NQO. Add the inoculum from each of the different colonies into each well of the plate containing sterile water and mix thoroughly. Spot three microliters of the cell suspension from each well onto EMM2 agar plates and add appropriate concentrations of 4-NQO to the plates.

After growing the cells at 32 degrees Celsius for three to four days, identify the colonies that show growth in different concentrations of 4-NQO as putative suppressors. To further validate the suppressors, grow the selected suppressors and appropriate control strains in EMM2 medium containing 225 micrograms per milliliter of adenine and uracil at 32 degrees Celsius overnight with shaking. After the end of the incubation, dilute the cells to an optical density of 0.3 in fresh EMM2 medium and grow them while shaking at 32 degrees Celsius until the mid-log phase.

Spot appropriate serial dilutions of cultures on EMM2 plates with required supplements containing either 0.4 micromolar 4-NQO or 0.01%MMS. For control, spot the strains on an EMM2 plate with required supplements but lacking any DNA-damaging agent. Incubate the plates at 32 degrees Celsius for three to five days to monitor growth.

Spot the mutant strains transformed with the full-length gene of interest or empty vector as positive and negative controls, respectively, along with the library transformants. Inoculate a single yeast colony in EMM2 medium containing 225 micrograms per milliliter of adenine and uracil and grow the cells at 32 degrees Celsius overnight with shaking at 200 to 250 RPM. At the end of incubation, harvest 10 milliliters culture of optical density 0.5 by centrifuging at 4, 000 times G for two minutes at room temperature.

Remove the supernatant and resuspend the cell pellet in 0.2 milliliters of lysis buffer. Add 0.2 milliliters of phenol chloroform isoamyl alcohol and 0.3 grams of acid-washed glass beads to the microcentrifuge tube. Mix by vortexing the tube for two minutes, followed by incubating on ice for one minute.

Centrifuge the tube at 10, 000 times G for 15 minutes at room temperature. Transfer the upper aqueous layer to a fresh microcentrifuge tube and add 200 microliters of phenol chloroform isoamyl alcohol. After centrifuging again at 10, 000 times G for 10 minutes, transfer the upper aqueous layer to a fresh tube.

Then, add two volumes of 100%ethanol and 1-by-10th volume of sodium acetate to the tube and incubate at minus 70 degrees Celsius for one hour. Precipitate the DNA by centrifugation at 10, 000 times G for 15 minutes at four degrees Celsius and remove the supernatant. Wash the DNA pellet with 70%ethanol and air dry at room temperature.

Resuspend the DNA in 20 microliters of sterile water and use three to five microliters of plasmid DNA to transform competent Escherichia coli cells. Using the standard protocol, transform the isolated yeast plasmids into Escherichia coli TOP10 strain and spread the cells on LB plates with required antibiotics. After isolating the plasmid from Escherichia coli transformants using the standard alkaline lysis protocol, follow the appropriate combinations of restriction enzymes to check for the release of the insert DNA fragment by restriction digestion.

Next, retransform the plasmids showing insert release after restriction digestion in the ell1 deletion strain to check for their ability to rescue the genotoxic stress-sensitive phenotype of the ell1 deletion strain. The ell1-deleted Schizosaccharomyces pombe strain showed sensitivity towards 4-NQO compared to the wild-type strain. A large number of colonies are obtained on the plate lacking 4-NQO.

However, fewer transformants were obtained on the 4-NQO plate as they act as punitive suppressors of the 4-NQO sensitivity of the ell1 null mutant. Of the 620 transformants, 74 showed growth on 0.4-micromolar 4-NQO. It was observed that only 16 out of 74 showed growth in the presence of 0.8-micromolar 4-NQO.

The ell1 deletion strain transformed with an empty vector and all the 16 transformants showed growth on the plate lacking 4-NQO. The ell1 deletion mutant containing only the empty vector showed no growth at 0.8-micromolar 4-NQO and six transformants exhibited growth at 0.8-micromolar 4-NQO, suggesting that the library clones present in them could suppress the genotoxic stress phenotype of the ell1 null mutant. Restriction digestion of suppressor plasmid 84 and 104 released an insert of one kilobase and 800 base pairs, respectively.

Reintroducing the suppressor plasmid clones into the ell1 null mutant resulted in the suppression of the 4-NQO-associated growth sensitivity. However, in the presence of MMS, the suppressor plasmids containing ell1 deletion mutant showed more growth than those with empty vector. These genetic screens can delineate potential resistance mechanisms and identify protein targets of novel antibacterial, antifungal, antiparasitic, or anticancer compounds.

They can also identify the mechanism of action of pharmaceutical drugs.