Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Networks of genetic interactions provide functional information about genes and delineate pathways and biological processes that these genes may be involved in in vivo. In addition, they may also provide insights into how different genes interact with one another, resulting in a specific phenotype^1,2,3. Over the years, a variety of genetic screens have been designed by researchers to answer fundamental biological questions and study human diseases. Screens for the identification of genetic interactions can be performed in multiple ways. Genetic interactions identified in different genetic screens can represent distinct mechanistic relationships between genes. Furthermore, studies have revealed that a common set of genetic interactions are shared by genes that encode proteins belonging to the same pathway or complex^4,5. Thus, genetic interaction networks can be used to establish the functional organization in a cell, wherein genes sharing the most similar profiles belong to the same complex or pathway, those genes sharing somewhat less similar profiles belong to the same biological process, and those genes exhibiting overlapping but more diverse profiles reflect members belonging to the same cellular compartment⁶.

Genetic interaction screens based on dosage suppression ('high-copy or multicopy suppression') are one of the commonly used approaches. These screens can be performed by transforming a query mutant strain with a high-copy-number genomic or cDNA library, followed by suitable assays/selection techniques to identify suppressing or enhancing genetic interactions^7,8,9. To ensure a comprehensive genome-wide coverage, these screens have also been carried out by overexpressing a specific gene of interest in a collection of genome-wide loss-of-function mutant or by overexpressing a high-copy-number plasmid-encoded genomic or cDNA library in a loss-of-function query mutant^{9,10,11,12,13,14,15}. The multicopy strategy could also work using a dominant/overexpression approach using a regulatable promoter.

The main advantages of using suppressor-based screens are that suppression of a preexisting phenotype in a mutant strain by another gene establishes a genetic relationship among these two gene products that may not have been demonstrated using other approaches. Second, it has been observed that the presence of a preexisting mutation sensitizes a particular pathway, allowing additional components of that pathway to be identified by the isolation of suppressors, which may have not been identified by more direct genetic selections. Moreover, this screen can be used to identify suppressors that have different mechanisms of suppression¹⁶. Suppressor interactions usually occur between genes that are functionally related and can be used to elucidate hierarchies in pathways. The exact underlying mechanism of suppression may differ based on several factors, including the type of query mutant used in the screen, experimental conditions, and the level of gene expression. One of the common dosage suppression mechanisms involves genes encoding products that function together in the same complex or in parallel in the same cellular/biological process. The results of such screens in simpler model organisms such as yeast can be extended to higher eukaryotic organisms since most fundamental biological pathways and processes are conserved across evolution.

These genetic screens can also be modified in several ways to answer different biological questions. For example, orthologous genes from different organisms that can suppress the phenotype of the query mutant strain can be identified. It has also been used to delineate potential resistance mechanisms and determine protein targets of novel antibacterial^17,18, antifungal^19,20, antiparasitic²¹, and anticancer²² compounds. This screen has also been exploited to identify suppressors of the activity of pharmaceutical drugs whose mechanism of action is not known. Thus, in principle, these multicopy suppressor screens can be optimized and used in a variety of applications in different organisms. Although most of the genetic screens employed by yeast researchers have been initially developed in S. cerevisiae, S. pombe has emerged as a complementary model system for carrying out various genetic screens and assays²³. Moreover, genomic organization and biological processes in S. pombe, such as occurrence of introns in more genes, complexity of origins of DNA replication, centromere structure, organization of the cell cycle and presence of the RNAi machinery, show greater resemblance between S. pombe and higher eukaryotes^23,24, underscoring the importance of designing and using genetic tools in S. pombe.

This paper describes a protocol for identifying genetic interactors based on 'dosage suppression' of a loss-of-function mutant phenotype in S. pombe. The basis of this protocol is that it is a rapid and efficient method to screen a cDNA library overexpressing wild-type genes either on a multicopy plasmid and/or from a strong promoter. This protocol has four main steps: transformation of the library into a query mutant strain, selection of plasmid clones that suppress the desired phenotype of the query mutant strain, retrieving the plasmid(s) from these suppressor clones, and identification of the gene responsible for the suppression of the phenotype. As is true for any method based on the selection and identification of cDNAs from a library, the success of the screen is dependent on using a high-quality and high-complexity library as the screen can retrieve only those cDNA clones that are present in the library.

Using this protocol, we have successfully identified two novel suppressors of the genotoxic stress-sensitive phenotype of the query S. pombe ell1 null mutant. The ELL (Eleven Nineteen Lysine Rich Leukemia) family of transcription elongation factors suppress transient pausing of RNA polymerase II on DNA templates in in vitro biochemical assays and are conserved across various organisms, from fission yeast to humans²⁵. Earlier work has provided evidence that an S. pombe ell1 null mutant shows genotoxic stress sensitivity in the presence of 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS)²⁶. Therefore, we transformed a S. pombe plasmid-encoded multicopy cDNA library into the query S. pombe ell1 null mutant and identified two putative clones that exhibited the ability to suppress the genotoxic stress sensitivity of the S. pombe ell1 null mutant in the presence of 4-NQO, a compound that induces DNA lesions. Subsequent sequencing of the insert present in the plasmid clones identified that the genes encoding rax2⁺ and osh6⁺ were responsible for suppressing the genotoxic stress sensitivity of ell1 null mutant when overexpressed in the ell1 null mutant.

1. Transformation of the cDNA library into the query S. pombe mutant strain to screen for multicopy suppressors

NOTE: The Standard Lithium-Acetate method²⁷ was followed to transform the S. pombe cDNA library into the query S. pombe ell1Δ strain with a few modifications:

1. Grow the S. pombe ell1Δ strain at 32 °C on a YE medium (Table 1) plate supplemented with 225 µg/mL each of adenine, leucine, and uracil. Inoculate a loop full of inoculum of ell1Δ strain from the above plate in 15-20 mL of YE medium supplemented with 225 µg/mL each of adenine, leucine, and uracil. Incubate it overnight at 32 °C with shaking (200-250 rpm).
2. Next day, dilute the overnight culture in 100 mL of fresh YE medium with the desired supplements to an OD_{600 nm} (optical density at 600 nm) of 0.3 and incubate it at 32 °C with shaking at 200-250 rpm until the OD_{600 nm} reaches mid-log phase.
3. Divide the 100 mL culture into two 50 mL centrifuge tubes, followed by centrifugation at room temperature for 10 min at 4,000 × g.
4. Discard the supernatant and wash the cell pellet with 1 mL of sterile water, i.e., resuspend the cells in 1 mL of sterile water and centrifuge at 4,000 × g for 5 min at room temperature.
5. Discard the supernatant and wash each cell pellet with 1 mL of 1x LiAc-TE (100 mM lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) solution as described in step 1.4.
6. Remove the supernatant and resuspend each cell pellet in 250 µL of 1x LiAc-TE. Use these S. pombe competent cells (500 µL) for transformation with the DNA of interest. Transfer 125 µL of the competent cells into four different sterile microcentrifuge tubes for subsequent transformation steps.
7. Boil carrier DNA from Salmon testes or herring sperm for 1 min and immediately place on ice. For each transformation, add 10 µL of 10 mg/mL denatured, single-stranded carrier DNA to the microcentrifuge tube containing 125 µL of the competent cells, followed by gentle mixing using a micropipette tip. Subsequently, add 50 µg of the S. pombe cDNA library to the competent cells-carrier DNA mixture-containing microcentrifuge tube.
8. Incubate the microcentrifuge tubes at room temperature for 10 min, and then add 260 µL of polyethylene glycol-lithium acetate solution (40% [w/v] PEG 4,000, 100 mM lithium acetate, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to the tubes and mix gently with the help of a micropipette tip.
9. Incubate the microcentrifuge tubes containing the transformation mixture at 32 °C for 2 h without shaking. Add 43 µL of prewarmed dimethyl sulfoxide (DMSO) to the microcentrifuge tube and mix gently. Subsequently, expose the tubes to heat shock at 42 °C for 5 min.
10. Pellet the cells at 4,000 × g for 5 min at room temperature. Discard the supernatant and remove the residual PEG- LiAc-TE solution with the help of a micropipette tip.
11. Resuspend the cells in 100 µL of sterile water and plate on one EMM2 (Table 1) plate (150 mm) with required supplements and 0.2 µg/mL of 4-NQO.
12. Incubate the plates at 32 °C for 5-6 days to allow colonies to appear on the plates. Streak the obtained colonies on the same medium plate in the presence of 0.2 µg/mL of 4-NQO and use for further screening.
13. For one library transformation experiment, use 500 µL of competent cells (125 µL of competent cells x 4 microcentrifuge tubes) for transformation as described in steps 1.8 to 1.14 above.
14. As a control, plate 1/10^th of the transformation mixture on one EMM2 plate (150 mm) with required supplements, but lacking 4-NQO, to calculate the total number of library clones obtained after transformation of the library, and screen for isolation of the suppressor clones.

2. Test and validate the rescue/suppression of the phenotype associated with the query mutant strain by the putative suppressor

NOTE: Stress spot assays were carried out as described below to test and validate the rescue/suppression of the ell1 deletion-associated 4-NQO stress sensitivity by the putative suppressor(s).

1. Add 100-200 µL sterile water in each of the different wells of a sterile 96-well microtiter plate.
2. Pick a small amount of inoculum from each of the different colonies obtained after cDNA library transformation on the plate containing 4-NQO using a sterile toothpick or a 20-200 μL micropipette tip. Add the inoculum from each of the different colonies into separate independent wells of the microtiter plate containing 100-200 µL of sterile water. Mix thoroughly.
3. Spot 3 µL of the cell suspension from each well onto EMM2 agar plates containing adenine (225 µg/mL) and uracil (225 µg/mL) but lacking leucine (the selectable auxotrophic marker present on the plasmid vector used for construction of the library used in this work). Add appropriate concentrations of 4-NQO to the plates as required.
4. Incubate the plates at 32 °C for 3-4 days to allow the cells to grow. Identify the colonies that show growth in the presence of different concentrations of 4-NQO as putative suppressors.
5. To further validate the suppressors, grow the selected suppressors along with appropriate control strains overnight at 32 °C with shaking (200-250 rpm) in EMM2 medium containing 225 µg/mL each of adenine and uracil but lacking leucine.
6. Next day, dilute the cells to an OD_{600 nm} of 0.3 in fresh EMM2 medium and grow them at 32 °C with shaking until mid-log phase (approximately OD_{600 nm} of 0.6-0.8).
7. Spot appropriate serial dilutions (1:10 or 1:5) of cultures on EMM2 plates with required supplements containing either 0.4 µM 4-NQO or 0.01% MMS. For control, spot the strains on an EMM2 plate with required supplements but lacking any DNA-damaging agent.
8. Incubate the plates at 32 °C for 3-5 days to monitor growth.
   NOTE: Suitable assays based on the phenotype associated with the query mutant strain should be used to test the rescue or suppression of the phenotype. For example, if suppressors of a cold-sensitive phenotype of a query mutant strain need to be identified, the assay for testing and validation of the suppressor clones would involve growing transformants at low temperature.
9. Spot the mutant strains transformed with full-length gene of interest (Spell1 in this case) or empty vector as positive and negative controls, respectively, along with the library transformants.

3. Isolation of the plasmid from the S. pombe suppressor clones

NOTE: Plasmid isolation from S. pombe was carried out by following the protocol described in Fission yeast: a laboratory manual²⁸ with a few modifications.

1. Inoculate a single yeast colony in EMM2 medium containing 225 µg/mL of adenine and uracil and grow the cells overnight at 32 °C with shaking at 200-250 rpm. Next day, harvest 5 O.D. cells (i.e., 10 mL culture of O.D. 0.5) by centrifuging for 2 min at 4,000 × g at room temperature.
2. Remove the supernatant and resuspend the cell pellet in 0.2 mL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris HCl (pH 8.0), and 1 mM Na₂EDTA).
3. Add 0.2 mL of phenol:chloroform:isoamyl alcohol (25:24:1) and 0.3 g of acid-washed glass beads to the microcentrifuge tube. Vortex the microcentrifuge tube for 2 min at 4 °C and incubate on ice for 1 min. Repeat this step 6x.
4. Centrifuge the tube at 10,000 × g for 15 min at room temperature. Transfer the upper aqueous layer to a fresh microcentrifuge tube and add 200 µL of phenol:chloroform:isoamyl alcohol (25:24:1).
5. Centrifuge the tube at 10,000 × g for 10 min. Transfer the upper aqueous layer to a fresh tube and add two volumes of 100% ethanol (~400 µL) and 1/10 volume of sodium acetate (3 M, pH 5.8) (~20 µL) to the tube. Incubate the microcentrifuge tube -70 °C for 1 h.
6. Precipitate the DNA by centrifugation at 10,000 × g for 15 min at 4 °C and remove the supernatant. Wash the DNA pellet with 70% ethanol (~500 µL) and keep it at room temperature to air-dry.
7. Resuspend the DNA in 20 µL of sterile water. Use 2-5 µL of plasmid DNA for transformation of competent E. coli cells.
   NOTE: Plasmid can also be isolated from yeast cells using any of the commercially available yeast plasmid isolation kits.

4. Identification of the gene encoded by the suppressor clone

1. Transform the isolated yeast plasmids into E. coli Top10 strain [F-mcrA (mrr-hsdRMS-mcrBC) 80LacZM15 lacX74 recA1 ara139 (ara-leu)7697 galUgalKrpsL (StrR) endA1 nupG] using the standard protocol²⁹ and spread the cells on LB (Luria Broth) plates with required antibiotics.
2. Isolate the plasmid(s) from E. coli transformants using the standard alkaline lysis protocol²⁹, and follow the appropriate combinations of restriction enzymes to check for the release of the insert DNA fragment by restriction digestion.
   NOTE: Suppressor plasmid 84 was digested with BamHI restriction enzyme, and Suppressor plasmid 104 was digested with PstI/BamHI restriction enzymes.
3. Retransform the plasmids showing insert release after restriction digestion in the ell1Δ strain to check for their ability to rescue the genotoxic stress-sensitive phenotype of the ell1Δ strain.
4. Select the plasmid clones showing suppression of the genotoxic stress sensitivity and sequence the insert DNA fragment present in these plasmid clones using vector-specific forward and reverse primers.
   NOTE: In this study, the adh1 promoter-specific universal forward primer (5'CATTGGTCTTCCGCTCCG 3') was used³⁰.
5. To identify the gene responsible for the suppression, align the sequence obtained using NCBI nucleotide blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch) or Pombase sequence alignment tool (https://fungi.ensembl.org/Schizosaccharomyces_pombe/Tools/Blast?db=core). Select the sequence showing maximum alignment and identify it as the gene for the multicopy suppressor plasmid.

Screening for multicopy suppressor(s) of ell1 deletion-associated genotoxic stress sensitivity in S. pombe
We performed the genetic screen using the protocol described above to identify multicopy suppressors of the loss-of-function phenotype of the query ell1 deletion mutant strain. The growth-related sensitivity of the ell1 deletion strain observed in the presence of the 4-NQO genotoxic agent was adopted as the loss-of-function phenotype to select for multicopy suppressors. Figure 1 shows a schematic representation of the genetic screen. To begin the screen, the genotoxic stress sensitivity of the ell1 deletion mutant (h- ura4-D18 leu1-32 ade6-M210 can1-1 ell1Δ :: kanMX6) was first confirmed with respect to its isogenic wild-type strain (h- ura4-D18 leu1-32 ade6-M210 can1-1) in the presence of 4-NQO (Figure 2A). Subsequently, the query S. pombe ell1 null mutant was transformed with a high-copy-number S. pombe cDNA library to select for those plasmid clones that could suppress/rescue the 4-NQO growth-related sensitivity of the ell1 null mutant. This library contains ~6 × 105 S. pombe cDNA fragments cloned in the pLEV3 S. pombe overexpression vector under the control of the adh1 promoter30. As a control, 1/10th of the transformation mixture was also plated on EMM2 plates lacking 4-NQO to calculate the total number of recombinant clones obtained after transformation of the library, an indication of the coverage of the library, and screened for isolation of the suppressor clones. This is critical, as screening fewer clones will reduce the possibility of identifying the suppressor clones.

Figure 2B shows representative images of the control and 4-NQO-containing plates. As shown in the figure, a large number of colonies are obtained on the control plate lacking 4-NQO showing a good coverage of the library. As expected, fewer transformants were obtained on the 4-NQO plate as these transformants likely represent the putative suppressors of the 4-NQO sensitivity of the ell1 null mutant. Next, the different transformant colonies obtained on EMM2 plates containing 0.2 µM 4-NQO were further streaked or spotted on EMM2 plates containing 0.2 µM 4-NQO to confirm the ability of these transformants to grow in the presence of 4-NQO. It was observed that 620 transformant colonies were able to grow on 0.2 µM 4-NQO-containing EMM2 plates.

Validation of the ability of the putative suppressor clones to restore the growth of ell1 deletion mutant under genotoxic stress conditions
To confirm the putative suppressors, it is important to test the rescue of the desired phenotype under higher stringency conditions as shown in the schematic representation (Figure 3A). Therefore, the 620 shortlisted transformants were spotted on EMM2 plates with 0.4 µM 4-NQO. As can be seen in Figure 3B, only 74 transformants showed growth at 0.4 µM 4-NQO. Subsequently, these 74 transformants were spotted on EMM2 plates containing 0.8 µM 4-NQO, and it was observed that only 16 showed growth in the presence of 0.8 µM 4-NQO (Figure 3C). To further confirm these observations, these 16 transformants were spotted on EMM2 plates with required supplements, containing either 0.8 µM 4-NQO or no 4-NQO (control), along with the ell1 deletion mutant transformed with empty vector. Results of these spotting assays revealed that, as expected, the ell1 deletion strain transformed with empty vector, as well as all the 16 transformants, showed growth on the plate lacking 4-NQO. In comparison, while the ell1 deletion mutant containing only the empty vector showed no growth at 0.8 µM 4-NQO, six transformants exhibited growth at 0.8 µM 4-NQO (Figure 3D), suggesting that the library clone(s) present in them had the ability to suppress the genotoxic stress phenotype of the ell1 null mutant.

Identification of the gene encoded by the suppressor clone
We next selected 02 genetic suppressors of the ell1 deletion strain, sup 84 and sup 104, showing complete suppression of the ell1-associated growth sensitivity in the presence of 4-NQO. The library plasmid clone(s) was isolated from these two yeast suppressors and transformed into competent E. coli cells. Subsequently, plasmid was isolated from E. coli cells using standard protocols31. The two isolated plasmids, labeled as 84 and 104, were subjected to restriction digestion, which revealed the presence of approximately 1,000 bp and 800 bp insert DNA fragments, respectively (Figure 4A,B). To further validate the results of this suppressor screen, these plasmids were retransformed into the ell1 deletion mutant and checked if they could restore the growth of the ell1 null mutant in the presence of 4-NQO. The stress spot assays revealed that the suppressor plasmid clones resulted in the suppression of the 4-NQO-associated growth sensitivity when reintroduced into the ell1 null mutant, suggesting that the suppression was plasmid-dependent (Figure 4C). Furthermore, we decided to determine whether these suppressor clones could also suppress the growth-related sensitivity of the ell1 deletion strain in the presence of another DNA damage-inducing compound, MMS. The spot assays demonstrated that transformation of the suppressor plasmid clones into the ell1 null mutant resulted in more growth on MMS-containing medium than transformation of the ell1 deletion mutant with empty vector, indicating that these plasmid clones could suppress the genotoxic stress sensitivity of the ell1 deletion mutant in the presence of both genotoxic agents, i.e., 4-NQO and MMS (Figure 4D).

Next, to identify the gene present in these suppressor plasmid clones, sequencing of the insert DNA fragment was carried out using the adh1 promoter-specific universal forward primer30. The nucleotide sequence obtained was searched by using 'BLAST at Ensembl' tool available on the PomBase website (https://www.pombase.org), which revealed that the two plasmid clones 84 and 104 contained rax2+ and osh6+ genes, respectively, providing evidence that Rax2 and Osh6 were responsible for suppressing the genotoxic stress-sensitive phenotype associated with absence of ell1+ in S. pombe. Knowledge about the functions of both these proteins in S. pombe is limited. Rax2 has been shown to regulate the localization of For3p to control the cell polarity during vegetative growth in S. pombe32. Osh6 is a lipid transport protein that belongs to the oxysterol-binding protein-related protein family. It is involved in the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane33.

Figure 1: Schematic representation of the genetic multicopy suppressor screen. This protocol has four main steps: transformation of the library into a query mutant strain, selection of plasmid clones that suppress the desired phenotype of the query mutant strain, retrieving the plasmid(s) from these suppressor clones, and identification of the gene responsible for the suppression of the phenotype. Abbreviation: 4-NQO = 4-nitroquinoline 1-oxide. Please click here to view a larger version of this figure.

Figure 2: Screening for multicopy suppressor(s) of ell1 deletion-associated genotoxic stress sensitivity in S. pombe. (A) Stress spot assay showing 4-NQO sensitivity of ell1-deleted S. pombe strain as compared to its isogenic wild-type strain. Serial dilutions (1:10) of appropriate strains were spotted on EMM2 medium containing 225 µg/mL each of adenine, leucine, and uracil with or without (control) 0.4 µM 4-NQO. Plates were then incubated for 3-5 days at 32 °C. (B) A high-copy-number cDNA library was transformed in ell1-deleted S. pombe strain, and transformed colonies were plated on EMM2 medium lacking leucine but containing 0.2 µM 4-NQO. A small aliquot of the transformation mixture was also plated on EMM2 plates lacking 4-NQO, as a control. Plates were incubated at 32 °C for 5-6 days and photographed. Panel B shows a representative image of these plates. Abbreviation: 4-NQO = 4-nitroquinoline 1-oxide. Please click here to view a larger version of this figure.

Figure 3: Validation of the ability of putative suppressor clones to suppress the genotoxic stress sensitivity of the ell1 deletion mutant. (A) A schematic representation of the screening of the library transformants by testing suppression of the desired phenotype. Growth of 620 transformants was tested on increasing concentrations of 4-NQO by spotting different transformants on EMM2 plates lacking leucine and containing either (B) 0.4 µM 4-NQO or (C) 0.8 µM 4-NQO. Plates were incubated at 32 °C for 5-6 days and photographed. Representative images of plates have been shown. (D) Sixteen transformants that were showing growth on 0.8 µM 4-NQO were spotted on EMM2 plates lacking leucine but containing either 0.8 µM 4-NQO or not containing 4-NQO (control), along with the ell1 deletion strain transformed with empty vector. Plates were incubated at 32 °C for 5-6 days and photographed. Only six colonies exhibited the ability to rescue ell1 deletion associated-genotoxic stress sensitivity. Abbreviation: 4-NQO = 4-nitroquinoline 1-oxide. Please click here to view a larger version of this figure.

Figure 4: Identification of the suppressor plasmid clones. Agarose gel images of (A) restriction digestion of suppressor plasmid 84 with BamHI restriction enzyme released an insert of ~1 kb. (B) Suppressor plasmid 104 digested with PstI/BamHI restriction enzymes released an insert of ~800 bp. (C) 1:10 serially diluted ell1 null mutant transformed with plasmids, sup 84 or sup 104, were spotted on EMM2 plates lacking leucine and containing either 0.4 µM 4-NQO or (D) 0.01% MMS. In control plates, no DNA-damaging agent was added. Plates were incubated at 32 °C for 4-5 days and photographed. Please click here to view a larger version of this figure.

Table 1: Composition of media for growth of S. pombe. Please click here to download this Table.

Yeasts have been widely used to investigate the basic biological processes and pathways that are evolutionarily conserved across eukaryotic organisms. The availability of genetic and genomic tools along with their amenability to various biochemical, genetic, and molecular procedures make yeasts an excellent model organism for genetic research^34,35,36. Over the years, various genetic screens have been designed by yeast researchers to identify genetic interactions that would shed light on the functions of individual gene(s), as well as the pathways and biological processes in which they may be involved^10,11,17,37. One of the common and useful screens to identify genetic interactions is known as 'multicopy or high copy number suppression'.

This screen requires a query mutant strain(s) exhibiting a loss-of-function phenotype, followed by transformation of this mutant query strain(s) with a high-copy-number genomic or cDNA library. Subsequently, the transformants obtained are screened to identify those genes, which when overexpressed, suppress the phenotype associated with the query mutant strain(s). This type of genetic interaction between the specific mutant strain and gene(s) present in the plasmid library clones result in the isolation and identification of gene products that are likely to act in the same pathway or affect the same biological process⁴. Thus, this screen provides insights into the functions of the gene(s) of interest and their functional organization into pathways and biological processes in vivo. Traditionally, this screen has been extensively used in S. cerevisiae^{38,39,40,41,42}.

The goal of the present work is to describe a simple and straightforward protocol for carrying out a multicopy suppression genetic screen in S. pombe. We carried out the screen using an S. pombe strain carrying a deletion of the ell1 transcription elongation factor. ELL is a family of transcription elongation factors, which are present in a wide range of organisms, from fission yeast to humans. The functions of Ell1 are only beginning to be understood in S. pombe. Earlier work had highlighted the role of Ell1 in optimal growth during normal growth conditions and in survival during genotoxic stress conditions²⁶. We have employed the multicopy suppressor screening as one of the approaches to uncover the molecular mechanism underlying the role of Ell1 in genotoxic stress sensitivity in S. pombe²⁶. A multicopy plasmid-encoded S. pombe cDNA library was transformed into the S. pombe ell1 null mutant that exhibits sensitivity to 4-NQO. Subsequently, those transformants that could grow in the presence of 4-NQO were selected.

Isolation of plasmids from these transformants and sequencing of inserts present in these plasmids led to the identification of two novel genetic interactors of ell1⁺ in S. pombe, rax2⁺ and osh6⁺. A previous study using a similar genetic screen in S. pombe has also identified the anti-silencing protein, epe1⁺, as a multicopy suppressor of the ell1 deletion-associated phenotypes⁴³. We have also used this method in identifying the multicopy suppressors of the non-essential Rpb9 subunit of S. pombe RNA polymerase II (unpublished observations). Collectively, these results validate the utility of the genetic multicopy suppressor screening approach as a method to identify putative genes/proteins that exhibit genetic interaction with a desired gene of interest, elucidating the function, pathways, and processes in which the gene of interest may be involved.

To use the screen described in this work, it is important to first have a query mutant strain that exhibits a clear phenotype associated with the mutation, which can be used to isolate the multicopy suppressor(s). Moreover, the success of this screen will depend upon the availability of a high-quality and good representative library containing clones representing the maximum number of wild-type genes or their corresponding cDNAs. Traditionally, both genomic and cDNA libraries have been used in these screens, but cDNA libraries have the advantage of being made from mRNAs expressed in the cell/organism. Furthermore, a high efficiency of its transformation into the yeast cells is imperative for the success of the screen because it is important to screen a large number of transformants, increasing the probability of identifying suppressors. If fewer transformants are obtained on the control plate after transformation of the library into the query mutant strain using the lithium acetate protocol, then electroporation can be used to transform the library.

It is also advisable to first standardize the transformation protocol with another plasmid before transforming the library. Otherwise, it will unnecessarily result in loss of the library. The protocol described here uses 4-NQO sensitivity as the mutant phenotype, but suitable screening methods/assays should be designed for selection of suppressors based on the specific mutant phenotype of interest. If no or very few transformants are obtained on the plate to select for suppressors after transformation of the library into the query mutant strain, then if possible, less stringent conditions/assays should be used when plating the library to select for suppressors so as to detect as many genetic suppressors as possible before confirming those under high-stringency conditions. In addition to using restriction digestion to test for the presence of the insert in the identified suppressor plasmid clones, PCR using vector-specific primers can be used to amplify the desired insert DNA fragment. Alternatively, the suppressor plasmid clones can be directly given for sequencing, avoiding the need for restriction digestion or PCR.

This screen may also be adopted for yeasts other than S. cerevisiae and S. pombe if a genetic tool kit with respect to appropriate mutant strains, plasmids, and promoter constructs is available that would facilitate overexpression of genes, allowing for the rescue of mutant phenotype present in the host yeast cell. Furthermore, with the availability of genome-wide resources, including collection of genome-wide deletion mutants⁴⁴ and overexpression libraries³⁷, this screen can also be performed by transformation of one or a few query plasmids into a systematic array of yeast mutant strain collection or by transformation of a systematic array of overexpression plasmid collection into one or a few mutant query strains⁴⁵. In conclusion, this approach can be clearly generalized to a wide variety of experimental questions in different organisms.