2.2. The Yeast Two-Hybrid System (Y2H)

A yeast Activation Domain (AD) two-hybrid (Y2H) library (“prey”) was screened in a haploid strain by mating with the opposite mating strain expressing the DNA binding domain (BD)-bait fusion protein (“bait”). Interaction of the BD-bait and AD-prey proteins was assessed based on the expression of the reporter genes in conditional autotrophs; this approach allowed capture of the AD plasmid(s) expressing the interacting proteins. The AD library and BD-bait were transformed into the yeast strain, Y187, and Y2HGold, respectively. The Y2HGold strain with full-length MsrA as bait was screened against an adult human brain cDNA library (Clontech, Mountain View, CA, USA) by Next Interactions Inc. (Richmond, CA, USA). The Y2H procedures were performed similarly to Clontech’s protocol of the Matchmaker Gold Yeast Two-Hybrid System. Constructs were made using Clontech’s pGBKT7 and pGADT7 plasmids that are components of the Matchmaker Gold Yeast Two-Hybrid System. The MsrA clone was purchased from DNASU Plasmid Repository (The Biodesign Institute, Tempe, AZ, USA). Constructs encoding the MsrA protein in pGBKT7 (“bait”, Trp1 selection marker of Y2H Gold yeast strain) and pGADT7 (“prey”, Leu2 election marker, Y187 yeast strain) were mated using the following procedure. An YPD-agar plate was used to streak the S. cerevisiae strains and then the strains were incubated in YPD medium overnight at 30 °C with rotation at 225 rpm. The S. cerevisiae/YPD culture was diluted 1:10 with fresh YPD medium and incubated for an additional 2 h at 30 °C with rotation (cell density: between 10⁷ and 3.0 × 10⁷ cells per mL). The cells were centrifuged at 580× g and the pelleted cells were resuspended in YPD medium and streaked onto agar plates, containing the standard selective media (SD) lacking tryptophan (Trp) and leucine (Leu). The plates were incubated for five days at 30 °C. Then, colonies from each plate were resuspended in H₂O and spotted onto agar plates lacking Trp and Leu or agar plates containing selective media lacking Trp, Leu, and histidine (His). Selection for positive clones was done for HIS3 reporter activation [low stringency, SD-Leu,-Trp, and -His (LTH)] and for HIS3 and ADE2 reporter activation [high stringency, SD-Leu, -Trp, -His, and -Adenine (LTHA)]. The initial selection was performed for 5 days. The equivalent of 1.07 × 10⁶ diploid cells was selected on SD-LTH and the equivalent of 7.4 × 10⁶ on SD-LTHA. Isolated colonies were picked and re-streaked for 2- to 3-day growth on high stringency medium (SD-LTHA) to ensure robust growth. Fifty-three colonies were finally obtained and prey cDNAs were amplified with colony PCR using a set of flanking primers. The resulting PCR products were sequenced in the forward direction as follows. Colonies from SD-LTH and SD-LTHA agarose plates were picked into a grid to grow larger quantities of cells. After regrowth, each colony was transferred into a 20 mM NaOH suspension and then boiled for 20 min. After cooling, lysates were clarified by centrifugation at >3000× g for 5 min. The supernatants were amplified by PCR using Mango Polymerase buffer, nucleotides mix, primers [NIXO-1838 (5′-gatGAAGATACCCCACCAAACC-3′) and NIXO-1839 (5′-acgatgcacagttgaAGTGAA-3′)], Mango Tag DNA polymerase (Bioline, London, UK), and HiFi Tag DNA polymerase (Clontech, Mountain View, CA, USA). Aliquots from each amplified sample were analyzed for DNA bands by electrophoresis using a 1% (w/v) TAE agarose gel and sequenced. DNA sequences were compared to the human proteome database (UniProtKB). Hits that did not map to the coding sequence of known protein sequences or were of bad quality were not analyzed further.