View - ResearchGate

262 Tikhmyanova et al.5. pGLS22-Ras—plasmid encoding cI-Ras, a negative control for activation and positivecontrol for interaction. Selection markers are HIS5 and Cm R .6. pGLS22-EE 12345L—a plasmid encoding cI-EE 12345L fusion, a strong positive controlfor activation. Selection markers are HIS5 and Cm R .7. pEG202-Krit—HIS3 plasmid encoding LexA-Krit, a strong positive control foractivation. The vector pEG202 is almost identical to pMW103 (yeast selectionmarker is HIS3), but the bacterial selective marker is Ap R .8. pEG202-Krev1—a plasmid encoding LexA-Krev1, a negative control for activationand positive control for interaction. Markers are HIS3 and Ap R .9. pJG4-5 (Origene Technologies, Inc., Rockville, MD, as a part of DKT100DupLEX-A Yeast Two-Hybrid System):Raf—library plasmid encoding a positivecontrol for interaction with Ras. Selection markers are TRP1 and Ap R .10. pJG4-5:Krit1—library plasmid encoding a positive control for interaction withKrev1. Selection markers are TRP1 and Ap R .11. pYesTrp:RalGDS—TRP1 library plasmid encoding a positive control for interactionwith both Ras and Krev1. This plasmid is similar to pJG4-5, but has anextended polylinker and a V5-epitope tag instead of a hemaglutinin tag. Selectionmarkers are TRP1 and Ap R .2.2. Strains1. Yeast strain PRT50 (MATα URA3 TRP1 HIS3 2LexA op-LEU2 3cI op-LYS2).2. Yeast strain PRT475 (MATa URA3 TRP1 HIS3 2LexA op-LEU2 3cI op-LYS2).2.3. Lithium Acetate Transformation of Yeast1. 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.1 Mlithium acetate, pH 8.0, sterile filtered.2. 10 mM Tris-HCl, 1 mM EDTA, 0.1 M lithium acetate, and 40% PEG4000, pH 8.0,sterile filtered.3. Dimethylsulfoxide (DMSO).4. 6 mg/mL freshly denatured (i.e., boiled for 5 min and chilled on ice) shearedsalmon sperm DNA (sssDNA).2.4. Minipreps/Polymerase Chain Reaction From Yeast1. Acid-washed sterile glass beads, 0.15–0.45 mm diameter (e.g., Sigma G-1145).2. Tris EDTA solution TE:10 mM Tris-HCl and 1 mM EDTA, pH 8.0.3. 1:50 β-glucuronidase type HP-2 (crude solution from Helix pomatia [Sigma]), 50 mMTris-HCl, 10 mM EDTA, and 0.3% (v/v) 2-mercaptoethanol (prepare fresh), pH 7.5.2.5. XGal/XGluc Overlay Assays1. 1% Low-melting agarose in 100 mM KHPO 4, pH 7.0 agarose; add 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (XGal) or 5-bromo-4-chloro-3-indolylbeta-D-glucuronidesodium salt (XGluc) (Diagnostic Chemicals, Oxford, CT) to0.25 mg/mL when cooled to approx 60°C.2. Chloroform (CHCl 3).