Drop out

For cultivation of E. coli cells terrific broth (TB) media from Carl Roth (Karlsruhe, DE) was used. For cultivation of S. cerevisiae cells d-Galactose, Peptone and Synthetic Complete Mixture (Kaiser) Drop-Out (-URA) were purchased from Formedium (Hunstanton, GB). Yeast nitrogen base (without amino acids) and Yeast extract were purchased from Carl Roth (Karlsruhe, DE). For P. pastoris cultivation methanol (99.9% Chromasolv purity grade) purchased from Honeywell Chemicals (Seelze, DE) was used as additional carbon source. PNGaseF and BsaI were purchased from New England Biolabs (Ipswich, US). BbsI and FastDigest AscI were purchased from ThermoFisherScientific (Waltham, US) and T4 DNA Ligase from Promega (Madison, US).

Using the Gateway™ recombination technology (Life Technologies) a human RPL7 cDNA was cloned into pActII in fusion with the Gal4p Activation Domain (Gal4AD), Gag and NCp7 cDNA were cloned into pGBKT7 in fusion to the Gal4p DNA binding domain (Gal4BD). All pActII and pGBKT7 constructs were introduced into the S. Cerevisiae strain AH109 (MAT a, trp 1-901, leu2-3, 112, ura3-52, his3-200, Δgal4, Δgal80, LYS2: GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3: MEL1UAS-MEL1TATA-lacZ) or Y187 (MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ, met–, gal80Δ, URA3::GAL1_UAS-GAL1_TATA-lacZ) respectively using a LiCl procedure [78 (link)]. The transformed cells were selected for Leu or Trp auxotrophy on minimal media plates (6.8 g L⁻¹ YNB w/o amino acids (Sigma ref Y0626), 0.6 g L⁻¹ of Drop OUT (ForMedium LTD, Hunstanton, England), 2 % glucose, 20 g L⁻¹ Bacto-agar (Difco ref 214010). To carry out the two-hybrid test, yeast cells of each mating type transformed with the studied constructs were mated overnight and the diploids selected on a minimal medium depleted for Leu and Trp. The interaction between the two proteins tested was assayed by a 5-days growth of diploid yeasts on a minimal medium depleted for Leu, Trp and His.