Strains and cultures
Yeast strains are shown on table 1. Strains BR450 and NI150 are
spontaneous ura3 - mutants of the respective type strains obtained
by plating strains on 5-FOA medium (Synthetic Complete Medium lacking
uracil, see below, to which is added a small quantity of uracil; 50mg/L,
and 5-fluoro-orotic acid, 1g/L). These alleles of ura3 were
sequenced by Eurofin GenomicsTM France. In C.
bracarensis , the ura3 gene, CABR0s13e0133 has a deletion
encompassing nt 240-657 with respect to the first nucleotide (nt) of the
start codon, and in C. nivariensis , the ura3 gene,CANI0s15e01067 has a deletion encompassing nt 488-610 with
respect to the first nt of the start codon. (Gene names from
http://www-archbac.u-psud.fr/genomes/nakaseomycetes/nakaseomycetes.html).
Yeast strains are grown in broth or on plates at 30°C, in YPD
(non-selective, 1 % Yeast Extract, 1 % Peptone, 2 % glucose), in
Synthetic Complete medium lacking uracil, methionine, and cysteine
(induction conditions for the MET3 promoter, SC-Ind, 1.67 %
Yeast Nitrogen Base without amino acids, 0.5 % ammonium sulfate, 2 %
glucose, supplemented with adenine and all amino acids except methionine
and cysteine), and SC-Ind supplemented with 2 mM each of methionine and
cysteine (repression conditions for the MET3 promoter, SC-Rep).
For growth of cells without the plasmid as controls in the induction
experiments, 2 mM uracil is added to the SC-Ind and to the SC-Rep
medium. When repression, but no selection is needed, cells are grown in
YPD supplemented with 2 mM each of methionine and cysteine (YPD-Rep).
For Synthetic Complete media, pH is brought to 5.8 by addition of drops
of 5M NaOH until correct pH is reached.