Ipid flippases.Supplies AND Techniques Genetic solutions and Hexaflumuron In Vivo growth assay Chemical compounds have been purchased from Wako Pure Chemical substances Industries (Osaka, Japan) unless otherwise described. Duramycin was bought from Sigma-Aldrich (St. Louis, MO). Yeast strains were cultured in wealthy YPDA [1 yeast Hygrolidin ADC Cytotoxin extract (Difco Laboratories, Detroit, MI), 2 bactopeptone (Difco), two glucose, and 0.01 adenine] or YPGA (1 yeast extract, two bacto-peptone, 3 galactose, 0.2 sucrose, and 0.01182 |T. Yamamoto et al.adenine) medium. When a tryptophan requirement was examined, YPDA was in addition supplemented with 200 mgml tryptophan (YPDAW). Standard genetic manipulations and plasmid transformation of yeast had been performed as described previously (Elble 1992; Guthrie and Fink 2002). Synthetic glucose (SD) medium containing the required nutrient (Guthrie and Fink 2002) was utilised for a genetic screen and fluorescent microscopy. To assay growth of PGAL1-3HA-CDC50 strains carrying TRP1-harboring or URA3-harboring plasmids, yeast transformants had been chosen on synthetic SGA-Trp [0.67 yeast nitrogen base wo amino acids (Difco), 0.five casamino acids (Difco), 3 galactose, 0.2 sucrose, 0.03 uracil, and 0.01 adenine] or SGA-Ura (0.67 yeast nitrogen base wo amino acids, 0.5 casamino acids, three galactose, 0.two sucrose, 0.03 tryptophan, and 0.01 adenine) medium, respectively, and then examined for growth on SDA-Trp (0.67 yeast nitrogen base wo amino acids, 0.five casamino acids, two glucose, 0.03 uracil, and 0.01 adenine) or SDA-Ura (0.67 yeast nitrogen base wo amino acids, 0.5 casamino acids, 2 glucose, 0.03 tryptophan, and 0.01 adenine) medium, respectively. For serial dilution spot assay, cells have been grown to early log phase in acceptable medium, washed with YP (1 yeast extract and two bactopeptone), and adjusted to a concentration of 0.1 OD600ml. From fivefold dilutions, 4 ml drops were spotted onto appropriate plates, followed by incubation under the indicated conditions. Yeast strains and plasmids Yeast strains used in this study are listed in Supplemental Material, Table S1. Regular molecular biological tactics (Sambrook and Russell 2001) were used for the construction of plasmids, PCR amplification, and DNA sequencing. Gene deletions of CFS1, KES1, FUN26, and PLB3 in the YEF473 (Bi and Pringle 1996) genetic background were performed as follows. The regions containing the KanMX4 disruption marker and the flanking sequences had been PCR-amplified using genomic DNA derived in the knockout strain inside the BY4741 (Brachmann et al. 1998) strain background (a gift from Charles Boone, University of Toronto) as a template. The amplified DNA fragments were introduced in to the suitable strains, and G418-resistant transformants were selected. Yeast strains carrying C-terminally green fluorescent protein (GFP)-tagged ENA1, C-terminally enhanced GFP (EGFP)-tagged CFS1, and C-terminally monomeric red fluorescent protein 1 (mRFP1)-tagged genes (DRS2, NEO1, and SEC7) had been constructed by the PCR-based procedure as previously described (Longtine et al. 1998). All strains constructed by the PCR-based procedure were verified by colony PCR amplification to confirm that replacement or insertion had occurred at the anticipated loci. The sec14-3 mutant inside the YEF473 genetic background was constructed by backcrossing the original mutant (a present from Randy Schekman) to our wild-type strain (YKT1066) 3 instances. The GFP-tagged Lact-C2 plasmid (pRS416-PGPD-GFP-Lact-C2) (Yeung et al. 2008) w.
