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| − | Btracting control values. The initial transport rate was calculated from the
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| − | Btracting control values. The initial transport rate was calculated from the radioactivity taken up by [https://britishrestaurantawards.org/members/neck40layer/activity/353350/ https://britishrestaurantawards.org/members/neck40layer/activity/353350/] proteoliposomes after 1.5 min (SLC25A33) or 3 min (SLC25A36) (in the initial linear range of substrate uptake). For efflux measurements, proteoliposomes containing 2 mM substrate were labeled with 5 M radioactive substrate by carriermediated exchange equilibration (18, 24). After 40 min, the external radioactivity was removed by passing the proteoliposomes through Sephadex G-75 columns pre-equilibrated with buffer A or buffer B for SLC25A33 or SLC25A36, respectively. Efflux was started by adding unlabeled external substrate or buffer alone (buffer A for SLC25A33 and buffer B forNOVEMBER 28, 2014 ?VOLUME 289 ?NUMBERSLC25A36) to aliquots of proteoliposomes and terminated by adding the inhibitors indicated above. Subcellular Localization--For the subcellular localization of SLC25A36 in Chinese hamster ovary (CHO) cells, these cells were co-transfected with 4 g of mtEBFP/pcDNAI (where EBFP stands for enhanced blue fluorescent protein) and 4 g of a modified pcDNA3 plasmid containing the coding sequence of SLC25A36 fused with the enhanced green fluorescent protein (EGFP) sequence at the C terminus (25). EGFP and EBFP fluorescence were detected as described (25). Yeast Strains, Media, and Growth Conditions--The strains used in this study are all in W303 genetic context (his3-11,15; ade2-1; leu2-3,112; ura3-1; trp1-1; can1-100). The wild-type W303 rho?haploid strain was produced as follows. Cells were grown at a density of 1 106 cells/ml on YPD medium for 24 h, then 0.05 M phosphate buffer, pH 6.5, and 50 g/ml ethidium bromide were added to 1 ml of culture. This culture was incubated at 28 for 24 h, then the cells were washed twice with H2O and plated on YPD medium. The lack of rho?cell growth was assessed on glycerol as the carbon source and the absence of mtDNA by 4 ,6-diamidino-2-phenylindole (DAPI; Sigma) staining. RIM2 diploid strain (RIM2/RIM2::kanMX) was generated using the PCR-mediated gene disruption technique (26) by replacing one of the two wild-type RIM2 copies with the kanMX cassette in wild-type W303 diploid strain (EUROSCARF). This strain was sporulated to obtain the wildtype and the RIM2 haploid strains. To obtain RIM2 straincontaining mtDNA, the RIM2 haploid strain was transformed with the RIM2-pRS42H, SLC25A33-pRS42H, or SLC25A36pRS42H plasmid and crossed with the wild-type haploid strain of the opposite mating type to obtain the diploid strains. Cell crossing was generated by bringing the cells near on plate with the needle of the Singer micromanipulator. The generation of zygotes was usually produced in 2 h. All the transformed RIM2 diploid strains were used for tetrad dissection to obtain transformed RIM2 haploid strains containing mtDNA (named RIM2 RIM2, RIM2 A33, RIM2 A36). Wild-type, wild-type rho? and deletion strains were grown at 28 in rich medium containing 1 Bacto-peptone and 1 yeast extract supplemented with 2 glucose (YPD) or 3 glycerol (YPG). 2.2 Bacto agar (Difco) was also present in solid media. In all cases the final pH was adjusted to 4.5. The presporulation medium contained 1 yeast extract, 1 Bacto-peptone, 0.17 yeast nitrogen base, 1 potassium acetate, 0.5 ammonium sulfate, and 0.05 M potassium phthalate, pH 5, and the sporulation medium contained 1 potassium acetate, pH 7.6.
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