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1.
FIGURE 3.

FIGURE 3. From: New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast.

Aequorin luminescence of S. cerevisiae mutants after high pH shock. The cch1, mid1, and ecm7 knock-out mutants and the wild-type W303-1A parent strain were transformed with aequorin expression plasmids and monitored for luminescence before and after dilution with pH 9 medium (at the 30-s time point). Relative luminescence units (RLU) were plotted after normalization for cell density.

D. Christian Martin, et al. J Biol Chem. 2011 March 25;286(12):10744-10754.
2.
FIGURE 6.

FIGURE 6. From: New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast.

Stimulus-dependent death of HACS-deficient cells. The indicated mutants of S. cerevisiae (A) and C. glabrata (B) were grown in synthetic complete medium and exposed to α-factor (+αF), tunicamycin (+Tm), or miconazole (+Mic) as indicated for ∼5 h. Live and dead cells were counted by flow cytometry after staining with propidium iodide. Bars represent the average of three independent cultures (±S.D.).

D. Christian Martin, et al. J Biol Chem. 2011 March 25;286(12):10744-10754.
3.
FIGURE 4.

FIGURE 4. From: New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast.

Expression and stability of Ecm7, Mid1, and Cch1 proteins in S. cerevisiae. The Ecm7, Mid1, and Cch1 proteins were tagged at their C termini in the indicated strains derived from W303-1A by chromosomal integration of the MYC epitope tag. Cells were grown to log phase with or without a 1-h exposure to cycloheximide (CHX), and then total cell lysates were fractionated by SDS-PAGE and analyzed by Western blotting. The top, middle, and bottom panels depict the Ecm7-MYC, Mid1-MYC, and Cch1-MYC proteins, respectively.

D. Christian Martin, et al. J Biol Chem. 2011 March 25;286(12):10744-10754.
4.
FIGURE 2.

FIGURE 2. From: New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast.

Quantitative Ca2+ uptake experiments on S. cerevisiae and C. glabrata mutants lacking Cch1, Mid1, or Ecm7. Log-phase cultures of BY4741 S. cerevisiae (A) and BG2 C. glabrata (B) were exposed to α-factor, tunicamycin (Tm), or miconazole (Mic) in the presence and absence of FK506 (FK) while growing in YPD medium containing tracer quantities of 45CaCl2. Bars indicate the average of three independent determinations of Ca2+ uptake per billion cells (±S.D.). Similar results were obtained for independently generate ecm7 mutants of S. cerevisiae W303-1A (data not shown).

D. Christian Martin, et al. J Biol Chem. 2011 March 25;286(12):10744-10754.
5.
FIGURE 5.

FIGURE 5. From: New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast.

Mating and fusion efficiencies of HACS-deficient mutants. Top, the indicated single and double mutants of strains BY4741 and BY4742 were allowed to mate for 4 h in low-calcium medium and then replica-plated to diploid selection medium. After 1 day of re-growth, relative mating efficiencies were visible. Similar results were obtained using high-calcium medium (not shown). Bottom, homozygous matings of the indicated strains were performed for a brief period of time and then stained with FM4–64. The zygotes were then counted as abnormal if cell wall or cell membrane material persisted within their fusion zones and are plotted as a percentage of the total number of zygotes. Bars indicate the averages of three-seven independent experiments (±S.D.).

D. Christian Martin, et al. J Biol Chem. 2011 March 25;286(12):10744-10754.
6.
FIGURE 1.

FIGURE 1. From: New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast.

Genome-wide screen for mutants with altered Ca2+ uptake. Individual mutants from the S. cerevisiae gene knock-out collection were exposed to tunicamycin (+Tm, blue symbols), α-factor (+αF, red symbols), or no stimulus (+0, black symbols) in the presence or absence FK506 (FK) while growing in YPD medium containing tracer quantities of 45CaCl2. Total cell associated radioactivity was measured, and the normalized data were plotted (A) as described under “Experimental Procedures.” Yellow diamonds indicate the cch1 mutant, and yellow squares indicate the mid1 mutant. After excluding mutants that failed statistical tests, the outliers were grouped by hierarchical clustering. Subclusters that contain known LACS- and HACS-deficient mutants were plotted (B). Black, white, and yellow arrows indicate mutants that lack transmembrane subunits, regulators, and putative new subunits of LACS and HACS, respectively.

D. Christian Martin, et al. J Biol Chem. 2011 March 25;286(12):10744-10754.

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