by4742 saccharomyces cerevisiae cells  (Zymo Research)

Journal: BMC Microbiology

Article Title: Characterization and ligand identification of a membrane progesterone receptor in fungi: existence of a novel PAQR in Sporothrix schenckii

doi: 10.1186/1471-2180-12-194

Figure Lengend Snippet: SsPAQR1 yeast-based assay. The agonist of SsPAQR1 was identified using a yeast-based assay as described in Methods. S. cerevisiae BY4742 was transformed with YEp353 ( FET3 - lacZ ) containing a fragment of the FET3 promoter fused to lacZ driven by a minimal CYC1 promoter and with pYES2CT w/wo the sspaqr1 gene insert. S. cerevisiae were grown in LIM-Fe medium containing 2% galactose and FET3 activity is measured using the FET3-lacZ construct as a reporter. Black bars show FET3-lacZ activity in yeast treated with the solvent only (H 2 O or ethanol) and gray bars show activity in yeast treated with different possible agonist; thaumatin, adiponectin or progesterone. FET3-lacZ activity was measured as the β-galactosidase activity expressed as the percentage of the untreated vector control. Panel ( A ) shows that SsPAQR1 does not repress FET3-lacZ when over-expressed in yeast by using the GAL1 promoter. Panel ( B ) shows β-galactosidase activity in cells expressing SsPAQR1 in the presence of 1 mM progesterone, panel ( C ) shows β-galactosidase activity in cells expressing SsPAQR1 in the presence of 50 μM thaumatin and panel ( D ) shows β-galactosidase activity in cells expressing SsPAQR1 in the presence of 0.1 μM adiponectin.

Article Snippet: S. cerevisiae strain BY4742 cells (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) co-transformed with plasmids, YEp353 ( FET3-lacZ ) and pYES2CT (1μg each) with the S.c. EasyComp ™ Transformation Kit (Invitrogen Corp. Carlsbad, CA, USA) was used for the ligand-binding assay.

Techniques: Transformation Assay, Activity Assay, Construct, Plasmid Preparation, Expressing