The progesterone receptor that we have identified in S. schenckii, brings to a close the search for a membrane progesterone receptor in fungi. Methods Strains and culture conditions S. schenckii (ATCC 58251) was used for all experiments. The yeast form of this fungus was obtained as described previously
[53]. S. cerevisiae strains AH109 and Y187 were used for the yeast two-hybrid screening and were supplied with the MATCHMAKER Two-Hybrid System (Clontech Laboratories Inc., Palo Alto, CA). S. cerevisiae learn more strain BY4742 for the yeast-based ligand-binding assay was obtained from Dr. Thomas J. Lyons, from the Foundation for Applied Molecular Evolution (Gainesville, FL). Nucleic acids isolation
DNA and RNA were obtained from S. schenckii yeast cells as described previously [54]. Poly A+ RNA was obtained from total RNA using the mRNA Purification Kit from Amersham Biosciences (Piscataway, NJ, USA) and used as template for cDNA Selleck GW786034 synthesis. Yeast two-hybrid MATCHMAKER Two-Hybrid System was used for the yeast two-hybrid assay (Clontech Laboratories Inc., Palo Alto, CA) using all 3 different reporter genes for the confirmation for truly interacting proteins as described previously [55]. For the construction of the bait plasmid, ssg-2 cDNA was obtained from poly A+ RNA, transcribed and amplified by RT-PCR using the Ready-to-Go™ Beads (Amersham Biosciences)
as described [55], cloned and used to transform competent S. cerevisiae yeast cells (Y187). Lazertinib mw Competent S. cerevisiae yeast cells were Arachidonate 15-lipoxygenase transformed using the YEASTMAKER™ Yeast Transformation System 2 from Clontech (BD Biosciences, Clontech Laboratories Inc.). Poly A+ RNA was isolated form total RNA extracted from logarithmically growing S. schenckii yeast cells. Double stranded cDNA was synthesized from RNA using SMART™ Technology Kit (Clontech Laboratories Inc.). The cDNAs were amplified using Long Distance PCR and size selected using the BD CHROMA-SPIN™+TE-400 columns (Clontech Laboratories Inc.) [55]. S. cerevisiae yeast cells AH109 transformed with SMART ds cDNA (20μl) were selected in SD/-Leu plates, harvested and used for mating with the bait containing S. cerevisiae strain Y187. Mating of S. cerevisiae yeast cells strains Y187 (Mat-α) and AH109 (Mat-a) was done according to the manufacturer’s instructions. The expression of three reporter ADE2, HIS3 and MEL1 genes in the diploids was used as confirmation for true interacting proteins. Diploids expressing interacting proteins were selected as described previously [55]. Colony PCR was used to corroborate the presence of both plasmids in the diploid cells using the T7/3′BD sequencing primer pair for the pGBKT7/ssg-2 plasmid and the T7/3′AD primer pair for the pGADT7-Rec library plasmid.
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