The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe

Figure 4

Ectopic mam2 expression in mam2Δ strain. (a) The schematic strategy of ectopic mam2 expression in endogenous mam2Δ strain. Left, mam2 gene (gray arrow) was driven on reporter plasmid. To complement ura4 gene, empty pSU1Z vector was introduced into ura4 locus on chromosome. Right, mam2 gene on pSU1Z vector was introduced into the ura4 locus. The gray pentagon depicted the promoter, which was nmt1 promoter, urg1 promoter or hCMV promoter. The two plasmids (reporter and receptor) were transformed at the same time. (b) The mam2 gene was expressed under the control of nmt1 promoter, urg1 promoter or hCMV promoter on chromosome or the reporter plasmid. OSP210-17 was used as a positive control. OSP230-17 was used as a negative control and did not express mam2 gene under the control of any promoter. The cells were exposed to 1 μM of P-factor and were incubated for 0 h (open box), 3 h (shaded box), 6 h (gray box), or 12 h (filled box) after the addition of P-factor. The strains including the nmt1 promoter were assayed with 0 (on) or 15 (off) μM of thiamine. The urg1 promoter was constitutively activated under the nitrogen starvation. The values were the means of triplicate determinations from a typical experiment. The error bars represent ± standard error. (c) Upper, the preculture medium of the strain OSP230-17 h2 expressing mam2 gene under the control of chromosomal hCMV promoter with pAL7-Usxa2-GFP-DSPBC4.01. Lower, the preculture medium of the strain OSP230-2 h2 expressing mam2 gene under the control of chromosomal hCMV promoter with pAL7-Umam2-GFP-LPI.