The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe
G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR path...
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| Format: | Article |
| Language: | English |
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Wiley
2012-01-01
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| Series: | Scientifica |
| Online Access: | http://dx.doi.org/10.6064/2012/674256 |
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| author | Shintaro Sasuga Toshiya Osada |
| author_facet | Shintaro Sasuga Toshiya Osada |
| author_sort | Shintaro Sasuga |
| collection | DOAJ |
| description | G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, the mam2 upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter. |
| format | Article |
| id | doaj-art-3eaf5dd69f18489c80b8cd00ce93907f |
| institution | Kabale University |
| issn | 2090-908X |
| language | English |
| publishDate | 2012-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Scientifica |
| spelling | doaj-art-3eaf5dd69f18489c80b8cd00ce93907f2025-02-03T01:26:09ZengWileyScientifica2090-908X2012-01-01201210.6064/2012/674256674256The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombeShintaro Sasuga0Toshiya Osada1Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-2 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, JapanDepartment of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-2 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, JapanG protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, the mam2 upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.http://dx.doi.org/10.6064/2012/674256 |
| spellingShingle | Shintaro Sasuga Toshiya Osada The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe Scientifica |
| title | The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe |
| title_full | The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe |
| title_fullStr | The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe |
| title_full_unstemmed | The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe |
| title_short | The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe |
| title_sort | reporter system for gpcr assay with the fission yeast schizosaccharomyces pombe |
| url | http://dx.doi.org/10.6064/2012/674256 |
| work_keys_str_mv | AT shintarosasuga thereportersystemforgpcrassaywiththefissionyeastschizosaccharomycespombe AT toshiyaosada thereportersystemforgpcrassaywiththefissionyeastschizosaccharomycespombe AT shintarosasuga reportersystemforgpcrassaywiththefissionyeastschizosaccharomycespombe AT toshiyaosada reportersystemforgpcrassaywiththefissionyeastschizosaccharomycespombe |