| Project/Area Number |
12672087
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Toho University (2001-2002)
Japan Advanced Institute of Science and Technology (2000) |
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Principal Investigator |
TAKAHASHI Tetsuo Toho University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (90133769)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Masakatsu Okazaki Natl. Res. Inst, National. Institute for Basic Biology, Associate Professor, 基礎生物学研究所, 助教授 (40124226)
UEDA Tetsuo Hokkaido University, Research Institute for Electronic Science Professor, 電子科学研究所, 教授 (20113524)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2002: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | unicellular flagellate / Chlamydomonas / Phototaxis / photoreceptor / retinal / eyespot / sensory rhodopsin / phoborhodopsin / ロドプシン / Acop-1 / Acop-2 |
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| Research Abstract |
In general, an attempt to clone homologs of plant or bacterial photoreceptors in fungi or in unicellular flagellates is unsuccessful because of their genetical diversity. We therefore combined several new PCR strategies in order to search for true archaeal-type rhodopsin phototaxis receptor(s) that had been physiologically well-established but had not been detected in molecular level.
Expressed Sequence Tag (EST) database of Chlamydomonas reinhardtii publicized in March 2000 from Kazusa DNA Research Institute contained a partial cDNA sequence that showed a little similarity in amino acid level to those of F-G helices of bacterial opsins. Using temperature-asymmetric PCR in combination with a highly degenerated primer, we have extended this sequence toward N-terminus using Chlamydomonas genomic DNA as a template. We found a reasonable similarity in the PCR product to DNA sequences corresponding to C-G helices of bacterial opsins. The complete DNA sequence was obtained and deposited in GenBank (accession numbers: AB058890 and AB58891). Using indirect immunofluorescence we also demonstrated the localization of one of these new archaeal-type opsins of Chlamydomonas in a cell-surface region close to the eyespot apparatus by which Chlamydomonas phototaxis is mediated. A homology modeling successfully indicated an archaeal-type folding of the proteins, accounting for previously established stereospecificity of retinal isomers in the phototaxis receptor(s).
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