| Project/Area Number |
17310127
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Living organism molecular science
|
|---|
| Research Institution | Kyoto Institute of Technology |
|---|
Principal Investigator |
KARATANI Hajime Kyoto Institute of Technology, Graduate School of Science and Technology, Professor (10169659)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
WADA Minoru The University of Tokyo, Ocean Resemrh Institute, Assistant Professor (70292860)
SASAKI Ken Kyoto Institute of Technology, Graduate School of Science and Technology, Associate Professor (20205842)
|
|---|
| Project Period (FY) |
2005 – 2007
|
| Project Status |
Completed (Fiscal Year 2007)
|
| Budget Amount *help |
¥8,580,000 (Direct Cost: ¥8,100,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥5,000,000 (Direct Cost: ¥5,000,000)
|
|---|
| Keywords | Bioluminescence / Bacterial luciferase / Fluorescent protein / Fusion protein / Fluorescence imaging / Bioluminescence imaging / Gene expression / cell division cycle / 生細胞イメージング / 遺伝子クローニング / 呼吸 |
|---|
| Research Abstract |
In this study, we have investigated into the intracellular space-time distribution of fluorescent protein of luminous bacterium and the molecular link with cell cycle mainly using Vibrio fischeri Y1 cells. The fluorescence images of the cells have proven that the fluorescent proteins responsible for bioluminescence (BL) color change is scattered in the vicinity of the cell membrane together with luciferase.
To clarify the reversible BL color change, the relationship between the BL color and the redox state of yellow fluorescent protein (YFP) has been studied in vitro. The results indicated that the YFP fluorescence ability depends on the redox states of YFP, and that the reduced YFP no longer has ability to cause yellow BL. It was also clarified that once the reduced YFP is reoxidized, the BL color changes from blue to yellow reversibly.
To characterize the space-time distribution of fluorescent proteins, the genes encoding YFP and another blue fluorescent protein (BFP) were cloned and their Escherichia coli expression systems were established. The fluorescence properties of the recombinant proteins were in good agreement with those of the authentic ones.
Subsequently, several fusion fluorescent proteins consisting of YFP and BFP have been designed and constructed. The fusion protein, of which N-terminus is YFP, was found to exhibit the bimodal fluorescence characteristic of both YFP and BFP. The appearance of bimodal fluorescences depended on the cellular conditions. Both fluorescences observed as small spots at the same site in growing cells, whereas the cells in the stationary phase appeared to emit mainly blue fluorescence. From the results, the fused fluorescent proteins were evaluated to be useful for collecting the intracellular information including the cell cycle. Furthermore, BL imaging of the luminous colony suggested that the bacterial colonies grow exhibiting the non-linear pattern.
|
