| Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2009: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2008: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
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| Research Abstract |
The detailed mechanisms of transcriptional regulation in malaria parasite, Plasmodium falciparum, remains almost unknown. Genome-wide analyses of transcription-associated proteins have revealed a paucity of putative regulatory transcription factors, suggesting that this parasite has unique regulatory transcription factors distinct from those of other eukaryotes. To identify unique regulatory transcription factors in P. falciparum, we tried purification and identification of nuclear factor which associates specifically with cis-acting enhancer region of pfl-cys-prx in this study. Nuclear extract (110 mL) was prepared from 33 L of parasite culture (5 X 10^<11> cells), then, the enhancer binding protein was purified from the extract by 5 steps of liquid chromatography. In each step, the DNA binding activity of each fraction was verified by electrophoresis mobility shift assay (EMSA). As results, 30 ! IL of fraction with the specific enhancer-binding activity was obtained. Then, this fraction was separated by SDS-PAGE and 3 bands were detected as possible candidates for the specific DNA binding protein. These bands were excised from the gel and analyzed by LC-MS/MS, then, about 20 parasite proteins were identified. Enhancer-binding activities of the recombinant proteins of these factors were analyzed by EMSA, then, one of these proteins revealed enhancer-binding activity. Recently, new plant-like transcriptional regulatory factors of Plasmodium, which termed AP-2 family, had been reported, however the factor detected in this study was not included in the AP-2 family. These results suggested that this factor is novel and unique, and possibly regulates transcriptional mechanism in malaria parasite.
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