The analysis of cell lineage specific expression of perforin gene

Research Project

Project/Area Number	15591013
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Hematology
Research Institution	Nagasaki University
Principal Investigator	MIYAZAKI Yasushi Nagasaki University, Hospital of Medicine and Dentistry, Lecturer, 医学部・歯学部附属病院, 講師 (40304943)
Co-Investigator(Kenkyū-buntansha)	UDONO Heiichiro RIKEN, Research center for allergy and immunology, Team leader, 免疫・アレルギー科学総合センター, チームリーダー (50260659)
Project Period (FY)	2003 – 2004
Project Status	Completed (Fiscal Year 2004)
Budget Amount *help	¥2,300,000 (Direct Cost: ¥2,300,000) Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000) Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Keywords	ETS gene / TAP method / MEF gene / ETS遺伝子
Research Abstract	MEF is a member of Elf-sub family of ETS transcription factors. MEF was shown to activate the expression of GN-CSF and IL-3 genes, which Elf-1, the closest gene to MEF, also transactivates. However, gene knock-out experiments demonstrated that MEF is indispensable for the expression of perforin gene in NK cells but not in suppressor T-cells, on the other hand, Elf-1 knock-out mice did not show clear phenotype. To elucidate the mechanisms of lineage specific expression of perforin gene, we planned two things : (1)determine the protein(s) that associate with MEF and (2)compare it with those of Elf-1. For this purpose, we performed "tandem affinity purification"(TAP) analysis using MEF as bait Cells trasfected with the expression plasmid for MEF were lysed and purified twice with Ig G cepharose beads and calmodulin regine, then associated proteins with MEW were fractionated by SDS-PGE. After visualized with protein staining, each bands was analysed with LC-MAS spectrometry. Proteins picked up were : Myb binding protein, nucleolin, RNA helicase, vimentin, nucleophosmin. Among those, nucleophosmin was shown to be mutated in one-third of acute myeloid leukemia recently, suggesting its important roles in hematopoietic cells. We are now in the process of the analysis for the direct physical and functional interactions between MEF and nucleophosmin.