The analysis of cell lineage specific expression of perforin gene

穿孔素基因细胞谱系特异性表达分析

基本信息

批准号：
15591013
负责人：
MIYAZAKI Yasushi
金额：
$ 1.47万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591013/
关键词：
ETS gene TAP method MEF gene ETS遺伝子

项目摘要

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.

MEF是ETS转录因子的ELF-SUB家族的成员。 MEF被证明激活了GN-CSF和IL-3基因的表达，ELF-1（最接近MEF）也会反式激活。然而，基因敲除实验表明，MEF对于NK细胞中孔蛋白基因的表达是必不可少的，但在抑制T细胞中不可或缺，另一方面，Elf-1敲除小鼠没有显示清晰的表型。为了阐明穿孔基因的谱系特异性表达的机制，我们计划了两件事：（1）确定与MEF关联的蛋白质，以及（2）将其与Elf-1的蛋白质进行比较。为此，我们对使用MEF作为诱饵细胞进行了“串联亲和力纯化”（TAP）分析（TAP）用MEF表达质粒染色的诱饵细胞被裂解，并用Ig G Gepharose珠和钙调蛋白进行了两次纯化，然后由SDS与MEW分解了相关的蛋白-pge。用蛋白质染色可视化后，用LC-MAS光谱法分析了每个条带。拾取的蛋白质是：Myb结合蛋白，核醇蛋白，RNA解旋酶，波形蛋白，核素蛋白。其中，核磷脂最近在三分之一的急性髓样白血病中突变，这表明其在造血细胞中的重要作用。现在，我们正在分析MEF和核磷脂之间直接的物理和功能相互作用。