文献类型: 外文期刊
作者: Guan, Zheng-Bing 1 ; Shui, Yan 2 ; Lu, Jian 1 ;
作者机构: 1.Jiangnan Univ, Sch Biotechnol, Wuxi 214122, Peoples R China
2.Chinese Acad Fishery Sci, Key Lab Genet Breeding & Aquaculture Biol Freshwa, Minist Agr, Freshwater Fisheries Res Ctr, Wuxi 214081, Peoples R China
关键词: ARE-binding protein;EST;Expression analysis;EMSA
期刊名称:MOLECULAR AND CELLULAR BIOCHEMISTRY ( 影响因子:3.396; 五年影响因子:3.282 )
ISSN: 0300-8177
年卷期: 2010 年 345 卷 1-2 期
页码:
收录情况: SCI
摘要: Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that can bind to and destabilize certain mRNAs containing AU-rich element (ARE) binding sites. In this study, a novel porcine cDNA has been isolated by expressed sequence tag assembly and subsequently confirmed by RT-PCR analysis, and designated porcine TTP (poTTP). The open reading frame of the poTTP cDNA is 981 bp, encoding 326 amino acids. The poTTP gene is approximately 2.5 kb in size and contains a single intron. Southern blotting analysis demonstrated that it is a single copy gene. Real-time quantitative PCR analysis revealed that the poTTP gene is constitutively expressed in all detected tissues, and with the highest mRNA level in lymphoid tissues spleen and thymus. Recombinant His6-tagged poTTP protein and its two zinc finger mutants (C146G and H127I) were efficiently expressed and purified from Escherichia coli BL21 (DE3), respectively. In vitro, RNA-electrophoretic mobility shift assay confirmed a direct interaction between poTTP protein and porcine TNF-alpha (poTNF-alpha) mRNA ARE probe; this interaction was eliminated when using either two zinc finger mutants of poTTP. Consistently, mutations within the ARE region prevented the binding interaction between recombinant poTTP protein and poTNF-alpha mRNA ARE probe. These results indicate that poTTP is an ARE-binding protein that might regulate the turnover of certain mRNAs in vivo.
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