摘要
探讨FⅧA基因表达的分子机制.凝胶阻滞实验(EMSA)结果证明,FⅧA基因第1内含子5′区的前12碱基与转录因子结合并由此调控基因表达.FⅧA基因第1内含子第12位碱基由C突变为A(内含子1(+12)C→A)导致与转录因子结合能力降低.构建不同的荧光素酶表达质粒Luc1、Luc2、Luc3、Luc4、Luc5、Luc6,并转染U937细胞和HepG2细胞.结果显示,如果Luc5(具有最高表达活性)的内含子1(+12)由C突变为A,启动子活性显著降低.与Luc5相比,突变后的Luc5的活性分别下降了52·9%(U937,P<0·01)和47·6%(HepG2,P<0·01).将Luc5与PN3 Sp1共同转染U937细胞和HepG2细胞后,Luc5的荧光素酶活性分别提高了42·4%(U937,与Luc5单独转染比较P<0·01)和54·9%(HepG2,与Luc5单独转染比较P<0·01),而突变后的Luc5(Mut)与PN3 Sp1共同转染则没有明显的改变.表明转录因子Sp1在FXA基因表达的重要性.这些结果也表明,内含子在FⅧA基因表达过程中起重要作用,为遗传性凝血因子Ⅷ发病机理的研究提供了新的证据.
The molecular mechanism of the F [ⅩⅢ] A gene expression was investigated in current experiments. The results from electrophoretic mobility shift assay （EMSA） demonstrated that the first 12 bp in 5＇-region of first intron of the F [ⅩⅢ] A gene was capable of binding transcription factors and hereby regulated the gene expression. The replacement of the F[ⅩⅢ] A first intron （ ＋ 12） C to A could lead to loss of capability binding transcription factors. Various luciferase expression plasmid Luc 1, Luc 2, Luc 3, Luc 4, Luc 5 and Luc 6 were constructed and transfected into U937 cell and HepG2 cells. Results showed that a substantial loss of promoter activity would be resulted if Luc 5 （the highest expressional ability） was mutated. Compared to Luc 5, the luciferase activity of mutated Luc 5 were reduced 52.9 % （U937, P 〈 0.01 ）and 47.6 % （HepG2, P 〈 0.01 ） respectively. After Luc 5 was co-transfected with PN3 Spl into U937 cells and HepG2 cells, luciferase activity were respectively increased 42.4 % （ U937, P 〈 0. O1 compared to Luc 5 alone ） and 54.9 % （ HepG2, P 〈 0.01 compared to Luc 5 alone）, indicating the importance of transcription factor Spl in expression of the F[ⅩⅢ] A gene. There were no obvious changes in luciferase activity of Mut group. The findings, which showed that intron also play an important role in expression of the F X[ⅩⅢ] A gene, provide a novel evidence for further study of the pathogenesis of hereditary factor [ⅩⅢ] deficiency.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2007年第5期338-343,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家教育部博士点项目基金(No.20030001028)
国家自然科学基金(No.30060037及30671856)
教育部科学技术研究重点项目(No.03147)
海南医学院重点学科项目(海医科研部[2005-46])~~
