x染色体调控元件的定位及dna亲和纯化技术的探索细胞生物学专业论文.docxVIP

摘要摘要 摘要 摘要 x染色体失活是哺乳动物维持雌雄性细胞中X染色体连锁基因剂量平衡的 一种机制。它存在随机失活(父性和母性来源的X染色体等机率失活)和印记 失活(父性来源的X染色体定向失活)两种方式，但是x染色体调控元件(Xce) 等遗传和表观遗传因素会导致X染色体偏斜式失活，即X染色体偏向父性或母 性一方失活。我们采用双荧光素酶报告基因检测系统和凝胶迁移实验比较了Mus m1．1sc“lu$museulus(1 29)和Mus musculus castaneus strain(cast)小鼠品系的五缸 majorpromoter，Xite，DXPas34，Xist minimalpromoter区域的活性，寻找出Xist minimal promoter是一个Xce，且一88位的A／C碱基差异是导致129和cast Xist minimalpromoter活性差异的主要原因。 另外，我们初步建立了DNA亲和纯化体系。我们用来源于Xist intron，的 2bl一3 DNA探针纯化和质谱鉴定出PTB，但经凝胶迁移实验超迁移和染色体免 疫共沉淀验证出PTB并不结合Xist 2b1．3。因此我们还需要通过提高蛋白纯化效 率、回收效率和胶分辨率等来优化DNA亲和纯化体系，寻找到特异DNA序列 上的结合蛋白。 关键词：偏斜式X染色体失活，x染色体失活调控元件，Xist minimalpromoter， DNA亲和纯化 AbstractAbstract Abstract Abstract X chromosome inactivation(XCI)is the mechanism of balancing the expression of X-linked genes between male and female in mammals．It includes two types of inactivation：imprinted XCI(Paternal X chromosome(Xp)is always inactivated)and random XCI(Paternal X chromosome and matemal X chromosome(Xpl iS randomly inactivated)．However,X-controlling element(Xce)and epigenetic factors will skew the inactivation rate between Xp and Xm(skewing toward Xp or Xm)．Through comparing the activities of居扔major promo胞r，Xite，DXPas34，Xist minimal promo跑r between Mus musculus musculus(1 29)and Mus musculus castaneus strain (cast)by Luciferase Assay and Electrophoretic Mobility Shift Assay(EMSA)，we found that Xist minimal promo纪r is a Xce，and the一88 site mutation(A／C)is the main reason of the different Xist transcriptional activity between X129 and X。8吼． In addition，we initially established the experimental system of DNA affinity chromatography．With a DNA probe from Xist intron J『，PTB Was purified and identified with mass spectrometry．However,EMSA Super-shift and Chromatin Immunoprecipitation(ChIP)results indicated that PTB does not bind to this DNA probe．In order to find the target protein，we need to improve our DNA affinity chromato铲aphX including the purification efficiency,the recovery efficiency of target protein and the resolution of SDS—PAGE gel