【作者】 徐海明；

【导师】 梁植权； 刘德培；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2000， 博士

【摘要】 随着人类基因组测序工作的顺利进行，基因组功能的研究已显得越来越紧迫。基因组的功能主要包括基因转录产物的功能、基因表达调节的功能以及基因组自身组织和进化的功能。其中基因实现有序、协调表达以及基因组形成高级结构是通过DNA与蛋白质的相互作用来完成的。发展新的研究思路，建立有效的技术方法，筛选、鉴定基因组中的DNA调节片段，是筛选DNA结合蛋白、揭示基因表达调节和基因组自身组织规律的基础。 目前，绝大多数基因的转录调节机制研究尚未开展。这主要是由于已有的研究方法难以大量筛选、鉴定DNA调节元件，大多数基因的DNA调节区仍未被确定。另外，由于转录调节因子在细胞中的表达水平很低，利用常规的纯化方法很难获得足以进行氨基酸序列分析或抗体制备的纯化蛋白质，目前已克隆的转录因子不多，对这些因子的表达模式和DNA-蛋白质相互作用规律尚缺乏足够的研究。利用现有的软件对DNA上的蛋白质结合位点进行分析，即使在体外实验证实没有蛋白质结合的DNA片段上，往往检索出大量的结合位点，难以获得有价值的信息。筛选、鉴定DNA调节片段和相应的转录调节因子，研究DNA与蛋白质相互作用的规律，仍然是当前转录调节研究的一个重要任务。 目前，已发展了多种方法筛选、鉴定DNA调节片段，主要包括以下几种。1) DNA连续删切分析：是最常用的方法，可初步确定DNA调节元件的大致位置、功能活性。但该方法对DNA片段进行连续删切时需要合适的限制性核酸内切酶位点。2) DNase Ⅰ敏感性分析：是初步确定体内重要调节区的常用方法，能较真实地反映体内调节机制，但该方法技术难度较大。另外，以上这两种方法不能克隆新的基因片段，只能对已经获得的基因片段进行分析：对于DNA更多还原

【Abstract】 As the sequencing of human genome goes on smoothly, it is imminent to investigate the function of genome. The main function of genome includes (a) the function of gene’s transcripts, (b) the regulation of gene expression, (c) the organization of genome and (d) the evolution of genome. Coordinated gene expression and highly ordered structure of genome results from the interaction between DNA and protein. So it is important to develop new strategy and to establish efficient techniques to isolate and to identify the genomic regulatory sequences, which is the basis for the identification of DNA binding protein and for the study of the regulation of gene expression.Presently there lack efficient methods to broadly isolate and identify DNA regulatory fragments. Only a few of transcription factors were cloned because they are expressed in low level and it’s difficult to obtain pure protein enough for amino acid sequencing or preparation of antibody. Their expression pattern and the interaction between transcription factor and DNA were not studied in detail. It’s difficult to obtain valuable DNA regulatory information by computer software analysis. Thus little is known about the transcriptional regulation of most genes. A main task to study the transcriptional regulation is still to isolate and identify the regulatory DNA fragments and the corresponding transcription factors, to study the interaction between DNA and proteins.Several methods to screen and to identify DNA regulatory fragments have been developed. They are: (a) expression assay with continuously truncted DNA constructs, which is widely used to analyze the position and the activity of regulatory fragments, but it needs suitable restriction enzyme sites for DNA deletion. (b) analysis of the sensitivity更多还原