摘要
目的 分离、克隆人基因组随机MARs片段 ,分析其序列特征 ,为进一步研究MARs在真核细胞DNA复制及基因表达调控中的作用与机制奠定基础。方法 用DNaseⅠ、高盐和蛋白酶K处理细胞核 ,分离人T细胞基因组随机MARs片段 ,并将其克隆进入PUC19质粒 ;用体外结合实验筛选能与核基质蛋白结合的克隆并测序 ,生物信息学分析其序列的特征。结果 从人T细胞基因组中分离得到大量的MARs片段 ,构建成MARs库 ,初步任意挑选 5 8个克隆 ,体外结合实验证实其具有与核基质蛋白结合活性 ,对其中 1个克隆的序列分析表明 ,其序列AT含量较高 ,不仅含有AC 和ATAT基序 ,还包含有多个转录 复制起点、增强子序列、弯曲DNA和扭结DNA基序以及多个反向重复序列。结论 分离获得的DNA片段具有MAR的特性 ,同一DNA序列中存在不同的元件组合 ,其参与基因表达调控的功能应该是复杂。
Objective To screen and clone the fragment the random matrix association regions (MARs) in human genome and analyze the characteristics of their sequences in order to provide proof for further investigation of the molecular mechanisms of MARs in the regulation of eukaryotes gene expression. Methods The fragment of functions and the random MARs of human genome, isolated by treatment of the nuclei using DNase I, high salt, and protein K, was cloned into the PUC19 vector. MARs which could bind with nuclear matrix proteins were identified by binding assay in vitro and sequenced consequently. The characteristics were analyzed by the bioinformatic method. Results A large number of MARs fragments were screened and obtained from human T cells successfully. A MARs library was constructed and 58 clones were selected randomly from the library. The results of the binding assay in vitro showed that the random MARs had binding activity with nuclear matrix proteins, and sequence analysis of one of the clones showed that it was consisted of rich A and T base pair, AC rich elements and ATAT motifs, many origin points of transcription/replication, enhancer, curved DNA, kinked DNA regions, and numerous reverse repeated base sequences. Conclusion The obtained DNA fragments have the characteristics of MARs and multiple cis function elements in a DNA sequence, suggesting that the functions of MARs in regulation of gene expression are complicated and multiform.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2004年第15期1342-1345,共4页
Journal of Third Military Medical University
