Wang M X, Church G M
Laboratory of Oncology Research, Wills Eye Hospital, Philadelphia, Pennsylvania.
Nature. 1992 Dec 10;360(6404):606-10. doi: 10.1038/360606a0.
The increasingly rapid pace at which genomic DNA sequences are being determined has created a need for more efficient techniques to determine which parts of these sequences are bound in vivo by the proteins controlling processes such as gene expression, DNA replication and chromosomal mechanics. Here we describe a whole-genome approach to identify and characterize such DNA sequences. The method uses endogenous or artificially introduced methylases to methylate all genomic targets except those protected in vivo by protein or non-protein factors interfering with methylase action. These protected targets remain unmethylated in purified genomic DNA and are identified using methylation-sensitive restriction endonucleases. When the method was applied to the Escherichia coli genome, 0.1% of the endogenous adenine methyl-transferase (Dam methylase) targets were found to be unmethylated. Five foreign methylases were examined by transfection. Database-matched DNA sequences flanking the in vivo-protected Dam sites all fell in the non-coding regions of seven E. coli operons (mtl, cdd, flh, gut, car, psp and fep). In the first four operons these DNA sequences closely matched the consensus sequence that binds to the cyclic AMP-receptor protein. The in vivo protection at the Dam site upstream of the car operon was correlated with a downregulation of car expression, as expected of a feedback repressor-binding model.
基因组DNA序列测定的速度越来越快，这就需要更高效的技术来确定这些序列的哪些部分在体内被控制基因表达、DNA复制和染色体机制等过程的蛋白质所结合。在这里，我们描述了一种全基因组方法来识别和表征此类DNA序列。该方法使用内源性或人工引入的甲基化酶来甲基化所有基因组靶点，但体内受蛋白质或非蛋白质因子保护而免受甲基化酶作用的靶点除外。这些受保护的靶点在纯化的基因组DNA中保持未甲基化状态，并使用甲基化敏感的限制性内切酶进行鉴定。当该方法应用于大肠杆菌基因组时，发现0.1%的内源性腺嘌呤甲基转移酶（Dam甲基化酶）靶点未被甲基化。通过转染检测了五种外源甲基化酶。体内受保护的Dam位点两侧与数据库匹配的DNA序列均位于七个大肠杆菌操纵子（mtl、cdd、flh、gut、car、psp和fep）的非编码区。在前四个操纵子中，这些DNA序列与结合环磷酸腺苷受体蛋白的共有序列紧密匹配。正如反馈阻遏物结合模型所预期的那样，car操纵子上游Dam位点的体内保护与car表达的下调相关。
