In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.
In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.
复制标题
体外顺式调节元件的 DNA 胞嘧啶甲基化调节体内 c-Ha-ras 启动子活性。
DOI：
10.1093/nar/17.13.5135
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发表时间：
1989
影响因子：
14.9
通讯作者：
Lapeyre,JN
中科院分区：
文献类型：
--
作者：
Rachal,MJ;Yoo,H;Becker,FF;Lapeyre,JN
利用PHrasCAT瞬时表达系统评价DNA胞嘧啶甲基化对启动子活性的影响。这个551bpHa-ras-1基因启动子区域富含84个CpG二核苷酸和6个功能GC盒，是许多基因启动子区域含有CpG岛的原型。细菌修饰酶Hhal甲基转移酶(MTase)和Hpall MTase单独或与人胎盘DNA甲基转移酶(HpMTase)一起用于甲基化CpG位点，包括不对称元件，如GC box CpG‘s，用于在启动子的不同类型的位点甲基化。Hhal和hpall位点的甲基化使CAT的表达减少了70%-80%，而在通用的cpG位点上用Hp MTase甲基化使启动子失活了95%。H-ras启动子活性的抑制不是由于甲基化引起的DNA摄取或细胞稳定性的差异，也不是由于质粒的拓扑形式，或者非启动子区域的甲基化效应。
The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacteria] modification enzymes Hhal methyl transferase (MTase) and Hpall MTase, alone or in combination with a human placenta] DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of Hhal and Hpall sites reduced CAT expression by −70% −80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by <95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.