RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.
RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.
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RglB 促进高度甲基化的真核 DNA 的克隆：人类 L1 转座子、植物 DNA 和体外用人类 DNA 甲基转移酶甲基化的 DNA。
DOI：
10.1093/nar/16.10.4465
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发表时间：
1988
影响因子：
14.9
通讯作者：
Smith,SS
中科院分区：
文献类型：
--
作者：
Woodcock,DM;Crowther,PJ;Diver,WP;Graham,M;Bateman,C;Baker,DJ;Smith,SS
In vitromethylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies inrglB+host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, therglB−derivative of DS410 showed no reduction in transformation efficiency with methylation while therglB−derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb Kpnl fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mCin vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with therglB−host strains than with therglB+hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (fromPetunia hybrida) when plated onrglB−hosts gave titres up to 222 times higher than on therglB+strains.
