Crowther P J, Cartwright A L, Hocking A, Jefferson S, Ford M D, Woodcock D M
Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Nucleic Acids Res. 1989 Sep 25;17(18):7229-39. doi: 10.1093/nar/17.18.7229.
We have used highly methylation tolerant host strains to clone hyper- and hypo-methylated genomic elements from different regions of the same family of long interspersed repetitive elements from human DNA, specifically the 1.8 kilobase (kb) and 1.2kb KpnI fragments from members of the L1 family of transposable elements in which respectively some 18% and 2.7% of cytosines are methylated in vivo in human spleen DNA. The consensus of the DNA sequences of the ends of 13 clones from the hypomethylated region of human L1 agreed exactly with the consensus derived previously from clones made using conventional host strains. However the sequences of 18 of our clones from the 5' end of the hypermethylated region differed significantly from the sequences of clones made using conventional hosts (P less than 0.0001). The 5' region of the 1.8kb L1 region is a CpG island which, in human somatic tissue, appears to be maintained in a highly methylated state, including methylation at sites other than CpG dinucleotides. The consensus sequence of this region also has features suggestive of a previously unrecognized open reading frame.
我们使用了高度耐甲基化的宿主菌株，从人类DNA中同一长散在重复元件家族的不同区域克隆高甲基化和低甲基化的基因组元件，具体来说，是来自转座元件L1家族成员的1.8千碱基（kb）和1.2kb KpnI片段，其中在人类脾脏DNA中，约18%和2.7%的胞嘧啶在体内被甲基化。来自人类L1低甲基化区域的13个克隆末端DNA序列的共识与先前使用传统宿主菌株制备的克隆得出的共识完全一致。然而，我们从高甲基化区域5'端获得的18个克隆的序列与使用传统宿主制备的克隆序列有显著差异（P小于0.0001）。1.8kb L1区域的5'区域是一个CpG岛，在人类体细胞组织中，它似乎保持在高度甲基化状态，包括在CpG二核苷酸以外的位点甲基化。该区域的共识序列也具有提示先前未被识别的开放阅读框的特征。
