Akiyama K, Nishi Y
Life Science Research Laboratory, Japan Tobacco, Inc., Kanagawa.
Nucleic Acids Res. 1991 Dec 25;19(24):6887-94. doi: 10.1093/nar/19.24.6887.
Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity.
对包含人神经母细胞瘤细胞系N - myc扩增子的DNA序列进行了克隆和物理图谱分析。使用酚乳液重缔合技术制备的探针分离出该区域内的多个λ噬菌体克隆。基于限制性图谱分析，它们被整合到8个重叠群中，大小为25 - 60 kb，总共覆盖了一个330 kb的区域。使用六边形场凝胶电泳鉴定出几个大小分别为100、420、480和520 kb的Notl片段扩增子，并将重叠群定位到这些Notl片段中。重叠群所覆盖的区域相当于鉴定为Notl片段的扩增子的约60 - 80%。为了比较细胞系中N - myc基因侧翼的扩增区域，将覆盖整个重叠群的噬菌体克隆用作探针进行杂交。结果表明，在所检测的细胞系中，整个重叠群的18 - 45%的部分也被扩增。这些结果使我们能够确定“重排位点”，这些位点在重叠群中分布较为均匀，每隔40 kb有一个。这些观察结果引发了这样一种观点，即扩增的DNA结构域是在多次重排后构建的，然后数量增加，最终导致形成具有序列同质性的扩增子亚群。
