Molecular Genetics Research Laboratory, Faculty-of-Science-Building No. 7, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, 113, Tokyo, Japan.
Plant Mol Biol. 1989 Feb;12(2):235-44. doi: 10.1007/BF00020508.
We isolated and cloned DNA fragments that exist as inverted-repeat structures in the genome of Pharbitis nil. The method used exploited the fact that if inverted repeat DNA is present in the DNA fragment, intramolecular double-stranded structures can be partly formed within single-stranded DNA molecules after denaturation and rapid renaturation of the fragment. The rapidly renaturing DNA fragments (termed snap-back DNA) were isolated by hybroxylapatite column chromatography and treatment with mungbean nuclease and were cloned into the pUC9 vector. Four snap-back DNA members out of thousands of independent clones obtained were characterized with respect to the reiteration frequency and the nucleotide sequences. When used as probes in Southern hybridization experiments, some of the members identified restriction fragment length polymorphism among the cultivars, suggesting that these sequences might be fluid in the genome. One of the four clones has regions of nucleotide sequence homology to those of inverted-repeat regions in the transposon Taml of Antirrhinum majus.
我们从牵牛花基因组中分离并克隆了以反向重复结构存在的 DNA 片段。所采用的方法利用了这样一个事实，即在 DNA 片段中存在反向重复 DNA 的情况下，在变性和片段快速复性后，单链 DNA 分子内可以部分形成分子内双链结构。通过羟磷灰石柱层析和绿豆核酸酶处理，快速复性的 DNA 片段（称为 snap-back DNA）被分离出来，并克隆到 pUC9 载体中。在数千个独立克隆中，有四个 snap-back DNA 成员在重复频率和核苷酸序列方面得到了表征。当作为 Southern 杂交实验中的探针使用时，其中一些成员在品种间鉴定出了限制片段长度多态性，这表明这些序列在基因组中可能是流动的。这四个克隆中有一个与金鱼草转座子 Tam1 的反向重复区具有核苷酸序列同源性。
