Iwasaki T, Ohki R, Kiyama R, Oishi M
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
FEBS Lett. 1995 Apr 24;363(3):239-45. doi: 10.1016/0014-5793(95)00325-4.
Essentially all eukaryotic cells contain circular extrachromosomal DNA as a result of excision from the chromosomes. To obtain insight into the nature of recombination associated with the occurrence of such DNA species and its biological significance, we analyzed a library enriched in recombination junctions which was constructed by a novel DNA subtraction technique; in-gel competitive reassociation (IGCR). Furthermore, we also introduced inverse PCR to characterize chromosomal DNA fragments containing the recombination junctions. At least 45% of the clones in the library constructed by the IGCR procedure comprised DNA with recombination junctions. Nucleotide sequence analysis of the recombination junctions indicated that three of four extrachromosomal DNAs thus analyzed were produced through recombination between sequences with a 3-5 bp homology in the chromosomes. One extrachromosomal DNA was apparently generated through non-homologous recombination, possibly by end-to-end joining. These results have demonstrated the usefulness of IGCR in concentrating recombination junctions, which provide the most direct evidence for the mechanism of the recombinational events involved, from highly complex genomes.
基本上所有真核细胞都含有由于从染色体上切除而产生的环状染色体外DNA。为了深入了解与此类DNA物种出现相关的重组性质及其生物学意义，我们分析了一个通过新型DNA消减技术——凝胶内竞争复性（IGCR）构建的富含重组连接点的文库。此外，我们还引入了反向PCR来表征包含重组连接点的染色体DNA片段。通过IGCR程序构建的文库中至少45%的克隆包含具有重组连接点的DNA。对重组连接点的核苷酸序列分析表明，如此分析的四个染色体外DNA中的三个是通过染色体中具有3-5个碱基对同源性的序列之间的重组产生的。一个染色体外DNA显然是通过非同源重组产生的，可能是通过端对端连接。这些结果证明了IGCR在从高度复杂的基因组中富集重组连接点方面的有用性，重组连接点为所涉及的重组事件机制提供了最直接的证据。
