Dalgaard J Z, Banerjee M, Curcio M J
Molecular Genetics Program, Wadsworth Center, David Axelrod Institute, Albany, New York, USA.
Genetics. 1996 Jun;143(2):673-83. doi: 10.1093/genetics/143.2.673.
We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24-retrotransposon insertions into two different mouse-derived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into a YAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units.
我们开发了一种用于基因组物理分析的强大新工具，称为Ty1介导的染色体片段化，并已使用该方法将24个逆转座子插入定位到两种不同的小鼠来源的酵母人工染色体（YAC）中。用逆转座指示基因ade2AI标记的质粒编码GAL1:Ty1融合元件的表达，导致很大一部分细胞发生单个用ADE2标记的Ty1插入。通过ADE2基因与URA3标记的YAC的共分离，鉴定出Ty1ADE2插入YAC的菌株。Ty1ADE2元件还带有一个核酸内切酶I-DmoI的位点，我们证明该位点在酵母基因组中任何地方都不存在。因此，I-DmoI在Ty1ADE2插入的独特位点切割单个染色体或YAC，从而允许快速定位整合事件。我们的分析表明，Ty1ADE2整合到YAC中的频率等于或高于基于随机插入预期的频率。值得注意的是，小鼠Steel基因座的50 kb转录单元被证明是Ty1整合的一个高度显著的热点。哺乳动物转录单元对Ty1插入的易接近性与酵母转录单元形成对比。
