Suzuki H, Ingersoll J, Stern D B, Kindle K L
Plant Science Center, Cornell University, Ithaca, NY 14853, USA.
Plant J. 1997 Apr;11(4):635-48. doi: 10.1046/j.1365-313x.1997.11040635.x.
Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin (oriA). Transformants were recovered only with the plasmid containing oriA, and all transformants contained an integrated plasmid copy at oriA, suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.
莱茵衣藻的异常叶绿体转化体最近被报道，其含有2000个叶绿体atpB基因突变体版本的拷贝，以染色体外串联重复的形式存在。本文开展了相关研究，以（i）探讨产生和维持扩增DNA的可能机制，以及（ii）确定是否可以利用叶绿体基因扩增来过量表达叶绿体基因或外源基因。本文提供的数据表明，高拷贝数转化体在叶绿体基因组大反向重复的两个拷贝中都存在特征性重排。这些重排似乎是维持扩增DNA的结果或必要条件。为了模拟叶绿体中染色体外DNA明显的自主复制，用与叶绿体基因组无同源性的质粒或携带假定叶绿体DNA复制起点（oriA）的同一质粒进行转化。仅用含有oriA的质粒获得了转化体，并且所有转化体在oriA处都含有一个整合的质粒拷贝，这表明染色体外串联重复的建立或维持需要本实验中未复制的条件。为了确定其他基因是否可以在叶绿体中以高拷贝数维持，将携带野生型atpB基因或细菌aadA基因的质粒导入高拷贝数转化体。令人惊讶的是，即使对导入基因施加强烈的选择压力，二次转化事件后质粒串联重复的拷贝数也迅速下降。因此，叶绿体转化既可以产生高拷贝数串联重复，也可以使其不稳定。
