Reams Andrew B, Neidle Ellen L
Department of Microbiology, University of Georgia, Athens, GA 30602-2605, USA.
J Mol Biol. 2004 May 7;338(4):643-56. doi: 10.1016/j.jmb.2004.03.031.
A system for studying gene amplification in the bacterium Acinetobacter sp. strain ADP1 was used to isolate 105 spontaneous mutants. The method selects for the elevated expression of neighboring transcriptional units in a parent strain lacking its normal transcriptional activators. Gene amplification can compensate for the activator loss by increasing the copy number of seven weakly expressed genes. Mutant colonies arose from the parent strain at a frequency of 10(-8) within three weeks. All but one of these mutants carried tandem head-to-tail repeats of a chromosomal segment (amplicon). These amplicons varied in size from approximately 12-290 kb and ranged in copy number from 3 to more than 30. Gene amplification involved a two-step process in which duplications formed independently of recA. Illegitimate recombination fused normally distant chromosomal regions to create novel DNA duplication junctions. These junctions were isolated from amplification mutants using an assay that exploits Acinetobacter natural transformability. Sequence analysis of 72 junctions revealed little identity in the recombining regions. Furthermore, multiple independently isolated mutants contained identical junctions. Six different junctions, each found in two to six mutants, revealed that some recombination events are site-specific. Several recurring junctions were studied using PCR. In each case, the identical duplication present in the mutant was estimated to have occurred in as many as one in a million cells in populations of strains never exposed to selective conditions. These duplications appeared to form spontaneously by a novel type of short homology-independent, site-specific process. However, in the absence of recA, mutant colonies were not selected from parent cells containing these duplications. Thus, the second gene amplification step most likely depends on homologous recombination to increase amplicon copy number. These studies support the theory that gene amplification is a driving force in the evolution of functionally related gene clusters.
利用一种用于研究不动杆菌属ADP1菌株基因扩增的系统分离出了105个自发突变体。该方法用于筛选在缺乏正常转录激活因子的亲本菌株中相邻转录单元的高表达情况。基因扩增可通过增加7个弱表达基因的拷贝数来补偿激活因子的缺失。在三周内，突变菌落以10(-8)的频率从亲本菌株中出现。除了一个突变体之外，所有这些突变体都携带了一个染色体片段（扩增子）的头尾串联重复序列。这些扩增子的大小在约12 - 290 kb之间，拷贝数在3到30以上。基因扩增涉及一个两步过程，其中重复序列的形成独立于recA。非法重组将通常距离较远的染色体区域融合，形成新的DNA重复连接点。利用不动杆菌的天然转化能力的检测方法从扩增突变体中分离出这些连接点。对72个连接点的序列分析表明，重组区域几乎没有同源性。此外，多个独立分离的突变体含有相同的连接点。六个不同的连接点，每个在两到六个突变体中被发现，表明一些重组事件是位点特异性的。使用PCR研究了几个重复出现的连接点。在每种情况下，估计突变体中存在的相同重复序列在从未暴露于选择条件的菌株群体中每百万个细胞中多达一个细胞中发生。这些重复序列似乎是通过一种新型的短同源性无关的位点特异性过程自发形成的。然而，在缺乏recA的情况下，未从含有这些重复序列的亲本细胞中筛选出突变菌落。因此，基因扩增的第二步很可能依赖于同源重组来增加扩增子的拷贝数。这些研究支持了基因扩增是功能相关基因簇进化中的驱动力这一理论。
