Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia.
Mol Biol Evol. 2012 Aug;29(8):1987-2000. doi: 10.1093/molbev/mss066. Epub 2012 Feb 14.
The subtelomeric regions of organisms ranging from protists to fungi undergo a much higher rate of rearrangement than is observed in the rest of the genome. While characterizing these ~40-kb regions of the human fungal pathogen Cryptococcus neoformans, we have identified a recent gene amplification event near the right telomere of chromosome 3 that involves a gene encoding an arsenite efflux transporter (ARR3). The 3,177-bp amplicon exists in a tandem array of 2-15 copies and is present exclusively in strains with the C. neoformans var. grubii subclade VNI A5 MLST profile. Strains bearing the amplification display dramatically enhanced resistance to arsenite that correlates with the copy number of the repeat; the origin of increased resistance was verified as transport-related by functional complementation of an arsenite transporter mutant of Saccharomyces cerevisiae. Subsequent experimental evolution in the presence of increasing concentrations of arsenite yielded highly resistant strains with the ARR3 amplicon further amplified to over 50 copies, accounting for up to ~1% of the whole genome and making the copy number of this repeat as high as that seen for the ribosomal DNA. The example described here therefore represents a rare evolutionary intermediate-an array that is currently in a state of dynamic flux, in dramatic contrast to relatively common, static relics of past tandem duplications that are unable to further amplify due to nucleotide divergence. Beyond identifying and engineering fungal isolates that are highly resistant to arsenite and describing the first reported instance of microevolution via massive gene amplification in C. neoformans, these results suggest that adaptation through gene amplification may be an important mechanism that C. neoformans employs in response to environmental stresses, perhaps including those encountered during infection. More importantly, the ARR3 array will serve as an ideal model for further molecular genetic analyses of how tandem gene duplications arise and expand.
从原生生物到真菌等生物的端粒区域经历的重排率远高于基因组其他区域。在对人类真菌病原体新型隐球菌的40kb 端粒区域进行特征分析时，我们在 3 号染色体的右端粒附近发现了一个近期的基因扩增事件，涉及一个编码亚砷酸盐外排转运蛋白（ARR3）的基因。3177bp 的扩增子存在于 2-15 个拷贝的串联阵列中，仅存在于具有新型隐球菌 var. grubii 亚群 VNI A5 MLST 图谱的菌株中。携带扩增子的菌株对亚砷酸盐表现出明显增强的抗性，与重复序列的拷贝数相关；通过对酿酒酵母亚砷酸盐转运突变体的功能互补，验证了增加抗性的起源与转运有关。随后在亚砷酸盐浓度不断增加的情况下进行实验进化，产生了具有 ARR3 扩增子的高度抗性菌株，该扩增子进一步扩增至 50 多个拷贝，占整个基因组的1%，使该重复序列的拷贝数与核糖体 DNA 一样高。因此，这里描述的例子代表了一个罕见的进化中间体——一个目前处于动态流动状态的阵列，与相对常见的、过去串联重复的静态遗迹形成鲜明对比，由于核苷酸分歧，这些遗迹无法进一步扩增。除了鉴定和工程化对亚砷酸盐具有高度抗性的真菌分离株，并描述新型隐球菌中首次报道的通过大规模基因扩增进行微进化的实例外，这些结果表明，通过基因扩增进行适应可能是新型隐球菌应对环境压力（包括感染期间遇到的压力）的重要机制。更重要的是，ARR3 阵列将作为进一步分析串联基因重复出现和扩展的分子遗传分析的理想模型。
