Colp Morgan J, Blais Cédric, Curtis Bruce A, Archibald John M
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada.
Institute for Comparative Genomics, Dalhousie University, Halifax, NS, Canada.
BMC Genomics. 2025 Apr 13;26(1):368. doi: 10.1186/s12864-025-11552-7.
The soil amoeba Acanthamoeba castellanii is an emerging model organism with which to study a wide range of biomedical, microbiological, and evolutionary phenomena. While transformation systems were established for this organism more than two decades ago, the fate of artificial transgenes has not been well characterized. In this study, artificial transformation experiments were performed to investigate how the A. castellanii genome responds to foreign DNA presented in both circular and linear plasmid form.
Nanopore sequencing was used as a high throughput method to screen for transgene DNA in the resulting transformant cultures, and candidate transgene integrations were identified. Molecular biology experiments were performed to validate the sequence data and provide additional context on the fate of transgenes. A method was devised to estimate the rate of read chimerism in nanopore sequencing runs and accurately account for the effects of read chimerism in identifying putative transgene integrations. Based on the experimental data in hand, a potential mechanism for transgene maintenance in A. castellanii is proposed, one in which incoming foreign DNA is tandemly duplicated and telomeres are added to the ends.
Our results suggest that transformation of A. castellanii with foreign DNA leads to linear molecules that are maintained as telomere-containing, transgene-bearing minichromosomes, which may facilitate chromosomal integration. This process may allow lateral gene transfer by expanding the window of opportunity for exogenous DNA to be taken up and integrated into the A. castellanii genome. Similar mechanisms exist in other eukaryote groups, suggesting this may be a widespread feature of eukaryote genome biology.
土壤变形虫卡氏棘阿米巴是一种新兴的模式生物，可用于研究广泛的生物医学、微生物学和进化现象。虽然二十多年前就已为这种生物建立了转化系统，但人工转基因的命运尚未得到很好的表征。在本研究中，进行了人工转化实验，以研究卡氏棘阿米巴基因组对外源DNA以环状和线性质粒形式呈现时的反应。
纳米孔测序被用作一种高通量方法，用于筛选所得转化体培养物中的转基因DNA，并鉴定候选转基因整合。进行了分子生物学实验以验证序列数据，并提供有关转基因命运的更多背景信息。设计了一种方法来估计纳米孔测序运行中的读数嵌合率，并在识别假定的转基因整合时准确考虑读数嵌合的影响。基于手头的实验数据，提出了一种卡氏棘阿米巴中转基因维持的潜在机制，即进入的外源DNA串联重复并在末端添加端粒。
我们的结果表明，用外源DNA转化卡氏棘阿米巴会产生线性分子，这些分子作为含有端粒、携带转基因的微型染色体得以维持，这可能有助于染色体整合。这一过程可能通过扩大外源DNA被摄取并整合到卡氏棘阿米巴基因组中的机会窗口来实现横向基因转移。其他真核生物群体中也存在类似机制，这表明这可能是真核生物基因组生物学的一个普遍特征。
