Gene Amplification: Acinetobacter baylyi as a bacterial model system

摘要本项目致力于一种新的细菌系统，用于研究涉及基因复制和进一步扩增的染色体重排。基因扩增是所有生物体中常见的过程，具有重大的后果。它对进化、遗传多样性和生物体适应多变环境的能力至关重要。基因放大也会导致严重的问题，如耐药性、癌症和微生物毒力。尽管它很重要，但基因扩增的许多方面仍然知之甚少。与其他类型的基因变化不同，放大是动态的和可逆的。它可能不会留下任何关于其位置或频率的证据。在这个研究项目中，一种土壤细菌的不同寻常的特征，贝利不动杆菌ADP1，促进了基因扩增的系统研究。这种细菌的关键特征是，它能以极高的效率从环境中提取DNA，并通过同源重组将其整合到基因组中。这种对DNA摄取的自然能力允许使用转化试验来检测自发产生的突变中重复染色体区域的精确终点。这些重复位点的DNA序列提供了关于潜在的基因重组事件的信息。这个项目建立在最初研究的耐人寻味的结果基础上，在这些研究中，发现了一种新型的位置特异性非法重组(PSIR)过程。PSIR事件缺乏典型的位点特异性重组的DNA特征。该项目的目标之一是确定PSIR的机制，这似乎是一种新颖的机制。此外，还将使用全基因组方法来确定自发复制的特征。有助于基因复制的DNA的重要特征将被研究，如DNA序列和基因组背景。这一策略将提高我们对一个基本的、常见的和重要的遗传过程的理解。学生将在连接遗传学、生理学、生物化学和计算的重要的多学科领域接受培训。该项目对各级学生开放，将涉及本科生和研究生。此外，一名博士后研究员将进行相关调查，并帮助指导项目参与者。佐治亚大学正在进行的项目将增加培训不同科学家群体的机会。这样的项目包括NSF支持的本科生研究经验(REU)原核生物学站点项目。这项研究的长期影响有可能通过更好地了解潜在的机制来抵消基因放大的有害影响。此外，基因扩增可以被开发用于有益的生物技术应用。理想情况下，染色体基因扩增可以用于所需的操作，以避免在基因工程中使用有问题的质粒和抗生素选择。此外，对染色体重排的计算分析具有预测价值，这将扩大储存在数据库中的DNA序列的用途。

AbstractThis project focuses on a new bacterial system for studying chromosomal rearrangements involving gene duplication and further amplification. Gene amplification, which is a common process in all organisms, has significant consequences. It is essential to evolution, genetic diversity, and the ability of organisms to adapt to variable environments. Gene amplification also contributes to serious problems such as drug resistance, cancer, and microbial virulence. Despite its importance, many aspects of gene amplification remain poorly understood. Unlike other types of genetic change, amplification is dynamic and reversible. It may leave no evidence of its position or frequency. In this research project, an unusual characteristic of a soil bacterium, Acinetobacter baylyi ADP1, facilitates the systematic study of gene amplification. The critical characteristic is that A. baylyi naturally takes up DNA from the environment with exceptionally high efficiency and incorporates it into the genome via homologous recombination. This natural competence for DNA uptake permits the use of a transformation assay to detect the precise endpoints of duplicated chromosomal regions in mutants that arise spontaneously. The DNA sequence of such duplication sites provides information about the underlying genetic recombination event. This project builds on intriguing results from initial studies in which a new type of position specific illegitimate recombination (PSIR) process was discovered. The PSIR events lack DNA features that characterize typical site-specific recombination. One objective of this project is to determine the mechanism of PSIR, which appears to be novel. Additionally, a genome-wide approach will be used to characterize spontaneous duplications. Important features of DNA that contribute to gene duplication will be investigated such as DNA sequence and genomic context. This strategy will improve our understanding of a fundamental, common and important genetic process.Broader Impacts. Students will be trained in important multidisciplinary areas that bridge genetics, physiology, biochemistry, and computation. The project is accessible to students at all levels and will involve undergraduate and graduate students. Additionally, a postdoctoral researcher will conduct related investigations and help mentor the project participants. Ongoing programs at the University of Georgia will enhance the opportunity to train a diverse group of scientists. Such programs include an NSF-supported Research Experiences for Undergraduate (REU) site program in prokaryotic biology. The long-term impact of this research has the potential to offset harmful effects of gene amplification through a better understanding of the underlying mechanisms. Moreover, gene amplification can be developed for beneficial biotechnology applications. Ideally, chromosomal gene amplification could be used for desired manipulations to avoid the problematic use of plasmids and antibiotic selections in genetic engineering. Furthermore, computational analyses of chromosomal rearrangements have predictive value that will expand the utility of DNA sequences deposited in databases.