Gene Amplification: Acinetobacter baylyi as a bacterial model system

摘要本项目的重点是一种新的细菌系统，用于研究染色体重排，包括基因复制和进一步扩增。基因扩增是所有生物体的共同过程，具有重大后果。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。基因扩增也会导致严重的问题，如耐药性、癌症和微生物毒力。尽管它的重要性，基因扩增的许多方面仍然知之甚少。与其他类型的遗传变化不同，扩增是动态和可逆的。它可能不会留下任何关于其位置或频率的证据。在本研究项目中，土壤细菌不动杆菌ADP 1的一个不寻常的特性，有利于基因扩增的系统研究。其关键特征是A. Baylyi以异常高的效率自然地从环境中摄取DNA，并通过同源重组将其整合到基因组中。这种DNA摄取的天然能力允许使用转化测定来检测自发产生的突变体中重复的染色体区域的精确终点。这些复制位点的DNA序列提供了关于潜在的遗传重组事件的信息。该项目建立在最初研究的有趣结果的基础上，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事件缺乏表征典型位点特异性重组的DNA特征。该项目的一个目标是确定PSIR的机制，这似乎是新的。此外，将使用全基因组方法来表征自发重复。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。这一战略将提高我们对一个基本的、共同的和重要的遗传过程的理解。学生将接受重要的多学科领域的培训，这些领域是遗传学，生理学，生物化学和计算的桥梁。该项目可供各级学生使用，并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。在格鲁吉亚大学正在进行的计划将提高培训科学家的多元化群体的机会。这些计划包括NSF支持的原核生物学本科生研究经验（REU）网站计划。这项研究的长期影响有可能通过更好地了解潜在机制来抵消基因扩增的有害影响。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，染色体基因扩增可用于所需的操作，以避免在基因工程中使用质粒和抗生素选择的问题。此外，染色体重排的计算分析具有预测价值，这将扩大存放在数据库中的DNA序列的效用。

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.