Mechanisms for Long-Term Coexistence of Selfish DNA Elements and Their Host Genomes: The Yeast Plasmid Paradigm

自私的DNA分子--如广泛存在于各种生物体中的质粒或病毒表体--利用策略确保它们传播给下一代，而不对宿主生物体有利或有害。芽殖酵母中的质粒所利用的一种策略是将自己附着在宿主染色体上，并搭便车到不断增长的细胞群体中的子细胞上。由该项目支持的研究将绘制出一个这样的酵母质粒连接的染色体位置，然后表征导致搭便车的质粒和宿主因素。实验方法将利用目前的全基因组方法，这些方法将由博士后研究员、研究生、本科生和高三学生(作为暑期实习生)执行。这项研究的发现将被纳入4-5节关于自私DNA的讲座，这将成为高年级本科生课程和一年级研究生课程的一部分。其中一些实验将在德克萨斯大学奥斯汀分校的新生研究计划下进行。通过“德克萨斯大学奥斯汀社区外展”，来自城市少数民族和社会经济地位较低地区的高三学生也将参与一个有监督的研究项目。与UTeach外联研究所合作，将开发一个学习模块，旨在唤起初中生对科学的好奇心，了解基因和基因组的自私性。总体而言，这项科学事业有望通过推进基础科学、开发新技术和促进人类健康而产生深远的社会影响。目前的证据表明，酵母2微米质粒的分割系统将通过复制形成的姐妹染色单体连接到姐妹染色单体，使它们能够像染色体一样1：1分离。然而，这种搭便车模式缺乏直接证据。根据研究人员的工作假说，质粒分离蛋白和宿主染色质结合蛋白之间的相互作用负责将质粒定位到染色体上。研究人员将通过两种独立但互补的高通量方法：CHIP-SEQ和4C-SEQ来鉴定和表征染色体上的质粒连接位点。预计这两种方法所富含的DNA序列之间将会有相当大的重叠。通过使用多个对照来提炼这两种方法的数据，并对其中一种方法的数据集进行筛选，研究人员将能够高度可信地确定染色体上的真实质粒系留位点。然后，他们将测试系留效率和质粒稳定性如何受到与系留有关的大量质粒和宿主蛋白突变失活的影响。这项研究将对搭便车模型进行最严格的检验，并将影响染色体偶联生存策略在自私DNA元件之间的收敛进化模型，这些自私DNA元件包括酵母、质粒体和哺乳动物病毒。因此，它将为对居住在进化遥远的宿主中的自私DNA元素的共同生活方式的机械论理解提供新的线索。它还将帮助设计简单、有效和创新的策略，在真核宿主细胞中保持有益的染色体外元素，并消除有害元素。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Selfish DNA elements—such as plasmids or virus episomes that are present in a wide range of organisms—utilize strategies to ensure their transmission to the next generation, without benefit or detriment to the host organism. One strategy utilized by a plasmid present in budding yeast is to attach itself to the host chromosomes and “hitchhike” to daughter cells within a growing cell population. Research supported by this project will map the chromosomal locations where one such yeast plasmid attaches, and then characterize the plasmid and host factors that contribute to hitchhiking. The experimental approaches will utilize current genome-wide methodologies that will be performed by post-doctoral fellows, graduate students, undergraduate students and high-school seniors (as summer trainees). The findings from this study will be incorporated into 4-5 lectures on Selfish DNA that will form part of an upper division undergraduate course and a first year graduate course. Some of the experiments will be carried out under the Freshmen Research Initiative Program at UT Austin. Through the ‘UT Austin Community Outreach,’ High School seniors from urban areas of minority and low socioeconomic status will also participate in a program of supervised research. In coordination with UTeach Outreach Institute, a learning module on ‘Selfishness of Genes and Genomes’ designed to awaken scientific curiosity among middle and high school students will be developed. Overall, this scientific undertaking is expected to have far-reaching societal impact by advancing basic science, developing new technologies and contributing to human health.Current evidence suggests that the partitioning system of the yeast 2-micron plasmid tethers plasmid sisters formed by replication to sister chromatids, enabling their chromosome-like 1:1 segregation. Yet direct proof of this hitchhiking model is lacking. According to the investigator's working hypothesis, interactions between plasmid partitioning proteins and host chromatin binding proteins are responsible for plasmid localization to chromosomal loci. The researchers will identify and characterize plasmid tethering sites on chromosomes by two independent but complementary high-throughput approaches: ChIP-seq and 4C-seq. The expectation is that there will be considerable overlap between DNA sequences enriched by the two methods. By refining the data from the two approaches using multiple controls, and by filtering the data sets from one against the other, the investigators will be able to define authentic plasmid tethering sites on chromosomes with high confidence. They will then test how tethering efficiency and plasmid stability are affected by mutational inactivation of a number of plasmid and host proteins that are implicated in tethering. This study will provide the most rigorous test of the hitchhiking model for plasmid segregation and will impact a model for the convergent evolution of chromosome-coupled survival strategies among selfish DNA elements as diverse as the yeast plasmid and mammalian viruses. It will thus shed new light on the mechanistic understanding of the shared lifestyles of selfish DNA elements that inhabit evolutionarily distant hosts. It will also help design simple, effective and innovative strategies for maintaining beneficial extra-chromosomal elements in eukaryotic host cells and for eliminating harmful elements.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.