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

自私的DNA元素，如质粒或病毒游离体，存在于广泛的生物体，利用策略，以确保其传输到下一代，没有好处或损害宿主生物体。芽殖酵母中存在的质粒利用的一种策略是将其自身附着到宿主染色体上，并在生长的细胞群中“搭便车”到子细胞上。由该项目支持的研究将绘制一个这样的酵母质粒附着的染色体位置，然后表征质粒和宿主因素，有助于搭便车。实验方法将利用目前的全基因组方法，将由博士后研究员、研究生、本科生和高中毕业生（作为暑期实习生）进行。这项研究的结果将被纳入4-5个讲座自私的DNA，将形成一个高年级本科课程和一年级研究生课程的一部分。一些实验将在UT奥斯汀的新生研究计划下进行。通过'UT奥斯汀社区外展'，来自少数民族和低社会经济地位的城市地区的高中毕业生也将参加监督研究计划。与UTeach外联研究所合作，将开发一个关于“基因和基因组的自私”的学习模块，旨在唤醒初中和高中学生的科学好奇心。总的来说，这项科学事业有望通过推进基础科学、开发新技术和为人类健康做出贡献而产生深远的社会影响。目前的证据表明，酵母2微米质粒的分配系统将通过复制形成的质粒姐妹篇束缚到姐妹染色单体中，使它们能够像染色体一样1：1分离。然而，这种搭便车的模式缺乏直接的证据。根据研究者的工作假设，质粒分配蛋白和宿主染色质结合蛋白之间的相互作用负责质粒定位到染色体位点。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来识别和表征染色体上的质粒连接位点。预期这两种方法富集的DNA序列之间会有相当大的重叠。通过使用多个对照对来自两种方法的数据进行精炼，并通过从一个对另一个的数据集进行过滤，研究人员将能够以高置信度定义染色体上的真实质粒连接位点。然后，他们将测试拴系效率和质粒稳定性如何受到涉及拴系的许多质粒和宿主蛋白质的突变失活的影响。这项研究将提供最严格的测试质粒分离的搭便车模型，并将影响自私的DNA元素之间的染色体偶联的生存策略的趋同进化模型，如酵母质粒和哺乳动物病毒。因此，它将为我们从机制上理解自私DNA元素的共同生活方式提供新的思路，这些自私DNA元素栖息在进化上遥远的宿主中。它还将帮助设计简单、有效和创新的策略，以保持真核宿主细胞中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知识价值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.