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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

System-Wide Studies of Gene Expression in Escherichia coli by Fluorescence Microscopy and High Throughput Sequencing

Chen, Huiyi 28 February 2014 (has links)
Gene expression is a fundamental process in the cell and is made up of two parts – the information flow from DNA to RNA, and from RNA to protein. Here, we examined specific sub-processes in Escherichia coli gene expression using newly available tools that permit genome-wide analysis. We begin our studies measuring mRNA and protein abundances in single cells by single-molecule fluorescence microscopy, and then focus our attention to studying RNA generation and degradation by high throughput sequencing. The details of the dynamics of gene expression can be observed from fluctuations in mRNA and protein copy numbers in a cell over time, or the variations in copy numbers in an isogenic cell population. We constructed a yellow fluorescent fusion protein library in E. coli and measured protein and mRNA abundances in single cells. At below ten proteins per cell, a simple model of gene expression is sufficient to explain the observed distributions. At higher expression levels, the distributions are dominated by extrinsic noise, which is the systematic heterogeneity between cells. Unlike proteins which can be stable over many hours, mRNA is made and degraded on the order of minutes in E. coli. To measure the dynamics of RNA generation and degradation, we developed a protocol using high throughput sequencing to measure steady-state RNA abundances, RNA polymerase elongation rates and RNA degradation rates simultaneously with high nucleotide-resolution genome-wide. Our data shows that RNA has similar lifetime at all positions throughout the length of the transcript. We also find that our polymerase elongation rates measured in vivo on a chromosome are generally slower than rates measured on plasmids by other groups. Studying nascent RNA will allow further understanding of RNA generation and degradation. To this end, we have developed a labeling protocol with a nucleoside analog that is compatible with high throughput sequencing.

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