Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

January 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Mark Johnston

Abstract

Organisms respond to their environment by altering patterns of gene expression. This process is orchestrated by transcription factors, which bind to specific DNA sequences near genes. In order to understand the regulatory networks that control transcription, the genomic targets of all transcription factors under various conditions and in different cell types must be identified. This remains a distant goal, mainly due to the lack of a high-throughput, in vivo method to study protein-DNA interactions. To fill this gap, I developed transposon "Calling Cards" for DNA-binding proteins. I endowed DNA binding proteins with the ability to direct the insertion of a transposon into the genome near to where they bind. The transposon becomes a "Calling Card" that marks the visit of a DNA-binding protein to the genome. I demonstrated that the Calling Card method is accurate and robust. I combined Calling Cards with "next generation" DNA sequencing technology to increase the sensitivity, specificity, and resolution of the method. This improved method: "Calling Card-Seq") allows for multiple transcription factors to be analyzed in a single experiment, greatly increasing sample throughput. I used Calling Card-Seq to study transcription factors of the yeast S. cerevisiae that have not been well-characterized, and I successfully identified DNA sequence recognition motifs and target genes for many of them. Calling Card-Seq will enable a systematic exploration of transcription factor binding under many different environments and growth conditions in a way that has heretofore not been possible. This dissertation describes my work developing this method, as well as several interesting results obtained using this method to study the gene regulatory networks of the yeast S. cerevisiae.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7MK69XS

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