Joseph, Maxim B. (2012) Development of methods for combinational approaches to cis-regulatory module interactions. PhD thesis, University of Warwick.

Preview

Text WRAP_THESIS_Joseph_2012.pdf - Submitted Version Download (14Mb) | Preview

Request Changes to record.

Abstract

The complexity and size of the higher animal genome and relative scarcity of DNA-binding
factors with which to regulate it imply a complex and pleiotropic regulatory system. Cisregulatory
modules (CRMs) are vitally important regulators of gene expression in higher
animal cells, integrating external and internal information to determine an appropriate
response in terms of gene expression by means of direct and indirect interactions with the
transcriptional machinery. The interaction space available within systems of multiple CRMs,
each containing several sites where one or more factors could be bound is huge. Current
methods of investigation involve the removal of individual sites or factors and measuring
the resulting effect on gene expression. The effects of investigations of this type may be
masked by the functional redundancy present in some of these regulatory systems as a
result of their evolutionary development. The investigation of CRM function is limited by a
lack of technology to generate and analyse combinatorial mutation libraries of CRMs,
where putative transcription factor binding sites are mutated in various combinations to
achieve a holistic view of how the factors binding to those sites cooperate to bring about
CRM function. The principle work of this thesis is the generation of such a library.
This thesis presents the development of microstereolithography as a method for
making microfluidic devices, both directly and indirectly. A microfluidic device was
fabricated that was used to generate oligonucleotide mixtures necessary to synthesise
combinatorial mutants of a CRM sequence from the muscle regulatory factor MyoD. In
addition, this thesis presents the development of the optimisation algorithms and assembly
processes necessary for successful sequence assembly. Furthermore, it was found that the
CRM, in combination with other CRMs, is able to synergistically regulate gene expression in
a position and orientation independent manner in three separate contexts. Finally, by
testing a small portion of the available combinatorial mutant library it was shown that
mutation of individual binding sites within of the CRM is not sufficient to show a significant
change in the level of reporter gene expression.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QD Chemistry Q Science > QH Natural history > QH426 Genetics
Library of Congress Subject Headings (LCSH):	Gene regulatory networks, Gene expression, Combinatorial chemistry, Microfluidic devices
Official Date:	March 2012
Dates:	Date Event March 2012 Submitted
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Covington, James A., 1973- ; Koentges, Georgy
Extent:	xxx, 360 leaves : illustrations.
Language:	eng

Request changes or add full text files to a record

Repository staff actions (login required)

View Item

Downloads