Studies on the evolution, structure, and function of homing endonuclease containing parasitic genetic elements

Date of Completion

January 2007

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

Inteins and introns are intervening elements that interrupt numerous essential genes and proteins in organisms belonging to all major domains of life, including viral and phage genes. Self splicing introns and inteins are often associated with homing endonuclease and generally do not appear to provide any benefit for their host. For these reasons, they and can be considered as parasitic genetic elements. ^ Mapping insertion sites of inteins and introns that encode homing endonucleases shows them to be predominantly inserted at highly conserved sites of their host proteins. Possible scenarios for the attraction of inteins and introns to conserved sites are discussed. ^ This thesis reports on the engineering of an intein that contains an internal Green Fluorescence Protein domain, while retaining splicing and endonuclease activity. The insertion of a marker gene into an intein allows monitoring of protein expression without changing the functionality of the intein's host protein, thereby promising to make intein-based protein purification and expression monitoring systems very attractive techniques in medical research, biotechnology, and molecular biology laboratories. ^ Using M13 phage and plasmids as vectors, a selection system is designed in Escherichia coli that utilizes the yeast V-ATPase (vma1) homing endonuclease. The system is designed to provide a selective advantage to those vectors that express a homing endonuclease that successfully cleaves a target site. The phage based in vivo selection system which is described in this thesis represents a step towards a new system of experimental evolution. Evolving homing endonucleases with altered recognition and cleavage specifications will prove useful for gene therapy and novel analytical applications. ^

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