[Identifying Strategies to Clone Viral Genes into Yeast Expression Vectors]

by

Acknowledgement

I would take this opportunity to thank my research supervisor, family and friends for their support and guidance without which this research would not have been possible (G, 2000,, 741).

DECLARATION

I, [type your full first names and surname here], declare that the contents of this dissertation/thesis represent my own unaided work, and that the dissertation/thesis has not previously been submitted for academic examination towards any qualification. Furthermore, it represents my own opinions and not necessarily those of the University (G, 2000,, 741).

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ABSTRACT

Saccharomyces cerevisiae Rep protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes. We have studied the substrate specificity of a recombinant Rep helicase (amino acid residues 400-1268 of the Rep protein). The term gene therapy describes any procedure intended to treat or alleviate disease by genetically modifying the cells of a patient. It encompasses many different strategies and the material transferred into patient cells may be genes, gene segments or oligonucleotides. The genetic material may be transferred directly into cells within a patient (in vivo gene therapy), or cells may be removed from the patient and the genetic material inserted into them in vitro, prior to transplanting the modified cells back into the patient (ex vivo gene therapy). Because the molecular basis of diseases can vary widely, some gene therapy strategies are particularly suited to certain types of disorder, and some to others. Retroviruses are distinct from other types of viruses due to their unique flow of genetic information. Once a retrovirus has gained entry into a host cell, the genetic material is converted via reverse transcription from RNA to DNA, a process that is essentially backwards from normal transcription in which a cell converts DNA into RNA. This "backwards" flow of genetic information is where retroviruses derive their name. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Rep protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Rep also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo.

Table of Contents

ABSTRACT4

CHAPTER I: INTRODUCTION6

Background6

Retroviruses7

Gag/pol env and LTR8

Gag/pol Expression in Yeast9

Example of Gene Cloning + Expression in Yeast9

The Value of Yeast to Express Large Amounts of Proteins10

Introduction10

Vectors for yeast and other fungi11

Gene delivery systems: nonviral vectors16

Yeast Expression- Saccharomyces Cerevisiae18

Cloning Gag Pol21

Adeno Associated Virus in Yeast Expression24

PUC19 PLASMID, TOPO TA cloning, Likers and PCR26

Problem Statement27

Scope of the Study28

Aims and Objectives29

Purpose of the Study29

CHAPTER II: LITERATURE REVIEW31

CHPATER III: METHODOLOGY35

CHAPTER IV: RESULTS & FINDINGS43

Comparison of Holliday junction unwinding by the recombinant Rep and RuvAB helicases51

DNase I footprinting of complexes between DNA and the recombinant Rep protein53

Length of DNA 3' overhang required for the binding and unwinding by the recombinant Rep protein57

Action of the recombinant Rep protein on ...