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Ph.D. Thesis

The abstract and chapters of my thesis are presented here. This thesis was submitted in October 1995, and successfully defended February 7, 1996. The internal examiner was Y. Nishioka (McGill Department of Biology), and the external examiner L. Malkus (National Institute of Health, Bethesda, MD). Excerpts available on request to torsten(nospam)@interchange.ubc.ca. Chapters 3 through 7 have been published; citations can be found on my .

Human Origins of DNA Replication

Identification, Analysis and Application

Torsten Nielsen

Department of Medicine
Division of Experimental Medicine
McGill University
Montreal, Canada

© Torsten Nielsen, 1995

Abstract

While replication origins, cis-acting sequences directing the initiation of DNA synthesis, have been well-characterized in many model organisms, the multiple sequence and protein components present at the chromosomal origins of higher eukaryotic organisms have not yet been fully defined. Genetic assays that identify origin function in cloned DNA fragments would provide a useful approach for the isolation and analysis of mammalian DNA replication origins.

In this thesis:

- Cloned fragments from a known mammalian origin, the ori-beta of the hamster 3' DHFR region, are demonstrated to replicate autonomously, both following transfection into human cells, and when used as templates in an in vitro replication system based on human cell extracts.
- Larger scale versions of these two assay methodologies are used to isolate over 40 novel putative origins of DNA replication from anticruciform purified human genomic DNA libraries.
- Transfection and in vitro autonomous replication assays are applied to demonstrate the potential origin function of a mitochondrial DNA sequence implicated in the insertional mutagenesis of a human genomic locus.
- An origin mapping strategy based on the in vitro assay is used to provide evidence for the existence of a replication origin in a cloned and sequenced portion of the human 15q11q13 chromosomal subdomain, a region associated with allele-specific replication timing, genomic imprinting, and genetic disease.
- Some of these autonomously replicating origins are cloned into a selectable YAC vector and are shown to permit the long term episomal maintenance, in human cells, of the transfected plasmid constructs.

These results consistently demonstrate that short mammalian genomic DNA fragments can replicate autonomously, supporting the applicability of the replicon model in humans, and could be extended to the search for an origin core consensus element, to the investigation of higher order organization and temporal control of human DNA replication origins, and to the construction of a complete human artificial chromosome.

Contents

CHAPTER 1
Introduction (includes overview, replication origins in model systems, origin structure in higher eukaryotes, chromosomal organization, and mapping replication origins)

CHAPTER 2
Thesis Goals

CHAPTER 3
Autonomous Replication In Vivo and In Vitro of Clones Spanning the Region of the DHFR Origin of Bidirectional Replication

CHAPTER 4
A Reproducible Method for Identification of Human Genomic Autonomously Replicating Sequences

CHAPTER 5
Autonomous Replication of a Human Mitochondrial DNA Sequence Inserted into Genomic DNA

CHAPTER 6
Identification of a Putative DNA Replication Origin in the GABA Receptor Subunit Gene Cluster on Chromosome 15q11q13, a Region Associated with Allele-Specific Replication Timing

CHAPTER 7
Circular YAC Vectors Containing Short Mammalian Origin Sequences are Maintained under Selection as HeLa Episomes

CHAPTER 8
General Discussion

torsten(nospam)@interchange.ubc.ca