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
publications
list.
Human Origins of DNA Replication
Identification, Analysis and Application
Torsten Nielsen
Department of Medicine
Division of Experimental Medicine
McGill University
Montreal, Canada
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