The nature of origins of DNA replication in mammalians has yet to be elucidated. The localization of the initiation sites and the identification of the corresponding sequences and/or structures is important to understand the process of initiation at the molecular level. The objective of the research in this thesis is to localize the initiation site of DNA synthesis at a specific locus on the chromosomes of mammalian cells, and to characterize the minimal sequence requirements for the function of this origin of DNA replication in vivo. A genomic clone was isolated using ors12, a mammalian autonomously replicating sequence (812 bp) that was previously isolated by extrusion of African Green monkey (CV-1 cells) nascent DNA from active replication bubbles. Ors12 associates with the nuclear matrix in a cell cycle dependent manner, and is present at the centromeric region of six CV-1 cell chromosomes and that of a marker chromosome. This genomic clone was used to generate PCR primers in a region encompassing ors12, in order to amplify nascent DNA strands isolated from asynchronously growing CV-1 and African Green monkey primary kidney cells. A competitive PCR-based mapping methodology was used, and revealed that DNA replication initiates preferentially in vivo in a region colocalizing with ors12, providing evidence of its function as a bona fide chromosomal origin of DNA replication. / In an attempt to define the sequence or structural elements that are important for mammalian origin function, a panel of deletion mutants of ors12 (812-bp) was generated. The deletion mutants were tested for their replication activity in vivo by the bromodeoxyuridine substitution assay, after transfection into HeLa cells, and in vitro by the DpnI resistance assay, using extracts from HeLa cells. A 215-bp internal fragment was identified as essential for the autonomous replication activity of ors12. When subcloned into the vector pML2 and similarly tested, this subfragment was capable of autonomous replication in vivo and in vitro. Several repeated sequence motifs are present in this 215-bp fragment, such as TGGG(A) and G(A)AG (repeated four times each); TTTC, AGG, and MA (repeated 3 times each); the motifs CACACA and CTCTCT, and two imperfect inverted repeats, 22-bp and 16-bp long, respectively.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.35043 |
| Date | January 1997 |
| Creators | Pelletier, Richard. |
| Contributors | Zannis-Hadjopoulos, Maria (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001601760, proquestno: NQ44551, Theses scanned by UMI/ProQuest. |
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