The objective of this thesis is to research at the molecular level the mechanisms involved in the initiation of mammalian DNA replication. Human WI-38 cells were synchronized to the G1/S border by serum starvation and aphidicolin block. Cells were relased from arrest and followed as they progressed into S phase by microfluorometric analysis. Early replicating DNA was extruded from replication bubbles, purified from the high molecular weight parental DNA and cloned into the NruI site of pBR322. The recombinant plasmids were surveyed for properties previously associated with origins of replication. In a random sample of 10 human origin enriched sequences (hors) that were analyzed, 5 were capable of autonomous replication in a transient BrdU substitution assay. Two clones contained DNA fragments that migrate anomalously on acrylamide gels suggestive of bent DNA. One clone contained a weak DNA unwinding element as judged by sensitivity to the single strand specific enzyme mung bean nuclease. Primary sequence analyses of five of the hors clones (hors 98, 106, 112, 129 and 133) revealed that they were enriched for the AP2-A, NF-1 related and iron response consensus sequences. The replicating clones also contained potential cruciform structures within 50 bp of an A/T rich region. A DNA binding activity was identified in HeLa nuclear extracts that binds to a sub-fragment of one of the replicating clones (hors106) by bandshift assays and it was partially purified by DEAE and PC-11 column chromatography.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.70264 |
| Date | January 1992 |
| Creators | McAlear, Michael A. |
| 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: 001265131, proquestno: AAINN74553, Theses scanned by UMI/ProQuest. |
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