Eukaryotic genomes possess significant amounts of highly tandemly-arrayed repetitive DNA sequences in their constitutive heterochromatin. Heterochromatin has been involved in processes, such as position-effect variegation (PEV), that disrupt normal expression of euchromatic genes. The role of highly repetitive DNA sequences, also termed satellite DNAs, in gene silencing has therefore aroused a great deal of interest. Previous plasmid transfection studies into tk-/neo- human cells had shown that the location (in cis) of a 1.797 kb human satellite II DNA sequence next to the 5' end of the neo gene, exerted a severe negative effect on the recovery of stable tk+/neo + transfectants, in the presence of HAT and G418 drugs. As shown in this thesis, positioning of the same satellite II DNA sequence at another location, between the 3' ends of the tk and neo genes, led to the loss of this negative effect. To identify a possible size limit to the silencing effect exerted by the satellite II DNA, fragments of 813 and 983 by of the 1.797 kb DNA sequence were also inserted near the 5' end of the neo gene. These sequences were still able to exert a drastic negative effect on the recovery of stable tk+/neo + transfectants. The effect was found to be dependent on the orientation of the satellite II DNA fragments relative to the marker genes. Further reduction in the size of the satellite II DNA sequence, to fragments of 354 to 620 bp, revealed a significant reduction in the negative effect. Selection of the transfected cells in media containing single drugs (i.e. HAT or G418) showed that the neo gene in the plasmids was more affected than the tk gene. Analysis of the integration pattern of the plasmid vector and a plasmid containing the 1.797 kb satellite II DNA indicated that the satellite II DNA did not influence the plasmid site of integration.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.36699 |
| Date | January 1999 |
| Creators | Saint-Dic, Djenann. |
| Contributors | DuBow, Michael S. (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 (Department of Microbiology and Immunology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001754124, proquestno: NQ64659, Theses scanned by UMI/ProQuest. |
Page generated in 0.0018 seconds