Spontaneous mutations occur in all genomes as the result of normal cellular mechanisms and interactions with the environment. In addition to their important contribution to genetic diversity and evolution, spontaneous mutations are associated with several genetic disorders and certain cancers. Spontaneous mutational events comprise an impressive heterogeneity of DNA alterations which can be categorized into two major classes: "moderate" and "potent" mutations. Spontaneous moderate mutations are defined here as comprising mutations with a subtle (i.e. leaky, reversible) effect on the encoded phenotype of a gene. In contrast, potent mutations can severely and permanently alter a gene's phenotype and may result in more important DNA alterations. The selective enrichment for the isolation and characterization of potent mutations can be hindered by the frequent occurrence of more moderate mutations. / A selection procedure was thus developed to enrich for spontaneous potent mutations in the KT cell line, comprising a chromosomally-integrated, single-copy plasmid pSV2neoKT, which contains the $neo sp{ rm R}$ and the HSV-1 thymidine kinase (tk) genes. Spontaneous $tk sp-$ mutants were selected on the basis of their resistance to nucleoside analogues (acyclovir (ACV), trifluorothymidine (TFT), and ganciclovir (DHPG)). In order to enrich for potent mutations bearing severe consequences for the function of the tk gene, over the "background" of more simple moderate mutations, an enrichment procedure was established and verified. Combinations of nucleoside analogues reduced the apparent mutation frequency in the tk gene and allowed the enrichment for potent mutations with a severe impact on the TK phenotype. The different potencies of the compounds and their potential synergisms during the selection for $tk sp-$ mutants is also discussed. / Mutants resistant to ACV + TFT + DHPG demonstrated a stable TK$ sp-$ phenotype, as they did not revert to wild type at a detectable frequency, and the severe effect on gene function was exemplified by the absence of a TK protein, discernible by Western blotting, in $>$85% of these multiple drug resistant mutants. In contrast, the moderate $tk sp-$ mutants resistant to TFT only, demonstrated phenotype reversal at a high frequency and a detectable TK polypeptide. Between 14.5% and 25% of the potent $tk sp-$ mutants resistant to combinations of drugs underwent a major DNA alteration, consisting mostly of partial, or complete, deletions of the tk gene. Complex rearrangements of the tk gene were also obtained, and an intragenic deletion/duplication event is presented and characterized. However, it was observed that more than 75% of the spontaneous potent mutational events implicated less than 5 bp of the tk gene, yet presented a null phenotype of stable complete alteration of the TK phenotype. The level of mutated tk transcripts was affected in 64% to 70% of the small potent $tk sp-$ mutants, as demonstrated by Northern blotting. Extensive DNA methylation and multidrug resistance (MDR) seemed not to be implicated in the generation of the TK$ sp-$ phenotype observed in the small potent $tk sp-$ mutants. / Thus, the KT cell line, combined with the use of multiple nucleoside analogues, allows the enrichment for, and the characterization of, potent (and infrequent) spontaneous mutations in a human chromosomal context. The mutational events characterized in this study may then reflect the diversity, the complexity, and the dynamics of the human genome.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.41558 |
| Date | January 1994 |
| Creators | Brisebois, Josée J. |
| 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: 001397310, proquestno: NN94597, Theses scanned by UMI/ProQuest. |
Page generated in 0.0024 seconds