In mice, natural resistance to infection with murine cytomegalovirus (MCMV) is controlled by a dominant chromosome 6 locus, Cmv1, which is expressed at the level of Natural killer (NK) cells. To characterize the molecular mechanism underlying MCMV-resistance, our laboratory initiated a positional cloning approach for the identification of Cmv1, and localized the host resistance locus to a 0.35 cM genetic interval, corresponding to 1.6 Mb genomic DNA.
This dissertation presents the results leading to the cloning and characterization of the Cmv1 gene. First, I constructed a transcription map for the Cmv1 interval, which included 19 genes comprising the full Ly49 gene cluster and other novel candidate genes. In an attempt to narrow down the localization of Cmv1, I determined haplotypes in the vicinity of the Cmv1 interval in a panel of 17 inbred mice strains. This approach demonstrated the presence of three major classes of independent haplotypes. Close inspection of allele sharing among those haplotypes excluded Ly49e, Ly49f and Ly49d as Cmv1 candidates.
As an alternative approach to cloning Cmv1, I took advantage of a recombinant inbred strain, the BXD-8 strain. The BXD-8 line was of particular interest because it is susceptible to MCMV infection while harboring an MCMV-resistance C57BL/6 haplotype at Cmv1. Using STS content and PFGE analysis, I showed the presence of a 23 kb deletion in BXD-8. Subsequent gene expression analysis of the full Ly49 gene cluster revealed that the deletion of Ly49h is associated with MCMV susceptibility in BXD-8 mice.
Finally, to test the role of Ly49h in host resistance to MCMV infection, I introduced the Ly49h gene into a susceptible background. Investigation of Ly49h gene expression by RT-PCR and FRCS analysis demonstrated a strong correlation between the level of Ly49h mRNA and protein expression and the control of MCMV replication, providing conclusive evidence of the allelism between Cmv1 and Ly49h.
In conclusion, I identified the Ly49h gene encoding a NK activation receptor as Cmv1 supporting a specific role for NK cells in antiviral immunity. Transgenic mice expressing Ly49H will be instrumental for understanding of mechanism of NK mediated resistance and possible pleiotropic effects of Cmv1.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/29133 |
| Date | January 2004 |
| Creators | Lee, Seung-Hwan |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 202 p. |
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