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Genomic and functional analysis of mouse Ly49 natural killer cell receptor genes McQueen, Karina Lee


The overall objective of my research has been to better understand the genomic complexity of the mouse Ly49 multigene family, which encodes receptors for MHC class I molecules on natural killer (NK) cells. When I began my study, nine Ly49 genes, Ly49a-i, had been identified in the C57BL/6 (B6) mouse, though Southern blot analysis suggested the existence of additional Ly49 genes. To characterize and localize new Ly49 genes, we isolated and mapped PI genomic clones hybridizing to an Ly49c-related probe. The relative order of all Ly49 genes within the clones was determined, and five new Ly49 genes, Ly49j-n, were identified. Three of these new genes, Ly49j, k and n, belonged to the Ly49c-related subset of the Ly49 family. To determine whether the Ly49j, k and n genes were transcribed, RT-PCR was performed using gene-specific primers. A full length cDNA for Ly49j was detected and shares 96% nucleotide identity with Ly49c and i. Many different sized Ly49k and n transcripts were observed, although they likely do not encode functional proteins due to severe truncations in the open reading frame. Interestingly, the most abundant Ly49j transcript detected lacked the transmembrane domain, yet maintained the reading frame. Further studies revealed the presence of Ly49i transmembrane-less transcripts, although at a much lower frequency than observed for Ly49j. Finally, we examined the 5' and 3' regions of the closely related Ly49c and j genes, to determine if they contained cis-acting elements involved in gene regulation. Luciferase reporter assays in EL-4 cells indicate that the 5' regions of Ly49c and j contain promoter elements and repressor sequences, and that Ly49j contains an active promoter in the first intron. Finally, comparisons of the 3' non-coding regions of Ly49c and j revealed that the sequence of Ly49j diverges completely from Ly49c downstream of the termination codon, resulting in a longer 3' untranslated region (UTR). When the Ly49j 3' UTR was used to provide the polyadenylation signal for the GFP reporter gene, expression of GFP was reduced two-fold. These results suggest that both internal promoters and 3' regions play a role in regulating Ly49 gene expression.

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