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Genomic and Functional Analysis of Mouse Ly49 Natural Killer Cell Receptor Genes

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Genomic and Functional Analysis of Mouse Ly49 Natural Killer Cell Receptor Genes GENOMIC AND FUNCTIONAL ANALYSIS OF MOUSE LY49 NATURAL KILLER CELL RECEPTOR GENES by KARINA LEE McQUEEN B.Sc, The University of Guelph, 1995 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Medical Genetics; Genetics: Graduate Program) We accept this thesis as ccjiibH»ipg to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 2001 © Karina Lee McQueen, 2.00\ In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of meci^QaX Gyg.ng.'KcS. The University of British Columbia Vancouver, Canada Date YAOLCCK Ql-, QLQOI Abstract 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 /. 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 czs-acting elements involved in gene regulation. Luciferase reporter assays in EL-4 cells indicate that the 5' regions o£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. Ill Table of Contents GENOMIC AND FUNCTIONAL ANALYSIS OF MOUSE Ly49 NATURAL KILLER CELL RECEPTOR GENES i Abstract ii Table of Contents iii List of Figures vii List of Tables ix Acknowledgements x List of Abbreviations xi Chapter 1 Introduction 1 1.1 Natural Killer Cells 2 1.1.1 Overview 2 1.1.2 Ontogeny 2 1.1.3 Roles of NK Cells in the Body 4 1.1.4 Mechanism of Cytotoxicity 6 1.2 Missing Self Hypothesis 7 1.2.1 A Historical Perspective 7 1.3 Major Histocompatibility Complex (MHC) 11 1.3.1 Roles and Structure 11 1.3.2 Genomic Organization 13 1.4 NK cell Receptor Families 16 iv 1.4.1 Overview 16 1.4.2 Ig Superfamily Members 19 1.4.3 C-type Lectin-like domain (CTLD) Superfamily 23 1.5 Mouse Ly49 Receptors 35 1.5.1 Overview 35 1.5.2 Functional role of Ly49 35 1.5.3 Polymorphisms and Evolution 38 1.5.4 Genomic Organization 40 1.5.5 Regulation of the Ly49 gene family 40 1.6 Thesis Objectives and Organization 49 Chapter 2 Localization of five new Ly49 genes, including three closely related to Ly49c 51 2.1 Introduction 52 2.2 Materials and Methods 53 2.2.1 PI Bacteriophage Library Screening and Isolation of PI DNA 53 2.2.2 Probes and hybridizations 53 2.2.3 PI clone Restriction Enzyme Mapping 54 2.2.4 Exon PCR, Subcloning, and Sequencing 56 2.3 Results 57 2.3.1 Complexity of Ly49c-related genes in C57BL/6 mice 57 2.3.2 Isolation and mapping of PI clone DNA 59 2.3.3 Gene Localization 61 2.3.4 Sequence Analysis 63 2.4 Discussion 69 Chapter 3 Expression analysis of the new Ly49c-related genes 75 V 3.1 Introduction 76 3.2 Materials and Methods 77 3.2.1 Preparation of NK cell cDNA 77 3.2.2 PCR reactions 77 3.2.3 Sequencing 78 3.2.4 Probes and hybridizations 79 3.2.5 Ly49J expression cloning constructs 79 3.2.6 Antibodies 79 3.2.7 Cell lines 80 3.2.8 Transfections and flow cytometry 80 3.2.9 Cell adhesion assay 80 3.3 Results and Discussion 82 3.3.1 Analysis of Ly49j, k and n cDNAs 82 3.3.2 Transcripts Lacking the Transmembrane Domain 85 3.3.3 Protein Expression of Ly49J 90 3.3.4 Alternative Splicing of C-type Lectin Family Members 90 Chapter 4 Functional analysis of 5' and 3' regions of the closely related Ly49c and j genes 95 4.1 Introduction 96 4.2 Materials and Methods 97 4.2.1 Cell lines 97 4.2.2 Sequencing of putative regulatory regions 97 4.2.3 Generation of luciferase reporter gene constructs 97 4.2.4 Determining intron/exon boundaries for Ly49c 98 4.2.5 Luciferase assays 100 4.2.6 RT-PCR analysis 100 4.2.7 Probes and hybridization 101 4.2.8 3' Race for Ly49j 101 vi 4.2.9 Generation of GFP reporter gene constructs 102 4.2.10 Transfections and Flow Cytometry 103 4.3 Results and Discussion 104 4.3.1 Intron/exon boundaries of Ly49c 104 4.3.2 Sequence and comparison of the 5' region ofLy49c and j 104 4.3.3 Promoter activity of the 5'region 107 4.3.4 Sequence comparison of a putative promoter region in intron 1 ofLy49a, candy 109 4.3.5 Intron 1 promoter analysis 110 4.3.6 Analysis of transcripts from the intron 1 promoter 113 4.3.7 Different 3' untranslated regions in Ly49c and j 115 Chapter 5 Summary 121 Bibliography 123 Vll List of Figures Figure 1-1 The missing self hypothesis 8 Figure 1-2 Schematic representation of the MHC class I molecule 12 Figure 1 -3 Schematic map of the human and mouse MHC 14 Figure 1-4 Schematic representation of KIR (Ig-SF) and Ly49 (CTLD-SF) inhibitory and activating receptors 17 Figure 1-5 Schematic map of the leukocyte receptor complex (LRC) on human chromosome 19ql3.1-ql3.3 20 Figure 1-6 Schematic map of the natural killer cell gene complex (NKC) in human and mouse 27 Figure 1-7 Ontogeny of expression of Ly49 receptors by splenic NK cells 42 Figure 1-8 Models of differential Ly49 expression 48 Figure 2-1 Southern blot analysis of genomic DNA derived from inbred mouse strains..58 Figure 2-2 Southern blot analysis of eight PI clones, digested with EcoRI or Hindlll, and hybridized to the Ly49c-related probe under stringent conditions 60 Figure 2-3 A physical map showing the relative location of some of the known and potentially new Ly49 genes in the B6 genome 62 Figure 2-4 Comparison of exon 2 sequences 64 Figure 2-5 Comparison of exon 4 sequences 67 Figure 2-6 Comparison of exon 7 sequences 68 Figure 2-7 The genomic organization of the Ly49 gene cluster in the B6 mouse 71 Figure 3-1 Amino acid sequence of the full-length Ly49J protein, compared to the closely related Ly49C and I proteins 84 Figure 3-2 Products from RT-PCR performed on total NK cell RNA using Ly49c, i- and y'-specific primers 86 Figure 3-3 Nucleotide sequence of the exon 3 splice donor (SD) and exon 3 and 4 splice acceptor (SA) sites for Ly49c, i and j 89 Figure 3-4 COS cell expression of the Ly49j cDNA 91 Figure 4-1 Genomic organization of the Ly49a and c genes 105 Vlll Figure 4-2 Comparison of 5' sequence upstream (and including) exon 1 from Ly49j, c and a 106 Figure 4-3 Promoter activity of the 5' region measured by the firefly luciferase assay system 108 Figure 4-4 Comparison of partial intron 1 sequence from Ly49j, c and a Ill Figure 4-5 The promoter activity of intron 1 112 Figure 4-6 Semi-quantitative RT-PCR on approximately 5, 10, 50 and 100 ng of NK cell cDNA to identify transcripts produced from the putative intron 1 promoter in Ly49j 114 Figure 4-7 The 3' region of Ly49c and j 116 Figure 4-8 Expression of GFP constructs containing the 3' UTR of either Ly49c orj .. 118 ix List of Tables Table 1-1 NK cell receptors belonging to the immunoglobulin superfamily (Ig-SF) 24 Table 1-2 NK cell receptors of the C-type lectin (CTLD) superfamily 33 Table 1-3 MHC class I binding specificities of the known Ly49 receptors 37 Table 1-4 Nucleotide identity between known Ly49 gene coding regions in B6 mice....39 Table 2-1 Nucleotide sequences of Ly49 gene-specific oligonucleotide probes 55 Table 2-2 The presence or absence of an ITIM in the known and new Ly49 molecules .73 Table 3-1 Alternatively spliced products observed for the Ly49g, h,j, k and n genes 83 Table 3-2 Estimate of the frequency of full-length versus transmembrane-less transcripts of the Ly49c, i and j genes 88 Table 3-3 Members of the CTLD-SF for which exon-skipping creates both membrane- bound and putative cytoplasmic protein isoforms 94 Table 4-1 Primers used to amplify the putative regulatory regions of Ly49a, c and j 99 Acknowledgements I would like to begin by thanking my whole family for their love, support and encouragement over the last few years.
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