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Functional Analysis of the Mouse RBBP6 Gene Using Interference RNA

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Functional Analysis of the Mouse RBBP6 Gene Using Interference RNA FUNCTIONAL ANALYSIS OF THE MOUSE RBBP6 GENE USING INTERFERENCE RNA Ashley Pretorius A thesis submitted in fulfilment of the requirement for the degree of Doctor Philosophiae in the Faculty of Science, University of the Western Cape. Supervisor: Prof D. Jasper G. Rees November 2007 ABSTRACT Functional analysis of the mouse RBBP6 gene using interference RNA A. Pretorius PhD thesis, Department of Biotechnology, Faculty of Science, University of the Western Cape A novel hamster gene homologous to the human cDNA clone 21C4 was identified in a genetic screen to identify new genes involved in the MHC class I antigen processing and presentation pathway. The identified gene however showed no significant matches to sequences in the Genbank Database and was subsequently named the Domain Without No name (DWNN). The corresponding human gene was found to be located on chromosome 16p12.2, upstream of the previously identified RBBP6/PACT/P2P-R gene. Analysis of cDNA sequences showed that the sequence coded for the previously unidentified N-terminus of the RBBP6 protein (Dlamini et al, in prep), which was named the DWNN domain. RBBP6 is one of the few proteins identified that has been shown to interact with both p53 and Rb, suggesting a possible model for the integration of the regulation of transcription, cell cycle control and apoptosis. Over-expression of the mouse homolog P2P-R has been shown to lead to cell cycle arrest and apoptosis. The conserved mechanisms of I programmed cell death play a fundamentally important role in tissue homeostasis, embryogenesis and cellular defense and had only recently been subjected to molecular analysis. The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene. Genetic analysis showed the presence of two promoters for RBBP6 namely Promoter 0 (P0) and Promoter 1 (P1) both being responsible for the different RBBP6 transcripts. The Enhanced Green Fluorescent Protein (EGFP) and the Red Fluorescent Protein (DsRed1) were both placed under the transcriptional control of P0 and P1. Promoter activities were measured using FACS and Real-Time qRT-PCR. The results showed P0 to have a higher level of transcriptional activity than P1 before and after camptothecin-induced apoptosis. RNAi was used to target the expression of the RBBP6 gene. Several small interference RNA (siRNA) constructs were designed that would result in the expression of two distinct siRNA oligonucleotides designated DWNN-A and DWNN-B targeting different regions of the gene. Two stable siRNA-expressing cell lines were established, namely RU6A and GU6B expressing DWNN-A and DWNN-B respectively. Fluorescence microscopy and Real-Time qRT-PCR analysis were used to evaluate the effect on RBBP6 expression following (i) transient transfections of the siRNA constructs into the parental cell line NIH 3T3 cell line (ii) as well as expression in the stable cell lines RU6A II and GU6B. The silencing effect of the DWNN -A oligonucleotide appeared to be more potent than that exerted by DWNN-B. Both stable lines were used in assays to determine the effect of RBBP6 silencing on apoptosis. The stable cell lines proved to be significantly more resistant to apoptosis induced by camptothecin compared to the parental NIH 3T3 cell line. Furthermore complementing the stable cell lines with the full-length DWNN-200 cDNA, restored sensitivity to camptothecin and the degree of cell death observed in the parental cell line. In addition, over-expression of the RBBP6 protein showed an increase in the apoptotic population following camptothecin-induced apoptosis. Apoptosis mediated through RBBP6 was shown to be dependent on p53 expression and possibly follows an intrinsic pathway. Finally, the siRNA stable expressing cell lines RU6A and GU6B were also shown to be restricted in the G1 phase of the cell cycle implicating the RBBP6 gene in cell cycle progression. From the study the RBBP6 Promoters showed an increase in transcriptional activity following the induction of apoptosis. Furthermore the involvement of the RBBP6 was implicated in the processes of apoptosis and the cell cycle although the exact mechanisms have not been fully elucidated. Keywords: Apoptosis Real-Time qRT-PCR III Cell cycle Interference RNA (RNAi) Homologous recombination Promoter p53 Retinoblastoma PACT RBBP6 IV DECLARATION. I declare that “Functional analysis of the mouse RBBP6 gene using Interference RNA” is my own work that has not been submitted for any degree or examination in any other university and that all the sources I have used or quoted have been indicated and acknowledged by complete references. Ashley Pretorius November 2007 Signed:……………………………. V TABLE OF CONTENTS ABSTRACT...........................................................................................................................................................I DECLARATION. ................................................................ ............................................................................... V TABLE OF CONTENTS..................................................................................................................................VI LIST OF TABLES.............................................................................................................................................. X LIST OF FIGURES...........................................................................................................................................XI ABBREVIATIONS......................................................................................................................................XVIII ACKNOWLEDGEMENTS ..........................................................................................................................XXI CHAPTER 1: LITERATURE REVIEW......................................................................................................... 1 1.1 INTRODUCTION........................................................................................................................................ 1 1.2 THE IMMUNE SYSTEM.............................................................................................................................3 1.2.1 Classes of T lymphocytes ..............................................................................................................4 1.2.2 MHC class 1 presentation pathway...............................................................................................7 1.3 CTL KILLING .........................................................................................................................................10 1.3.1 Granule mediated exocytic killing ..............................................................................................11 1.3.2 Receptor mediated CTL killing (see figure 1.2).........................................................................13 1.3.3 The role of Caspases in CTL killing ...........................................................................................14 1.4 APOPTOSIS.............................................................................................................................................16 1.4.1 Introduction................................................................................................................................... 16 1.4.2 Characteristics of Apoptosis........................................................................................................17 1.4.3 Components of the Apoptotic pathway.......................................................................................19 1.4.4 The Apoptotic pathways ..............................................................................................................22 1.4.5 Regulation of the Apoptotic Proteins..............................................................................................29 1.5 THE P53 GENE........................................................................................................................................30 1.5.1 Functions of the p53 gene................................................................................................................31 1.5.2 p53-activating pathways ..............................................................................................................33 1.5.3 Control of p53...............................................................................................................................34 1.6 RETINOBLASTOMA (RB) GENE..................................................................................................................36 1.6.1 RB function in the cell cycle .......................................................................................................37 1.6.2 Function of Rb in apoptosis.........................................................................................................39 1.7 THE DWNN GENE FAMILY ..................................................................................................................40 1.7.1 Identification of the DWNN domain..........................................................................................40 1.7.2 Structure of DWNN ....................................................................................................................43 1.7.3 DWNN/RBBP6 homologues.......................................................................................................45
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