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Somatic Hypermutation and Germline Evolution of Immunoglobulin Variable Genes Harald Sebastian Rothenfluh University of Wollongong

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Somatic Hypermutation and Germline Evolution of Immunoglobulin Variable Genes Harald Sebastian Rothenfluh University of Wollongong University of Wollongong Research Online University of Wollongong Thesis Collection University of Wollongong Thesis Collections 1994 Somatic hypermutation and germline evolution of immunoglobulin variable genes Harald Sebastian Rothenfluh University of Wollongong Recommended Citation Rothenfluh, Harald Sebastian, Somatic hypermutation and germline evolution of immunoglobulin variable genes, Doctor of Philosophy thesis, Department of Biological Sciences, University of Wollongong, 1994. http://ro.uow.edu.au/theses/1053 Research Online is the open access institutional repository for the University of Wollongong. For further information contact Manager Repository Services: [email protected]. Somatic hypermutation and germline evolution of immunoglobulin variable genes A thesis submitted in fulfilment of the requirements for the award of the degree Doctor of Philosphy from [UNIVERSITY OF WOLLONGONG LIBRARY. The University of Wollongong by Harald Sebastian Rothenfluh, B.Sc.(UNSW) Grad. Dip. Ed.(SCAE) B.Sc.Hons.(UW) Department of Biological Sciences 1994 Declaration This thesis is submitted in accordance with the regulations of the University of Wollongong in partial fulfilment of the requirements for the award of a Doctor of Philosophy. It does not incorporate any material previously published or written by another person except where due reference is made in the text The experimental work described in this thesis is original work and has not been previously submitted for a degree or diploma in any university. H. Rothenfluh July, 1994 i Abstract The work presented in this thesis was aimed at further defining the mechanism of somatic hypermutation and analysing in detail the patterns of sequence variability observed in germline heavy chain variable gene segments (VH) of the mouse. A number of mechanisms have been invoked to explain somatic hypermutation of immunoglobulin variable (IgV) region genes. Some of these mechanisms predict the presence of multiple and differentially mutated DNA or RNA copies of the rearranged IgV region. In order to detect these, the RNA and DNA was isolated from the same pool of antigen specific B cells that were isolated from the spleen of hyperimmunized mice. Although insufficient sequences were collected to allow identification of DNA and RNA from the same cell, it was found that identical N regions and VH-D or D-JH joins were present in B cells that were not clonally related. This suggests that certain CDR3 sequences that are not encoded in the germline may be selected during B cell ontogeny and/or the germinal center reaction. A major hurdle in elucidating the mechanism of somatic hypermutation is the fact that it has not yet been reproduced in vitro. In a preliminary experiment it was attempted to induce antigen-specific splenic B cells, both singly and in groups of 10, to undergo somatic hypermutation during in vitro culture. The cells were isolated using flow cytometry, and cultured according to a recently developed B cell activation system method which utilizes the membranes of activated T cells. Some B cells were successfully induced to secrete Ig and/or proliferate, however none of the proliferating cells that were analyzed displayed evidence of having accumulated mutations during in vitro culture. Previous reports have failed to determine the precise 5' boundary for somatic hypermutation in rearranged IgVpj regions. In order to allow a more accurate definition of where this boundary lies, the 5' flanking region sequences of a number of previously characterised B cell hybridomas were determined. These sequences were added to all previously published sequences. This data set indicates that almost 97 % of somatic mutations were found downstream of the transcription start site (cap) site, and that the mutation frequency distribution around IgVn regions is asymmetrical, with a single mode centered on the rearranged VH region and a long tail extending into the J - C intron. Two classes of model are consistent with the new data: those where transcription products are the direct mutational substrates, and those where the mutational machinery operates directly on the DNA. Although the polymerase chain reaction (PCR) has revolutionized the study of genetic information, a number of in vitro artifacts can result from the use of this technique: Nucleotide misincorporations and the production of hybrid DNA molecules. The fidelity of the Taq and Pfu DNA polymerases was assessed by sequencing multiple clones of PCR amplified DNA fragments. In this way it was demonstrated that Pfu DNA ii polymerase has a 12-fold lower error-rate than Taq DNA polymerase. A number of experiments involving the restriction analysis of PCR products that were amplified from mixtures of well characterized cloned DNA revealed that under the PCR conditions used in the work carried out for this thesis, hybrid DNA molecules are produced below detectable limits. Thus the DNA sequences presented in this thesis are free of significant levels of in vitro generated artifacts. A number of laboratories previously reported the presence of hypervariable regions corresponding to the complementarity determining regions (CDR) in germline IgV genes. However, the murine germline VH gene sequences presented in this thesis also include significant amounts of non-transcribed 5' flanking region sequence. Nucleotide and amino acid variability plots clearly illustrate the similarity between the germline VH genes and their somatically rearranged and mutated counterparts. Statistical analysis indicates that the sequence patterns are significantly different from those expected under a random point mutator model, and that there is a significant deficit of stop codons generated by nucleotide substitutions. Phylogenetic analysis revealed that the putative transcription/coding units evolved differently and more rapidly than the non-transcribed 5' flanking regions, suggesting that hyper-recombination events targeted to the putative transcription/coding regions contributed to the evolution of germline VH genes. A number of evolutionary models that have been proposed to account for the evolution of the IgV multigene family will be evaluated on the basis of how well they can explain the new data. iii Acknowledgments The time spent gathering and interpreting the data presented in this thesis was a very rewarding experience. This is in no small way due to the enthusiastic involvement of my supervisor, Associate Professor Ted Steele, at all stages of the work. I would also like to thank Ted for all of his support, and for giving me the opportunity to experience the different facets of science and the scientific process. I am also very grateful to Dr Phil Hodgkin and Alusha Mamchak, who made me welcome in their laboratory and who were very patient with me throughout the work involving flow cytometry and tissue culture. Many thanks also to Drs. Gerry Both, Linda Taylor and Stefan Eick, who were involved in many critical discussions during the early phases of this work, and to Professor Adrian Gibbs for confirming the phylogenetic analyses and Professor Bob Blanden for his very insightful involvement in discussions about the germline analyses. Several members of staff at the Department of Biological Sciences also need to be thanked: Dr. Mark Walker for helpful advice, and the technical staff who are always ready to help out with materials, in particular Wendy Forbes who re-sequenced the DNA that I PCR amplified from the single splenic B cells. However, none of this work would have been possible without the love, understanding and support I received from my wife Josie. Many thanks also to my family, whose support has been invaluable. iv Contents Declaration i Abstract ii Acknowledgments iv Contents v Abbreviations x Chapter 1. Introduction 1 1.1 Clonal Selection 1 Confirmation of Burnet's clonal selection theory 1 Affinity maturation of the immune response 2 Antigenic selection of B cells 3 1.2 The generation of antibody diversity 4 a The germline 4 b. Rearrangement and junctional diversity 6 Ordered versus stochastic rearrangement of H and L chain loci 6 Allelic exclusion 8 D gene reading frames 9 Rearrangement signalling sequences 9 Transcription control elements in rearrangement 10 Joining of the genetic elements 11 Junctional diversity 12 VH replacement and secondary VK rearrangement 13 Recombination activating genes (RAG) 1 and 2 14 Preferential utilization of VH genes 14 c. Somatic hypermutation and the germinal center 16 Characteristics of somatic hypermutation in murine systems 16 Timing of somatic hypermutation 17 Boundaries of somatic hypermutation 17 Somatic hypermutation, antigenic selection and 3-dimensional conformation of V regions 18 Recruitment of new B cell clones 21 Germinal centers are the site of somatic hypermutation 23 The germinal center reaction 25 Transgenic models for somatic hypermutation 27 Somatic hypermutator models 30 Somatic hypermutation due to error-prone DNA synthesis/repair 30 Somatic hypermutation due to gene conversion events 32 Somatic hypermutation due to an error-prone DNA-»RNA-»DNA loop.33 Chapter 2. Aims of this thesis 36 2.1 Comparison of RNA and DNA sequences isolated from the same pool of anti-NP splenic B cells 36 2.2 In vitro analysis of splenic antigen-specific B cells 37 2.3 Determination of the 5' boundary for somatic hypermutation in VH regions 37 2.4 Minimization of PCR generated artifacts 38 2.5 Molecular and phylogenetic analysis of related germline VH genes 39 3. Materials and Methods 40 3.1 DNA, bacterial strains
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