Doctor of Philosophy
University of Wollongong. Dept. of Biological Sciences
Rothenfluh, Harald Sebastian, Somatic hypermutation and germline evolution of immunoglobulin variable genes, Doctor of Philosophy thesis, University of Wollongong. Dept. of Biological Sciences, University of Wollongong, 1994. http://ro.uow.edu.au/theses/1053
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 (VW of the mouse. A number of mechanisms have been invoked to explain somatic hypermutation of immunoglobulin variable (lgV) 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 IgVH 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 IgVH regions is asymmetrical, with a single mode centered on the rearranged VH region and a long tail extending into the J - Cintron. 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 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 germ line 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 multi gene family will be evaluated on the basis of how well they can explain the new data.