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Klasen, Maik (2004): Cis and trans-acting elements in somatic hypermutation. Dissertation, LMU München: Faculty of Biology
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Abstract

Somatic hypermutation in the Ig genes is a paradigm of a site-specific, stage-specific, lineage-specific "mutator system" that generates point mutations at high rates at the Ig locus in an AID mediated and transcription dependent manner during affinity maturation of activated B-cells. The questions in the field of hypermutation today are (i) what targets the mutator factors to the Ig genes? And (ii) what are these factors? To address some aspects of these questions, I engineered and compared retroviral vectors to monitor hypermutation. Retroviral vectors combine a high transduction rate with integration at random sites within the host cell genome, thus equalizing positional effects on the reporter gene. The vectors contain a reporter gene with a premature TAG termination codon; upon reversion, a full-length fluorescent protein is expressed. The number of fluorescent cells can be easily measured in flow cytometry, and thus mutation frequencies can be determined with accuracy. I tested the green and yellow fluorescence proteins (GFP and YFP); and various proteins with red fluorescence (dsRed). Furthermore, I tested various selection markers to select for transduced cells. I found that GFP as a reporter, combined with a drug selection marker, gave the most consistent and convenient mutation rate measurements. DsRed is a good alternative to GFP, but variants with greater fluorescence intensity are needed when combined with green fluorescence measurements. To study the activity of enhancers on transcription and hypermutation, I introduced various cis-acting enhancer elements into the reporter construct. Using such constructs I found that no immunoglobulin specific sequence is needed to target hypermutation. Also, depending on their position in these ectopically expressed constructs and on the transcription rate, enhancers can have positive or negative effects on hypermutation. I also applied the indicator system to investigate whether or not mismatch repair influences AID mediated hypermutation in a non-B lymphocyte line. To do so MLH 1 expression, which is essential in mismatch repair, was regulated in a non-lymphocyte cell line that had been transduced by an AID containing vector. Whether or not MLH1 was expressed, we found no difference in mutation rates of an indicator gene. This is in contrast to activated B cells, where the absence of mismatch repair results in a reduction of hypermutation. I, therefore, conclude that in order to contribute to hypermutation, mismatch repair needs additional factors that are present in activated B lymphocytes but absent in the cell line investigated. During my cloning efforts to create DsRed containing indicator vectors, I found that a single base substitution that does not change the amino acid sequence in the gene encoding DsRed2 resulted in an increase in relative fluorescence intensity of the protein. Surprisingly, the mutated codon is slightly less favored than the original one and, therefore, would be expected to have a negative effect on fluorescence intensity, if any at all. I suggest that in highly expressed genes, the tRNA with the anticodon corresponding to a commonly used codon might become depleted. As a consequence, overall less protein is synthesized, but mRNAs using less frequent codons at some positions may be translated more efficiently.