Gene conversion of the mouse MHC class II

The major histocompatibility complex (MHC) is the most polymorphic vertebrate gene loci known to exist. Gene products of the MHC are responsible for presentation of antigen derived peptides to T cells of the immune system, which thereby become activated and involved in the elimination of particles recognised as non-self. The mechanism of acquiring this extensive polymorphism is not known, but gene conversion has been suggested as one possible molecular genetic mechanism employed to generate new MHC alleles. A strict definition of gene conversion, based on the phenomenon in yeast, involves the transfer of a DNA fragment from a donor gene to a homologous acceptor gene without the donor gene being changed in the process. For practical reasons, the term gene conversion in higher organisms is rather used in the context of itemplated segmental mutationi. This thesis describes the first direct evidence of gene conversion events between two endogenous MHC class II genes in a mouse system using sperm as a representative for future potential individuals. A PCR assay was developed using primers for the acceptor gene as well as the donor gene to obtain a detectable product only in cells where a gene conversion event had occurred. Interchromosomal gene conversion between a MHC class II Ab and Eb gene located on separate chromosomes was found to occur at a frequency of 1/500.000 sperm, whereas no gene conversion was detected in somatic liver cells. Intrachromosomal gene conversions between Ab and Eb genes located on the same chromosome were detected at frequencies ranging from 1/35.000 sperm to 1/830.000 sperm depending on the alleles in ves gat ed. This difference in frequency was found although the investigated alleles coexisted in the same cell, which indicates that there is sequence restraints acting on the genes involved in a gene conversion event. Both inter- and intra-chromosomal gene conversions transferred DNA fragments which varied in length, but the transfer breakpoints investigated were usually in regions where the donor and acceptor genes showed a high nucleotide similarity. Investigation of gene conversion during male gametogenesis showed that most gene conversion events detected between MHC class II genes were completed already in the premeiotic spermatogonia cell stage, which indicates that gene conversion relies on other molecular genetic mechanisms than normal meiotic recombination. Sequence analysis of several MHC mutants proposed to have been caused by gene conversion events revealed that both donor and acceptor sequences resided in regions where normal mammalian CpG suppression was absent. CpG dinucleotides have been shown to be relatively mutation prone, which accordingly suggests that regions with a high CpG content could be targeted for mutation events. Moreover, gene conversion could be induced and increased up to 15 times of the background level in a cell line by DNA damage, which indicates that the DNA repair system is involved in the gene conversion mechanism. From an evolutionary point of view, gene conversion thus is a very potent mechanism for creation of new MHC alleles by shuffling of already existing and functional parts of genes into new homologous locations. This mechanism can efficiently produce new functional MHC variants, which could give the population a selective advantage in terms of the ability to cope with various pathogens as well as being a mean to avoid inbreeding that could result in a reproductive loss.