Jan Geliebter

Recombination and the concerted evolution of the murine MHC

Abstract The concerted evolution of the murine major histocompatibility complex (MHC) class I multigene family has resulted in the diversification as well as homogenezation of its member genes. Micro-recombinations between class I genes have resulted in the extreme diversification of the H-2 genes. Different regions of the MHC have drastically expanded and contracted due to unequal crossovers.

Microrecombinations generate sequence diversity in the murine major histocompatibility complex: analysis of the Kbm3, Kbm4, Kbm10, and Kbm11 mutants.

The mechanism that generates spontaneous mutants of the Kb histocompatibility gene was analyzed. Nucleotide sequence analysis of four mutant genes (Kbm3, Kbm4, Kbm10, and Kbm11) revealed that each mutant K gene contains clustered, multiple nucleotide substitutions. Hybridization analyses of parental B6 genomic DNA and cloned class I genes with mutant-specific oligonucleotide probes, followed by sequence analyses, have identified major histocompatibility complex class I genes in the K, D, and Tla regions (K1, Db, and T5, respectively) that contain the exact sequences as substituted into mutant Kb genes. These data provide evidence for the hypothesis that the mutant Kb genes are generated by a microrecombination (gene conversion) mechanism that results in the transfer of small DNA segments from class I genes of all four regions of the major histocompatibility complex (K, D, Qa, and Tla) to Kb. Many of the nucleotides substituted into the mutant Kb genes were identical to those found in other naturally occurring K alleles such as Kd. Thus, we propose that the accumulation of microrecombination products within the K genes of a mouse population is responsible for the high sequence diversity among H-2 alleles.

The H-2Kkml mutation: A single nucleotide substitution is responsible for multiple functional differences in a class I MHC molecule

Nucleotide sequence analysis of mRNA from the H-2K locus of the CBA.M523 mouse, which has the class I murine MHC mutation H-2Kkml, has established the only alteration to be at the codon for amino acid position 152 as compared to the sequence of standard Kk from both the AKR and CBA inbred mouse lines. Complete sequence information for the nucleotides coding for amino acids 1-292, which includes all of the extracellular protein domains, demonstrated an A----C alteration in the codon for amino acid 152 as compared to the standard Kk sequence, changing Asp (GAT) in Kkml. The GCT codon occurring in Kkml may be the result of a gene conversion in Kkml. The GCT codon occurring in Kkml may be the result of a gene conversion event because a potential donor gene, the pH-2III pseudogene of H-2k, is transcribed in the CBA.M523 mouse and has a GCT codon at amino acid position 152. This sequence information obtained for Kkml also demonstrates that Kk gene transcripts from two genetically distinct inbred mouse lines, CBA and AKR, are completely identical. Finally, several other murine and human class I MHC variants have similar alterations at amino acid position 152 which result in altered biological functions. This information suggests that amino acid 152 is an important part of a T-cell-recognized antigenic determinant on MHC class I antigens.

The H-ZKkm1 mutation: Nucleotide sequence and comparative analysis

Abstract Nucleotide sequence analysis of mRNA from the class I murine MHC mutant H-2Kkml has established a site of mutation to be at the codon for amino acid position 152. Complete sequence information for the nucleotides coding for amino acids 136–163 demonstrates an A → C alteration at the codon for amino acid 152, changing Asp (GAT) in Kk to Ala (GCT) in Kkml . Several other murine and human class I MHC variants have similar alterations at amino acid position 152, resulting in altered biological activity. Finally, the pH-2III pseudogene of the H-2k haplotype has a GCT codon at amino acid position 152, suggesting that the GCT codon occurring in Kkml is the result of a gene conversion event.

Analysis of the H-2Kbm8 mutant: Correlation of structure with function

The gene for H-2K class I major histocompatibility antigen on the bm8 variant was cloned and the DNA sequence compared with the parental gene. Sequence analysis demonstrated that seven nucleotides were changed with respect to the parental gene sequence spanning 24 nucleotides. These changes represent an alteration of four amino acids from the parent protein. As this mutation occurred in a single generation, a potential donor gene for such a complex mutation was suggested and identified. The Q4 gene class I-like molecule has a stretch of 95 nucleotides of identity in the region of the bm8 mutation. Genomic Southern analysis of the mutant and parental DNA with a gene-specific oligonucleotide demonstrated that the potential donor gene Q4 is a likely candidate sequence for such an event. The amino acid alterations for the H-2Kbm8 mutation are discussed in consideration of hte three-dimensional structure of the characterized human class I glycoprotein.

Diversification of H-2 Genes through Recombination: A Study of Kb Mutants

H-2 genes, members of the MHC class I multigene family, exhibit extreme allelic sequence diversity. Spontaneous, histogenic mutants of the H-2Kb gene have been found to arise at a relatively high frequency. Sequence analysis of the Kb mutants indicate that they consist of clustered, multiple nucleotide substitutions that are identical to nucleotide sequences in other class I genes. Such characteristics suggest that the mutant Kb genes are generated by recombination of the Kb gene with other class I genes. Oligonucleotide probes were utilized to detect donor genes in the K, D, and Qa regions of the MHC with the exact sequence substituted into the mutant Kb genes. Recombination between Kb and donor genes involves short stretches of DNA and can be explained by gene conversion or double crossovers. Genealogical analyses indicate that some of the Kb mutants were generated by mitotic recombination in germ cells. Thus, the Kb mutants serve as a model system to understand the generation of diversity in the MHC.

The Analysis of H-2 Mutants: Molecular Genetics and Structure/Function Relationships

The class I genes of the murine major histocompatibility complex are located in four regions (K, D, Qa, Tla) along chromosome 17. The K and D regions contain the genes encoding the classical H-2 transplantation antigens (K, D, L). Alleles of each of the K, D and L loci exhibit substantial sequence diversity (1–3). Further, H-2 loci are highly polymorphic, with over 100 alleles identified per locus (3). The high sequence diversity and polymorphism of H-2 genes are thought to play a role in the function of their products as antigen presenting molecules, thus enabling a population to respond to many different pathogens. In contrast, Qa and Tla region loci are much less polymorphic and alleles of each locus appear to be much more conserved (4,5). Qa and Tla gene products have a limited tissue distribution and their function is unknown.