Gertrude M. Pfaffenbach

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2bm6, B6.C-H-2bm7, and B6. C-H-2bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2bm5 (bm5) and B6-H-2bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2Kb molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2bm6 (bm6), B6.C-H-2bm7 (bm7), and B6.C-H-2bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the Kb molecule.

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2 bm5 and B6-H-2 bm16

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6 H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tryosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2Kb mutants analyzed.

Biochemical analysis of related, independently arising histocompatibility mutants: bm17 and KB-98 enlarge the "bg series" of H-2Kb mutants.

The “bg” series of MHC mutations is the most prevalent type of mutations of Kb in C57BL/6 mice screened by reciprocal tail skin grafting. The basis for identification of this series of mutations is the incompatibility of grafts between the parental B6 and the mutant. This series takes the longest to reciprocally reject the skin grafts. The series can be subdivided into “bg 1” and “bg 2” groups based on Kb-restricted recognition of virus-infected mutant target cells. The biochemical basis for these mutations are amino acid substitutions at residues 116 and 121 of the Kb transplantation antigen. These substitutions do not alter monoclonal antibody binding sites. The structural basis of MAb binding and the genetic basis of the mutation are discussed.

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.