Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse.

A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2bc). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2k, H2b, and H2bc, and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6- Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species.

H2-M3 presents a nonformylated viral epitope to CTLs generated in vitro.

Most CTL responses to epitopes from influenza virus are restricted by MHC class Ia molecules. However, a synthetic peptide corresponding to residues 173 to 190 of influenza A/JAP/305/57 hemagglutinin (HA) can induce, in vitro, a CTL response to peptide presented by a mouse class Ib molecule encoded by a gene telomeric to H2-Q. Here, we identify the molecule as H2-M3 and show that the last five residues of HA173-190, MLIIW, is the minimal epitope for CTL recognition. Cells that express M3wt, from C57BL/6 or BALB/c mice, are sensitized by both MLIIW and the longer peptide HA173-190, whereas cells that express M3f, from A.CA or B10.M mice, are sensitized only by MLIIW; a single amino acid change at residue 31 (V-->M) of M3 accounts for this difference. Although M3-restricted CTLs preferably recognize N-formylated epitopes, i.e., those of mitochondrial or prokaryotic origin, our findings show that M3-restricted primary CTL responses can be generated in vitro against nonformylated epitopes.

BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-mog-D17Tu42-M3-M2.

A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2(b)) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M. It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al. 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1. A bacterial artificial chromosome (BAC) contig from the 129 (H2(bc)) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2. Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen - Znf173 - Tctex5 - Mog - D17Tu42 - D17Mit232 - H2-M3 - D17Leh525 - H2-M2 - Tel. The gene order of Znf173 - Tctex5 - Mog - D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A.

On naming H2 haplotypes: functional significance of MHC class Ib alleles.

George D. Snell began defining and naming the H2 haplotypes many years ago by histogenetic typing. Since then, a few haplotypes have been given an additional letter, such as bc for strain 129, to show that they are minor variants from the prototype (b). But by and large, differences in nonclassical class I antigens have been known (only?) to those in the field without being acknowledged by a separate haplotype symbol. Thus, strains BALB/c and NZB/BlNJ are both considered H2d and strains C3H/HeJ and B10.BR are both called H2k, although each pair differs in the TL and Qa1 antigens. In parallel with the interest in nonclassical class I antigens, the need for an appropriate haplotype nomenclature is growing. The haplotypes that require splitting are b, d, k, q, and s; the symbol bc should be retained and used, and, for the other haplotypes, the suffix 2 denotes a Qa1a haplotype with highly TL-positive thymocytes.

Genomic evolution of the distal Mhc class I region on mouse Chr 17.

A 5-Mb YAC contig, partly supplemented with BAC contigs, was created from the distal Mhc class I region on mouse Chr 17. The gene order of Znf173-Tctex5-Mog-D17Tu42-D17Leh 89 is conserved between mouse and human but not the physical distance, supporting the independent expansion of Mhc class I genes in the so-called accordion model of Mhc evolution. The distal H2-M region includes the breakpoint of conserved synteny between mouse and human as well as the In(17)4 t-inversion. The H2-M region is rich in L1 repeats, implying that the insertion of L1 repeats may be associated with the evolutionary flexibility to break a chromosome.

Physical mapping of the human and mouse MOG gene at the distal end of the MHC class Ib region.

Myelin/oligodendrocyte glycoprotein (MOG) is expressed specifically in the central nervous system (CNS) by myelinating glial cells, the oligodendrocytes. The external location of MOG on myelin sheaths and its late expression during myelinogenesis argue for a role of MOG in the completion of myelin and maintenance of its integrity. MOG is a target autoantigen in demyelinating diseases, such as experimental autoimmune encephalomyelitis (EAE) in animals and multiple sclerosis (MS) in humans. We previously located the gene encoding MOG to the major histocompatibility complex (MHC), both in human, by cytogenetics, and in mouse, by analysis of recombinants. To refine the position, we have now selected yeast artificial chromosome clones (YAC) which contain the MOG gene. Physical mapping of the human MOG and the mouse Mog genes by characterization of these YAC clones indicated that the gene is located at the distal end of the major histocompatibility complex (MHC) class Ib region in both species. The human MOG gene lies 60 kilobases (kb) telomeric to HLA-F in a head-to-head orientation; the mouse Mog gene lies 25 (kb) telomeric to H2-M5 in a tail-to-head orientation. These orthologous genes provide markers for comparative analysis of the evolution of the MHC in the two species. The physical mapping of MOG should facilitate analysis of its role in hereditary neurological diseases, and the YAC clones identified here will permit the identification of new genes in the region.

Peptide antigen presentation by non-classical MHC class I molecules.

The mouse major histocompatibility complex contains more than 40 class I genes in addition to those encoding the classical class I molecules. Many of these encode functional class I heavy chains that are expressed and displayed on the cell surface in association with beta 2-microglobulin. Several laboratories, using genetic, biochemical, and molecular analysis, RMA-S mutant cells, and synthetic peptides, have recently shown that mouse non-classical or class Ib molecules present peptide antigens to T lymphocytes with alpha beta or gamma delta receptors. The class Ib genes, with their limited polymorphism, may have evolved to serve specialized presentation functions.

Cellular location of thymus-leukemia (TL) antigen as shown by immuno-cryoultramicrotomy.

Thymus-leukemia (TL) antigen is a class I molecule of the major histocompatibility complex that is expressed on the surface of mouse cortical thymocytes. Though not expected, it has been reported that TL antigen can be found on isolated mitochondria of TL+ cells. We used immuno-cryoultramicrotomy to look for TL on mitochondria in situ, thereby avoiding the plasma membrane contamination that occurs when isolating organelles. Establishing optimal fixation conditions was crucial, as mitochondrial structure was not preserved by the low concentrations of fixative needed for detection of antibody labeling. The plasma membranes of tissue culture and thymus cells were labeled well with anti-TL antibody and protein A-gold conjugate, while mitochondria within the cells were not labeled. Isolation of mitochondria on a one-step Ficoll gradient resulted in a purer organelle preparation than did isolation of mitochondria by centrifugation alone. Generally, mitchondria within this purer preparation were not labeled. Our data show that under conditions where contamination by plasma membrane is not a major concern, TL antigen cannot be detected on mitochondria.

Maternally transmitted antigen of mice: a model transplantation antigen.

Molecular identification proved Mta, the maternally transmitted antigen of mice, to be a model minor histocompatibility (H) antigen. It consists of a peptide, MTF, that is presented on the cell surface by an H-2 class-I molecule, HMT. MTF is derived from ND1, a mitochondrially encoded protein, and the amino-terminal N-formyl-methionine is essential for binding to HMT; conservative substitutions at the sixth residue causes MTF to be a minor H antigen. HMT is encoded by the M3 gene at the telomeric end of the H-2 complex. The peptide-binding site of HMT is hydrophobic, and allelic forms of the mature protein differ by only three amino acids. Homologues and analogues of the mouse Mta system have recently been identified in rats.