Qa-2 Antigen Research Articles

The expression pattern of the Qa-2 antigen in mouse preimplantation embryos and its correlation with the Ped gene phenotype.

The Qa-2 antigen, the product of the Ped (Preimplantation embryo development) gene, is a glycosylphosphatidylinositol-linked cell surface protein encoded in the Q region of the mouse major histocompatibility complex (MHC). Ped fast (Qa-2+) mouse strains have significantly higher preimplantation embryo cleavage rates both in vivo and in vitro than Ped slow (Qa-2-) mice. In this study, we determined whether the Ped fast phenotype of blastocysts is due to an increased number of blastomeres in the trophectoderm (TE), the inner cell mass (ICM), or both. We also analysed the Ped gene expression pattern, both at the mRNA and at the protein level, in these lineages. Blastocysts were collected from the congenic mouse strains B6.K2 (Qa-2 +) and B6.K1 (Qa-2-). We performed reverse transcription-polymerase chain reaction (PCR) and Immuno-PCR and found that the Ped gene is expressed at the mRNA and protein level in whole embryos and in isolated ICM cells. Lastly, we differentially stained embryos from these strains and found that B6.K2 blastocysts had significantly higher cell numbers (P < 0.05) in both the ICM and in the TE than B6.K1 blastocysts. These results suggest that Qa-2 expression in both the TE and the ICM of blastocysts directly contributes to the Ped phenotype.

Genetic regulation of preimplantation mouse embryo survival

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.

Increased resistance to Taenia crassiceps murine cysticercosis in Qa-2 transgenic mice.

We previously reported important differences in resistance to Taenia crassiceps murine cysticercosis between BALB/c substrains. It was suggested that resistance might correlate with expression of the nonclassic class I major histocompatibility complex (MHC) Qa-2 antigen; BALB/cAnN is Qa-2 negative and highly susceptible to T. crassiceps, whereas BALB/cJ expresses Qa-2 and is highly resistant. In this study, we investigated the role of Qa-2 in mediating resistance to cysticercosis by linkage analysis and infection of Qa-2 transgenic mice. In BALB/cAnN x (C57BL/6J x BALB/cAnN)F1 and BALB/cAnN x (BALB/cJ x BALB/cAnN)F1 backcrosses, the expression of Qa-2 antigen correlated with resistance to cysticercosis. Significantly fewer parasites were recovered from infected Qa-2 transgenic male and female mice than from nontransgenic mice of similar genetic background. These results clearly demonstrate that the Qa-2 MHC antigen is involved in resistance to T. crassiceps cysticercosis.

Qa-2 antigen, the product of the Ped gene, influences cell differentiation in murine blastocysts

The molecular weights of the fully glycosylated cell surface form and the unglycosylated polypeptide of five murine class I antigens (H-2Kb, Db, TL, Qa-1.1, and Qa-2) were compared by SDS-PAGE. Significant molecular weight diversity was observed for both forms among these molecules. The size of the fully glycosylated forms ranged from approximately 52,000 daltons (H-2Db) to 41,000 daltons (Qa-2), whereas the unglycosylated polypeptides ranged from 43,000 daltons (H-2Kb and TL) to 33,000 daltons (Qa-2). The magnitude of the size variation observed in the unglycosylated polypeptides implies that there are differences in the gene organization, RNA processing or post-translational modifications of various class I glycoproteins.

Role of the Ped gene and apoptosis genes in control of preimplantation development.

The properties of the mouse Ped gene and the genes that mediate apoptosis in mediating preimplantation embryonic survival were reviewed. Preimplantation mouse oocytes and embryos were evaluated microscopically and biochemically for rate of development, degree of fragmentation, and gene expression to correlate these characteristics with embryo mortality, Biochemical assays included PCR for DNA analysis, RT-PCR for mRNA analysis, immuno-PCR for protein analysis, and TUNEL assay for assessment of apoptosis. Using the mouse as a model system we have identified a gene that controls the rate of development, the Ped gene. The Ped gene product is a class Ib major histocompatibility complex protein called the Qa-2 antigen. Research to understand the molecular mechanisms of Ped gene action and to identify the human homologue of the Ped gene is under way. We have also shown using the mouse model, that fragmented embryos show the morphological and biochemical characteristics of apoptosis. Genes in the two major gene families that regulate apoptosis, the caspase and Bcl-2 families, are expressed in mouse oocytes and preimplantation embryos. Preimplantation embryonic survival depends on two major morphological parameters: rate of development and degree of fragmentation. A fast rate of development and a low degree of fragmentation lead to a better chance of producing live offspring. Both rate of development and degree of fragmentation are genetically controlled, the former by the Ped gene and the latter most likely by genes that mediate apoptosis. It seems probable that regulation of apoptosis will prove to be a major mechanism that mediates oocyte and preimplantation embryonic survival.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Identification of the Ped Gene at the Molecular Level: The Q9 MHC Class I Transgene Converts the Ped Slow to the Ped Fast Phenotype1

The Ped (preimplantation embryo development) gene, which maps to the Q region of the mouse major histocompatibility complex (MHC), controls the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival. Of the ten known MHC class I genes in the Q region of the mouse MHC, four--Q6, Q7, Q8, and Q9--are almost identical and encode similar proteins, all called the Qa-2 antigen. Previous studies have suggested that the Q9 gene encodes the Ped gene. To test this directly, one-cell embryos from the CBA/Ca strain (Ped slow) that is missing the Q9 gene and the Qa-2 antigen were injected with the Q9 gene from the C57BL/10 strain (Ped fast) that possesses the Q9 gene and expresses the Qa-2 antigen. The resulting Q9 transgenic mice were found to express the Qa-2 antigen. In addition, it was found that introduction of the Q9 gene converted the Ped gene phenotype of the recipient strain from slow to fast. Therefore, the Ped gene product is the Qa-2 antigen encoded by the Q9 gene.

A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides.

Unlike the highly polymorphic major histocompatibility complex (MHC) class Ia molecules, which present a wide variety of peptides to T cells, it is generally assumed that the nonpolymorphic MHC class Ib molecules may have evolved to function as highly specialized receptors for the presentation of structurally unique peptides. However, a thorough biochemical analysis of one class Ib molecule, the soluble isoform of Qa-2 antigen (H-2SQ7b), has revealed that it binds a diverse array of structurally similar peptides derived from intracellular proteins in much the same manner as the classical antigen-presenting molecules. Specifically, we find that SQ7b molecules are heterodimers of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. These peptides contain a conserved hydrophobic residue at the COOH terminus and a combination of one or more conserved residue(s) at P7 (histidine), P2 (glutamine/leucine), and/or P3 (leucine/asparagine) as anchors for binding SQ7b. 2 of 18 sequenced peptides matched cytosolic proteins (cofilin and L19 ribosomal protein), suggesting an intracellular source of the SQ7b ligands. Minimal estimates of the peptide repertoire revealed that at least 200 different naturally processed self-peptides can bind SQ7b molecules. Since Qa-2 molecules associate with a diverse array of peptides, we suggest that they function as effective presenting molecules of endogenously synthesized proteins like the class Ia molecules.

Antigenic heterogeneity of Qa-2 antigens in C57BL/6.

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of 125I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.

Modulation of preimplantation embryonic development by antisense oligonucleotides to major histocompatibility complex genes.

The Ped (preimplantation embryo development) gene, which controls the rate of mouse preimplantation embryonic cleavage division and subsequent survival of the embryo, maps to the Q region of the MHC (major histocompatibility complex). Mouse embryos were treated with antisense oligonucleotides to mRNA for the Q region genes Q7/Q9, each of which encodes the Qa-2 antigen. The reverse transcription polymerase chain reaction (RT-PCR) was used to show that antisense treatment, but not sense treatment, decreased the level of mRNA for Qa-2 antigen in preimplantation embryos. Furthermore, both the expression of Qa-2 protein and the rate of embryonic cleavage division were decreased by treatment with antisense but not sense oligonucleotides. These results provide direct evidence that the Ped gene phenotype is at least partially encoded by the Q7/Q9 genes. It is likely that the mouse Ped gene has a human homolog, perhaps within HLA-F. Identification of genes--such as the Ped gene--that affect survival of the embryo may be vitally important for the enhancement of animal and human reproductive success.

Recognition of the Qa-2k tumor antigen by T cell receptor gamma/delta of an immunopotentiator-induced tumoricidal T cell of mice.

Tumor-specific expression of Qa-2k antigen coded by the Q5k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2-mice. Lymphocytes stimulated in vivo with P. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (LQ7b/Kb), which expressed the alpha 1 and alpha 2 domains of the Qa-2 antigen (Q7b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5' half of the Q7b gene and the 3' half of the H-2Kb gene (pQ7b/Kb). Using LQ7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes from P. acnes-stimulated (C3H/He x B6.K1)F1 mice (H-2k, Qa-2-) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the alpha 1/alpha 2 region of the Q7b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the gamma/delta type (TCR gamma/delta). The (C3H/He x B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2k+) derived from AKR mice (Qa-2-, H-2k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed LQ7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the alpha 1/alpha 2 region of the Qa-2k tumor antigen by TCR gamma/delta.

Removal of Qa-2 antigen alters the Ped gene phenotype of preimplantation mouse embryos.

Embryo survival is influenced by both genetic and environmental factors. Previous research in our laboratory has identified one gene associated with embryonic survival, the Ped gene, a gene that is linked to the major histocompatibility complex (MHC) of the mouse. The Ped gene has been shown to influence the rate of preimplantation embryonic cleavage division, as well as litter size, birth weight, and weaning weight. Genetic mapping of the Ped gene has located it in the Q region of the MHC and has suggested that possible Q region genes encoding the Ped gene are Q3, Q5, Q6, Q7, Q8, and/or Q9. Whereas the protein products of the Q3 and Q5 genes are unknown, the protein product of the very similar Q6, Q7, Q8, and Q9 genes is the Qa-2 antigen. Two forms of membrane-bound Qa-2 antigen are known: glycosylphosphatidylinositol (GPI)-linked and transmembrane bound. Only the GPI-linked form is sensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC). The first purpose of the present study was to determine the nature of the linkage of the Qa-2 antigen to the cell surface of preimplantation mouse embryos. It was found that all detectable Qa-2 antigen on the embryonic cell surface is sensitive to cleavage by PI-PLC and is therefore bound to the cell membrane by a GPI linkage. Furthermore, removal of Qa-2 antigen from the embryonic cell surface slows down the rate of development of preimplantation mouse embryos. These results suggest the likelihood that the Qa-2 antigen is the Ped gene product.

Detection of allogeneic Qa/TL and Ly specificities on murine tumor cells with IgD in tumor-regressor serum.

Serum from C3H/He mice, which show regression of MM2 tumor cells after transplantation and removal (regressor serum, RS) contains non-gammaglobulin components that cross-react with various tumor cells of mice [22, 23]. In addition to tumor cells, various allogeneic lymphocytes are also susceptible to an RS-dependent lymphocyte-mediated cytotoxic reaction. To identify tumor cell surface antigens that cause the cross-reactive host response, the serum components were analyzed by absorption of RS with allogeneic lymphocytes. RS components were found to recognize allogeneic lymphocyte antigens including Qa-2 and Ly6.2. Specificity for the Qa-2 antigen was further tested using Qa-2-congenic mice. The expression of Qa-2 antigen was detected on the surfaces of MM2 and other tumor cells derived from H-2k mice (seven among nine cell lines tested) by a membrane immunofluorescence method using a Qa-2-specific mAb. Physical characteristics of the Qa-2-specific component in RS were determined and found to differ from those of regular IgGs but to be similar to those of IgDs. Using an enzyme-linked immunosorbent assay with an IgD-specific mAb and Qa-2-lacZ fusion protein, the existence of IgD in RS with specificity for Qa-2 was confirmed. These results suggest that the RS component with Qa-2 specificity is an IgD, the specificity and physiological role of which are unknown.

Expression of the Qa-2k phenotype encoded by the Q5k gene on the surface of tumor cells derived from H-2k mice.

Immunological and biochemical characteristics of the Qa-2 murine nonclassical histocompatibility class 1 antigen expressed on tumor cells derived from H-2k (Qa-2-) mice were studied. It was found that the Qa-2 antigen on normal H-2b lymphocytes reacted with Qa-2-specific monoclonal antibodies (mAbs) 34-1.2, 59 (both specific to the alpha 1/alpha 2 region) and 141-15.8 (specific to the alpha 3 domain), and the Qa-2 antigen on H-2k tumor cells (Qa-2k antigen) reacted with mAbs 59 and 141-15.8, but not with 34-1.2. The normal Qa-2 antigen was susceptible to treatment with phosphatidylinositol-specific phospholipase C, but the Qa-2k antigen was insensitive to it. By Northern hybridization, polymerase chain reaction (PCR) studies on cDNA, Southern hybridization, Western blotting, and nucleotide sequence analysis, the Q5k gene was identified as the gene encoding the Qa-2k antigen expressed on BW5147 lymphoma cells derived from a mouse of AKR strain (H-2k, Qa-2-). The nucleotide sequence of PCR-amplified BW5147 Q5k cDNA showed complete agreement with the reported sequence of exons 1-5 of the Q5k gene of C3H/He. It also showed complete deletion of the region corresponding to exons 6 and 7, and a very short coding region in exon 8, resulting in very short cytoplasmic domain of the product compared with regular class 1 antigens. These characteristics were expected from the reported Q5k genomic sequence. These results revealed that the Qa-2k antigen was distinct from the normal Qa-2 antigen expressed on H-2b lymphocytes although it cross-reacted with some Qa-2-specific mAbs.

Cytotoxic T lymphocyte precursor cells specific for the major histocompatibility complex class I‐like antigen, Qa‐2, require CD4+ T cells to become primed in vivo and to differentiate into effector cells in vitro

Experiments were performed to determine whether CD4+ T cells are required for the generation of cytotoxic T lymphocytes (CTL) specific for the nonpolymorphic major histocompatibility complex (MHC) class I-like antigen, Qa-2. Splenic T cells from BALB/cBy (Qa-2b) mice that had been immunized with irradiated BALB/cJ (Qa-2a) splenocytes generated CTL following in vitro stimulation with BALB/cJ splenocytes. These CTL lysed all Qa-2+, but not Qa-2- targets, regardless of the H-2 haplotypes of target cells or their non-MHC backgrounds. This apparent MHC class I-unrestricted recognition of Qa-2 antigen was confirmed using Qa-2-specific CTL clones. The Qa-2-primed CTL precursor cells (CTLp) and CTL were found to be CD8+ T cells. Primed splenocytes depleted of CD4+ T cells prior to culture failed to generate CTL, but addition of lymphokines to the culture restored the CTL generation. Stimulation of primed splenic T cells with irradiated Qa-2+ T blast cells, instead of splenocytes or B blast cells, led to little to no CTL generation, suggesting that MHC class II molecules are involved in the presentation of Qa-2 antigen to CD4+ T cells. This was also supported by the results of experiments using Qa-2+, class II- thymoma cells of BALB/c origin. Stimulation of the thymoma-primed splenic T cells with the mitomycin C-treated thymoma cells resulted in no generation of anti-Qa-2 CTL, despite the fact that high levels of CTL specific for minor histocompatibility (H) antigens and H-2d were generated by immunizing the corresponding allogeneic hosts with the thymoma. However, the addition of lymphokines rendered thymoma-primed T cells capable of generating anti-Qa-2 CTL. Both CD4+ and CD8+ T cell populations, isolated from the BALB/cJ splenocyte-primed responder cells, proliferated in vitro in response to the Qa-2+ splenocytes, suggesting that Qa-2-reactive CD4+ T cells were present in the immunized mice. Depletion of CD4+ T cells from thymectomized BALB/cBy mice with anti-L3T4 monoclonal antibodies markedly reduced, but did not eliminate anti-Qa-2 CTL generation. In contrast, depletion of CD8+ T cells led to a complete abrogation of the CTL response. Addition of lymphokines to the culture of responder cells depleted of either T cell subset did not restore their reactivity. It is concluded that anti-Qa-2 CTLp need "help" from CD4+ T cells to become primed in vivo. Furthermore, primed CTLp also need "help" or lymphokines provided by CD4+ T cells to differentiate into effector CTL in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Physical and molecular genetic analysis of Qa-2 antigen expression: Multiple factors controlling cell surface levels

We have examined the surface expression of Qa-2 on lymphocyte subpopulations and on splenocytes from inbred mouse strains by a radioactive binding assay using purified anti-Qa-2 antibodies and antibody fragments (Fab). Quantitative measurements by Scatchard analysis revealed that spleen cells from Qa-2 high mice express (4–5) × 10 4 Qa-2 molecules/cell, whereas T lymphocytes have as high as (7–8) × 10 4 molecules/cell. In addition, it was determined that B lymphocytes express (5–6) × 10 3 molecules on their cell surface. The Qa-2 levels on anti-CD3-activated T cells is 1.0 × 10 5 molecules/cell. Previous experiments have shown that the quantity of Qa-2 varies in a strain-specific fashion and may be classified into three groups: Qa-2 high, Qa-2 medium and Qa-2 low. Our results indicated that Qa-2 high strains express (4–5) × 10 4 Qa-2 molecules/cell, Qa-2 medium strains (B6-K2, B10.A, A/J, BALB/c and DBA/2) express (1–1.7) × 10 4 molecules/cell, and Qa-2 low strains (SWR/J and DBA/I) express no more than 6 × 10 3 molecules/cell. Detection of Q7 or Q9 mRNA by the polymerase chain reaction revealed that Qa-2 high strains express two functional Qa-2 enconding genes, Q7 and Q9, whereas Qa-2 mediumand Qa-2 low strains express either Q7 or Q9. These results strongly suggest that Qa-2 gene dosage contributes in part to the variation of Qa-2 levels on the cell surface.

MHC gene Q8/9 d of the BALB/cJ mouse strain cannot encode a Qa-2,3 class I antigen

We have determined the nucleotide sequence of Q8/9d gene of the BALB/c strain of mice, isolated from Steinmetz's cosmid library. As for all other class I genes of the Qa region, the Q8/9d gene spans approximately 4.7 kilobases (kb) and consists of seven exons and six introns. A seven bases deletion in exon 3 results in the occurrence of an early termination codon. Thus the Q8/9d gene cannot encode a normal class I protein. Comparison of the nucleotide sequence of the Q8/9d gene with that of other class I MHC genes revealed a stronger homology to Q7 and Q8 than to K, D, L, TL, and other Q genes. However, the gene cannot originate from a mere fusion between Q8 and Q9 genes except if the ancestor to putative Q8d was markedly different from the present Q8b gene. Using polymerase chain reaction (PCR) technology, we have confirmed the presence of a Q8/9 gene, identical to that present in cosmid 46.1, in the genome of BALB/cJ (Qa-2low). Finally, it has been reported that cDNA clone 94-A, which codes for a Qa-2 antigen, could derive from a transcript of gene Q8/9d. The nucleotide sequences of gene Q8/9d and of cDNA clone 94-A are distinctly different in their 5' regions, in spite of an almost perfect matching in their 3' regions. Thus, clone 94-A cannot derive from an mRNA transcribed from the Q8/9d gene.

Analysis of litter size and weight in mice differing in Ped gene phenotype and the Q region of the H-2 complex

A gene has been described, Ped (Preimplantation embryo development), that influences the rate of cleavage of preimplantation mouse embryos. Previous studies of linkage of Ped gene phenotype (fast or slow embryo development) and H-2 haplotype ( H-2 b or H-2 k) in backcross embryos from the C57BL/10Sn and B10.BR congenic strains have shown that the Ped gene is located in the H-2 complex, the major histocompatibility complex (MHC) of the mouse. The present study was undertaken to localize the Ped gene to a particular subregion of the H-2 complex. Analysis of the B6.K1 and B6.K2 congenic strains, which differ at only the Q subregion of the MHC, was undertaken to test whether the Ped gene is located in the Q subregion of the MHC. Qa-2 antigen expression was used as a marker for the Q subregion and fast or slow development was used to assess Ped gene phenotype in backcross embryos generated from the mating of (B6.K1 × B6.K2)F 1 and B6.K1 mice. The results showed linkage of Ped gene phenotype and Qa-2 antigen expression, which strongly supports the idea that the Ped gene is located in the Q subregion of the MHC. In a further set of experiments, litter size and weight were investigated in the B6.K1 and B6.K2 mice. Pure line and reciprocal crosses were made using sires and dams of both the B6.K1 and B6.K2 genotypes. Traits measured on pups included birth weight, weaning weight and weight per day of age from birth to weaning. Litter traits measured were number born and weaned and survivability. Sex effects existed for weaning weight and weight gain per day of age. Males gained more and were heavier than female pups ( P < 0.05). Pups whose sire or dam were B6.K2 were significantly ( P < 0.05) heavier at birth than those pups whose sire or dam were B6.K1, and B6.K2 homozygous pups were significantly ( P < 0.001) heavier than all others. Litters whose dams were B6.K2 had significantly more pups at birth ( P < 0.05) than those litters whose dams were B6.K1. Results suggest that pups that are heterozygous or homozygous B6.K2 are more apt to be heavier at birth and be in larger litters suggesting that genes in the Q region of the H-2 complex are advantageous to reproductive performance.

Restriction fragment length polymorphisms in the major histocompatibility complex of the non-obese diabetic mouse

The inbred non-obese diabetic (NOD) mouse is a spontaneous model for insulin-dependent diabetes mellitus (IDDM). As in man and BB rats, IDDM in the NOD mouse has an autoimmune aetiology. The disease is controlled by several genes, one of which, Idd-1, has been mapped to the major histocompatibility complex (MHC) on chromosome 17. However, Idd-1 has not yet been identified. To facilitate the identification of Idd-1 we have further analysed the MHC region for restriction fragment length polymorphisms and we find that the NOD mouse has a distinct haplotype: H-2K1 nod K d A β nod A α d E β nod E α nod TNF- α b. In addition, the NOD mouse shows some similarities with the H-2 b haplotype in the Q region, in that either the Q7 or the Q9 gene seems to be like that in the b-haplotype and that the Qa2 antigen is expressed, while other parts of this region are distinct from the b- as well as the d-haplotype. In contrast, the sister strain, the non-obese normal (NON) mouse, derived from the same cataract-prone line of mice as the NOD mouse, has an MHC Class I region indistinguishable from the b-haplotype, but the MHC Class II region is distinct from the NOD mouse as well as the b-, d- and k-haplotype.

Biochemical differences in Qa-2 antigens expressed by Qa-2 +,6 + and Qa-2 +,6 − strains. Evidence for differential expression of the Q7 and Q9 genes

Cell surface forms of Qa-6 class I molecules are biochemically indistinguishable from Qa-2 although Qa-6 maps telomeric to Qa-2 with the recombinant strain B6.K2. Analysis of appropriate F 1 strains did not demonstrate the presence of a trans acting factor that could modify the Qa-2 molecule to produce the Qa-6 determinant. Also, neither a neighboring cell surface molecule nor oligosaccharides were found to block the recognition of the Qa-6 determinant in Qa-2 +,6 − strains. The 2-D gel profiles of neuraminidase or endoglycosidase treated anti-Qa-2 immunoprecipitates from lysates of cell surface iodinated Qa-2 +,6 + strains revealed an additional basic polypeptide which was absent from that of Qa-2 +,6 − strains. Thus, differential sialylation/glycosylation of Qa-2 molecules masks detection of Qa-2 antigen heterogeneity when cell surface forms are analyzed. Qa-6 + phenotype associated polypeptides were also found at various stages of post-translational processing in cells metabolically labeled in the presence and absence of tunicamycin. Northern analyses using Q7and Q9 specific oligonucleotide probes revealed appropriate sized transcripts for both genes in the Qa-2 +,6 + strain B6 but only Q9in the Qa-2 +,6 − strain B6.K2. These data demonstrate that there is structural heterogeneity in Qa-2 antigens expressed by Qa-2 +,6 + and Qa-2 +,6 − strains which results from differential expression of the Q7 and Q9 genes.