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Given the extensive allelic amino acid sequence polymorphism present in the first domain of Aa, Aß, and Eß chains and its profound effects on class II function, the minimal polymorphism in the mouse Ea chain (and in its human homologue DRa) is paradox. Two possible explanations for the lack of polymorphism in Ea are: (1) the Ea chain plays such a uniquely critical structural/functional role in antigen presentation, T-cell activation, repertoire selection, and/or pairing with Eß or other proteins for expression that it cannot vary, and mutations are selected against; (2) the Ea chain plays a less significant role than the outer domains of other major histocompatibility complex (MHC) proteins in determining the interactions with processed peptides or with T-cell receptor (TCR), so there is no selective pressure to maintain new mutations. To explore this question we compared the ability of transfectants expressing wild type (wt) EaEßd and mutant Ea wt Eßd proteins to present peptides and bacterial superantigens to T-cell hybridomas. Mutations at the Ea amino acid positions 31, 52, and 65&66, to residues that represent allelic alternatives in Aa chains, significantly reduced activation of peptide-specific T hybridomas, and mutations at 71 sometimes enhanced T-cell stimulation. None of the Ea mutations reduced, and some enhanced, superantigen stimulation of T-cell hybridomas. These results argue against the hypothesis that Ea chains are minimally polymorphic because mutations in Ea are functionally neutral.

Molecular and biochemical techniques have been used to explore the reasons behind low Ea chain expression in the Ea+Eß-I-region recombinant strain, A.TFR5. A.TFR5 (AfEk, ap5), a recombinant between A.CA (AfEf) and A.TL (AkEk), carries the Ek subregion. Previous results have shown that it expresses the Ea chain, but at reduced levels relative to Ea+Eß+strains. No Eß chains were detected, which is consistent with the A.TFR5Eß gene being derived from the A.CA parent, which carries the null Eßfallele. In this paper, the defect in Ea-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of Ea, in the region of the large intervening sequence of Eß. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the Ea message, but no Eß message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular Ea chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular Ea, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of Eß, the Ea chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.

Hamster alloantisera and a monoclonal antibody originally generated against antigens controlled by the murine I-Ek subregion, which cross-reacts with hamster cell surface antigens, have been used to define two distinct Ia-like complexes in the Syrian hamster. These complexes have been named a1: ß1 and a2:ß2 and are detected by hamster alloantisera or monoclonal antibody 14-4-4, respectively. For the three strains studied, a1:ß1, appears to be polymorphic in both a and ß chains, while the a2:ß2 complex is nonpolymorphic, as revealed by 2-D PAGE analyses. A third nonpolymorphic glycoprotein that appears to be the hamster's equivalent of the murine invariant chain (Ii) is associated with both the a1:ß1 and a2:ß2 complexes. In addition, we report the first biochemical detection of polymorphism between the closely related CB and MHA Syrian hamster strains.

The genetic and molecular requirements for cell-surface expression of Ia antigens precipitated by anti-I-E subregion sera have been examined. Inbred mice of thed, k, p, andr haplotypes synthesize and express on their lymphocytes the two I-region products normally found in anti-I-E-subregion immunoprecipitates, Ea and Ae (Eß). Cells from mice of theb ands haplotypes fail to synthesize Ea chains but do synthesize Ae chains, which remain in the cytoplasm as partially glycosylated precursors. Cells of thef andq haplotypes fail to synthesize either the Ae or Ea polypeptide chains, as shown by both genetic complementation tests and analyses of total cell proteins by two-dimensional polyacrylamide gel electrophoresis. The patterns of expression of the intact Ea:Ae complex are consistent with the theory that both the Ae and Ea polypeptide chains must be present in the cells for either chain to be expressed in normal amounts on the cell surface. The implications of these observations for the genetics ofI-region-controlled functions are discussed.