Free Ia Eα chain expression in the Eα+:Eβ−: recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E_a chain expression in the E_a⁺E_ß^-I-region recombinant strain, A.TFR5. A.TFR5 (A^fE^k, ap5), a recombinant between A.CA (A^fE^f) and A.TL (A^kE^k), carries the E^k subregion. Previous results have shown that it expresses the E_a chain, but at reduced levels relative to E_a⁺E_ß⁺strains. No E_ß chains were detected, which is consistent with the A.TFR5E_ß gene being derived from the A.CA parent, which carries the null E_ß^fallele. In this paper, the defect in E_a-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E_a, in the region of the large intervening sequence of E_ß. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E_a message, but no E_ß 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 E_a 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 E_a, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E_ß, the E_a 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.