Free Ia E alpha chain expression in the E+ alpha : E- beta recombinant strain A.TFR5.

Abstract

Molecular and biochemical techniques have been used to explore the reasons behind low E alpha chain expression in the E+ alpha E- beta I-region recombinant strain, A.TFR5. A.TFR5 (Af Ek, ap5), a recombinant between A.CA (Af Ef) and A.TL (AkEk), carries the Ek subregion. Previous results have shown that it expresses the E alpha chain, but at reduced levels relative to E+ alpha E+ beta strains. No E beta chains were detected, which is consistent with the A.TFR5E beta gene being derived from the A.CA parent, which carries the null Ef beta allele. In this paper, the defect in E alpha-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E alpha, in the region of the large intervening sequence of E beta. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E alpha message, but no E beta 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 alpha 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 alpha, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E beta, the E alpha 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.