Springer Online Journal Archives 1860-2000
Abstract Molecular and biochemical techniques have been used to explore the reasons behind low Eα chain expression in the E α + E β − I-region recombinant strain, A.TFR5. A.TFR5 (A f E k, ap5), a recombinant between A.CA (A f E f) and A.TL (A k E k), carries the E k subregion. Previous results have shown that it expresses the Eα chain, but at reduced levels relative to E α + E β + strains. No Eβ chains were detected, which is consistent with the A.TFR5E β gene being derived from the A.CA parent, which carries the null E β f allele. In this paper, the defect in Eα-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of Eα, in the region of the large intervening sequence of Eβ. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the Eα message, but no Eβ 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 Eα 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 Eα, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of Eβ, the Eα 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.
Type of Medium: