Reduction of class I expression in a bovine B-lymphoblastoid cell line is locus specific

Abstract

A bovine B-lymphoblastoid cell line, BL3.1.2, defective in MHC class I expression was isolated following γ irradiation and immunoselection of a previously mutated cell, BL3.1. Flow microfluorimetry demonstrated that BL3.1.2. cells expressed reduced amount of class I molecules on the cell surface when compared with the parent cells. 2D gel electrophoresis showed that two less class I gene products were synthesized by BL2.1.2 cells than the parent cells. However, no detectable RFLP difference was revealed between this mutant and the parent cell lines as analyzed by Southern blots using bovine class I cDNA and locus-specific oligonucleotide probes, suggesting no major deletions, insertions, or translocations within class I genes. Northern blot analysis, using class I cDNA probes, revealed a marked reduction of class I mRNA in BL3.1.2. cells. RNA unblots, probed with loci-specific oligonucleotides, and blot analysis demonstrated that absence of class I gene transcription was locus specific. This finding indicates there is separate regulation of each class I locus in bovine cells. Furthermore, recombinant IFN-γ upregulated class I gene expression on the BL3.1.2 cells, as indicated by flow cytometry and slot blot analysis. These findings indicate that the locus-specific MHC class I gene was functionally downregulated in its expression. The silent class I gene could be activated by cytokines known to activate the cis-interferon-responsive element for subsequent transcription and translation of the newly synthesized class I RNA. These studies provide evidence that absence of class I gene transcription in BL3.1.2 cells resulted from regulation of a locus-specific gene and suggest that a deficiency in locus-specific trans-acting factor(s) is instrumental in silencing the class I gene.