Molecular analysis and repair of a defective E(α)(f) gene

K. A. Donovan, S. K. Singh, C. S. David, L. R. Pease

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

The molecular basis for the defective expression of E(α)(f) was determined by analysis of the 5' region of a full length E(α)(f) gene. The gene was isolated from a genomic library prepared from the A.CA/SnDv mouse strain. DNA sequence analysis of the 5' portion of the E(α)(f) gene, which encodes the 5' regulatory sequences and the signal peptide, revealed the presence of a stop codon in the exon encoding the signal peptide. The remainder of the sequences were highly related to sequences found in previously characterized, functional E(α) alleles. Previous studies indicate that the f allele is transcribed at rates comparable to the rates of functional alleles and that mRNA accumulates in the cytoplasm. Primer extension analysis demonstrated that E(α)(f) transcripts initiate identically to the functional E(α)(f) allele, mapping the defect in the E(α)(f) gene of 3' of the transcriptional initiation site. To determine whether the stop codon in the signal peptide was the only major defect in this gene, reciprocal chimeric genes were constructed in which the 5' regions, including the first exons, of the defective f allele and the functional κ allele were exchanged. The hybrid genes were inserted into an SV40 promotor driven expression vector for co-transfection with an Eβ)((k) gene. Surface I-E expression was demonstrated using I-E specific mAb in Cos-7 and L cell lines transfected with the hybrid gene consisting of the 5' region of the κ allele and the 3' portion of the f allele. Therefore, the single stop codon present in the exon encoding the leader peptide of the E(α)(f) gene appears to be the only defect preventing this gene from expressing a functional E(α)-chain.

Original languageEnglish (US)
Pages (from-to)4034-4040
Number of pages7
JournalJournal of Immunology
Volume142
Issue number11
StatePublished - 1989

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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