Molecular analysis and repair of a defective Ef alpha gene.
The molecular basis for the defective expression of Ef alpha was determined by analysis of the 5' region of a full length Ef alpha 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 Ef alpha 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 alpha 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 Ef alpha transcripts initiate identically to the functional Ek alpha allele, mapping the defect in the Ef alpha gene 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 k allele were exchanged. The hybrid genes were inserted into an SV40 promotor driven expression vector for cotransfection with an Ek beta 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 k allele and the 3' portion of the f allele. Therefore, the single stop codon present in the exon encoding the leader peptide of the Ef alpha gene appears to be the only defect preventing this gene from expressing a functional E alpha-chain.
Donovan
K A
KA
Department of Immunology, Mayo Clinic, Rochester, MN 55905.
Singh
S K
SK
David
C S
CS
Pease
L R
LR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003062:Codon; D004260:DNA Repair; D004274:DNA, Recombinant / Q000302:isolation & purification; D005091:Exons; D005802:Genes, MHC Class II; D000949:Histocompatibility Antigens Class II / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D014162:Transfection
D003062:Codon; D004274:DNA, Recombinant; D000949:Histocompatibility Antigens Class II; C024138:I-E-antigen