The expression and characterization of rat IgE produced by construction of the epsilon-heavy chain gene from exon modules.
A system of exon "modules" was produced from the functionally rearranged epsilon-heavy gene isolated from the rat IgE-secreting immunocytoma IR162. The five individual exons, encoding the variable and constant region domains, were isolated and subcloned into the multiple cloning site of a pair of plasmid vectors with opposed orientation multiple cloning sites. The use of opposed orientation multiple cloning sites and the flanking restriction enzyme sites contained therein allows for the modular manipulation of the gene. These exon modules were initially used to reconstruct the epsilon-heavy chain gene into the native configuration to demonstrate the efficacy of the modular system for synthesis of IgE. Upon transfection into the rat myeloma cell line Y3, the reconstructed gene produced a polypeptide that associated with the endogenous light chain polypeptide and was secreted from the cell as tetrameric IgE. All physical and functional characterizations indicate that the IgE molecule produced is indistinguishable from native IR162 IgE. This modular system of exons will facilitate the manipulation of IgE structure through the systematic assembly of different epsilon-heavy chain mutant constructions. The resulting novel IgE proteins will be very useful to study the molecular nature of the interaction of IgE with its Fc receptor.
McMillan
D R
DR
Department of Biochemistry and Molecular Biology, Texas Tech University Health Sciences Center, Lubbock 79430.
Faust
C
C
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D006031:Glycosylation; D007073:Immunoglobulin E / Q000096:biosynthesis / Q000235:genetics*; D007139:Immunoglobulin epsilon-Chains / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D007139:Immunoglobulin epsilon-Chains; D012333:RNA, Messenger; D014415:Tunicamycin; D007073:Immunoglobulin E