Browsing by Subject "Immunoglobulin genes"
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Item Isolation and characterization of the alpha and beta subunit genes of the high affinity receptor for Immunoglobin E(Texas Tech University, 1992-05) Witthuhn, Bruce AAllergic responses are mediated through the high affinity Fc receptor for immunoglobulin E (FceRI) which is expressed exclusively on mast cells and basophils. These responses occur when receptor-bound IgE molecules are crosslinked on the cell surface via an interaction with an antigen. The FceRI receptor is a heterotetrameric protein structure, composed of three distinct polypeptides - one alpha, one beta and two gamma subunits, The rat alpha and beta subunit genes have been cloned in this study in order to better understand their tissue-specific expression, The alpha subunit gene was found to span eight kilobase pairs of DNA. A construction consisting of the entire coding region and the 5'- and 3'- flanking regions was electroporated into the mouse mastocytoma cell line, P815, which does not express the endogenous genes for the alpha or beta subunits of the FceRI. The transcription of the exogenous rat alpha subunit gene was demonstrated by Northern blot and polymerase chain reaction analyses. These results suggest that the tissue-specific nature of expression is conserved across species lines. The beta subunit gene composed of seven exons and six introns spans nine kilobase pairs of DNA. Analysis of the 5'-flanking region identifies putative transcriptional cis-control elements, including PyPyCAPyPyPyPy, TATA and CAAT consensus sequences. Also identified are the consensus binding sites for the GATA transcription factors, as well as potential interferon-y regulated consensus elements, Also noted are an 19 bp homopurine-homopyrimidine direct repeat and an 11 bp homopurinehomopyrimidine inverted repeat embedded in a 123 bp homopurinehomopyrimidine region. These elements may contribute in the tissuespecific expression of this gene. Alternative RNA processing involving exon three may explain the origin of the two previously reported beta subunit transcripts. The close correspondence of the structure of the carboxy-terminal coding exon to a recently identified functional cytoplasmic domain is noted for the full-length transcript. Some correlations of predicted structural features of the polypeptide to the other exons are also apparent.Item The expression and characterization of a series of rate IgE produced from in vitro reconstructed epsilon heavy chain genes(Texas Tech University, 1991-12) McMillan, Daniel RandyThe interaction between allergen-specific IgE and the high affinity Fc receptor on the surface of mast cells or tissue basophils forms the basis for the Type I Immediate Hypersensitivity reaction, or more commonly, the allergic response. In order to rigorously analyze this interaction, a series of exon modules have been produced to facilitate the production of mutant IgE. The functionally rearranged e-heavy chain gene isolated from the rat IgE secreting immunocytoma, IR162, was utilized for the creation of this system of exon modules. 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 in such a way as to preserve or reconstruct all the required mRNA splicing, cleavage and polyadenylation signals. The vectors utilized here permit the use of flanking restriction enzyme sites for the modular manipulation of the e-heavy chain gene. These exon modules were initially used to reconstruct the e-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. The immunoglobulin was indistinguishable fix)m authentic IgE, based on heavy chain specific mRNA initiation, overall e-heavy chain specific mRNA length, size of polypeptides recognized by anti-IgE antibodies and high affinity Fc epsilon receptor binding activity. The modular system of exons was then used to produce mutant IgE that were deficient in one or more whole domain(s) through the systematic assembly of different mutant e-heavy chain gene constructions. The data obtained with the full-length eheavy chain gene validates the efficacy of the modular approach and suggests that the use of mutant IgE, produced in such a manner, in receptor binding studies will provide information on the specific domain(s) involved in interaction with the receptor. In addition, a chimeric x-light chain gene coding for a variable region that contributes to specificity for the hapten, trinitrophenol, was produced and used to develop a second host cell line. Utilization of this cell line will permit the production of recombinant IgE with known antigen specificity for further purification and functional studies. Construction of the exon modules, full-length and mutant e-heavy chain genes and the physical and functional characterizations of the resulting RNA and polypeptide products of the full-length and mutant e-heavy chain genes are described.