The long term objective of the present proposal is to understand, in molecular detail, the assembly of MHC Class I products from their subunits, and the binding of peptides to MHC Class I molecules in genetically modified mice, in living cells and in cell-free systems. The proposed experiments will address the role of the MHC Class I subunits themselves and the accessory proteins required for their proper assembly. They will also address the question of how peptides are generated and delivered to the site where combination with Class I molecules takes place, and examine the specificity of their interactions with Class I molecules. Those questions will be approached with the methods of molecular biology (gene cloning; polymerase chain reaction; transgenic mice; mutant mice produced by the ES cell homologous integration technique; in vitro transcription/translation; site directed mutagenesis; transfection), cell biology and immunology (cell culture; hybridoma production; immunochemistry; cytofluorimetry; immunoelectron microscopy). Chemical synthesis will be used not only to generate peptides of defined sequence, but also to produce peptide libraries with which to investigate the specificity of MHC Class I peptide interactions. The results obtained in this project should contribute knowledge that will help provide a rational basis for manipulating the formation of specific MHC Class I-peptide combinations. this area of experimentation is relevant not only for understanding development of the T cell repertoire, but also for tumor immunology, autoimmunity and vaccine development.
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