One of the most important factors influencing transplantation and immune responsiveness is the structural variation of human leukocyte antigens (HLA). The highly polymorphic HLA proteins form complexes with a variety of peptides, and the HLA-peptide complexes are recognized in a very specific manner by T cell receptors. As a result of this specific interaction, subtle differences in HLA structure influence immune responsiveness, autoimmune reactions, and alloresponses following organ and tissue transplantation. The genetic basis for the extraordinary polymorphism of HLA loci, (i.e., the extent of polymorphism, the molecular mechanisms responsible for generating the polymorphism, the rate of evolution) as well as the physiologically important functions have intrigued investigators for many years. Although the existence of HLA polymorphism is well established, the molecular characterization is still in its infancy. This project will satisfy the need for more knowledge in this area by using new automated methods to determine the nucleotide sequences of large numbers of HLA genes. The sequence information will be used to develop new methods for analyzing HLA polymorphism using molecular biological techniques, e.g., sequence-specific oligonucleotide probe hybridization. The specific objectives will be (1) to determine the nucleotide sequences of class I HLA alleles, (2) to compare the polymorphic sequences with serological specificities in order to identify residues that may be involved in the formation of serological epitopes, (3) to determine the extent of class I HLA microheterogeneity, (4) to develop a practical and informative method for molecular analysis of HLA polymorphism, and (5) to use the data collected in conjunction with these studies to examine the molecular evolution of HLA polymorphism. This study is feasible for our laboratory because the basic techniques are already in use and large numbers of serologically typed samples from a variety of racial backgrounds are available for molecular analysis. This study will contribute to the long-term objectives of facilitating selection of donors for bone marrow transplantation and providing a basis for understanding HLA polymorphism and how it relates to the physiological role of the molecule. A further objective is to evaluate the feasibility of using oligonucleotide-based methods to complement or supplant conventional HLA typing methods that utilize serological and cellular methods.
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