Current knowledge of the human Major Histocompatibility Complex (MHC) will be used to discover new alleles or genes in the Class II region of chromosome 6p and to better define the extent of non-random association (linkage disequilibrium) of genes in the extended haplotypes. As there is a fixity of alleles of the MHC in extended haplotypes, it is possible to predict the presence of other unknown genes/alleles in this region, since a proportion of MHC-identical extended haplotype-matched individuals demonstrate alloreactivity. The new markers will be studied by restriction fragment length polymorphism (RFLP), mixed lymphocyte reaction, T cell clones, antibody blocking and biochemical techniques. These analyses will be done primarily with two extended haplotypes (HLA-B8,DR3,DQw2,DRw52,SC01) and (HLA-B18,DR3,DQw52,F1C30) which define two subtypes of HLA-D region gene products. These subtypes of HLA-DRw52, DR3 and DQw2 will be studied by: 1. Allostimulation between the extended haplotype-matched unrelated individuals and its correlation with RFLP patterns which will serve to develop programs for the selection of potential unrelated donors for bone marrow transplantation. 2. cDNA cloning and DNA sequencing to identify the sequences specific for the subtypes and to delineate the evolutionary mechanisms responsible for the generation of these subtypes. These results will clarify whether single or double gene conversion events and responsible for the generation of DR3 subtypes. 3. By antigen presentation studies and two-dimensional gel electrophoresis to determine the functional and structural equivalence or difference of the subtypes. Since these extended haplotypes are associated with insulin-independent diabetes mellitus and gluten-sensitive enteropathy, our studies will serve to identify, at the DNA level, markers of disease susceptibility. DNA sequence data will be useful in generating specific oligonucleotide probes for these disease-associated haplotypes. The mechanism regulating the differential expression of DQ antigens will be approached by: 1. Studying the expression, after transfection, of DQ gene constructs containing different lengths of 5' upstream sequences in leukemic cell lines representing different stages of myelomonocytic cell differentiation. 2. Cell- cell hybridization between DQ expressing and non-expressing cells. These studies will identify the possible involvement of negative regulatory sequences and transacting repressor mechanisms in the stable repression of Class II genes during myelomonocytic cell differentiation.
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