This proposal will involve the development of new systems for studying the expression of the thymus-leukemia antigens (TLa) during the differentiation of hematopoietic stem cells. TLa antigens are cell-surface glycoproteins, closely related to the major histocompetibility antigens H-2, which are found on surface of T-lymphocytes and are anomolously expressed on some leukemias in mice. Four alleles of the TLa locus have been described (TLa(a-e)). The genes for the TLa alleles from BALB/c, A/J, C57B1/6, P/J and A.CA mice will be cloned and characterized by DNA sequencing and mapping. These cloned TLa genes will be introduced into mouse fibroblasts in tissue culture by DNA-mediated gene transfer and their expression characterized. Then, a system for the study of the expression of cloned TLa genes in differentiated lymphocytes will be developed by inserting these genes into retrovirus vectors and introducing cloned TLa genes into established lymphoid cell lines by infection. The expression of cloned genes in functionally differentiated T cells will be studied and the DNA sequences necessary for physiological transcription localized. Non-physiological, constitutive expression may possibly be induced by promoter exchange with 5 feet flanking sequences of mouse H-2 genes. Genetic manipulation including In Vitro recombination and mutagenesis will be carried out to derive expressed TLa gene products which may be easily differentiated from the endogenous mouse gene products. Then, expression and function of the genetically engineered gene products will be studied in fibroblasts and T-cell expression systems. The functional sites on TLa genes which are recognized by monoclonal antibodies and cytolitic T-cells will be mapped within the protein structure. Finally, these cloned and modified genes will be introduced into intact animals by microinjection of mouse embryos or infection with retrovirus vectors containing cloned, modified TL genes. These studies may develop new strains of mice in which the development and function of the TLa antigens can be studied in intact animals. The long-term goals of this project are to understand the structure, function and expression of cell-surface recognition elements during development and to understand how the expression of these elements may be changed during malignant transformation.
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