The studies proposed in this application will investigate the molecular mechanisms of gene regulation during the differentiation of T lymphocytes from committed progenitor cells. DNA sequence elements involved in gene regulation will be localized in and around genes for T cell surface proteins which are activated at specific points in the T lymphocyte lineage. In the mouse, one of he earliest phenotypic changes is the appearance of the Thy-1 antigen on immature thymocytes. In previous studies, the Thy-1 gene has been cloned, the entire 6094 base pair DNA sequence determined, and several regions with tissue-specific transcriptional enhancer activity identified by the ability to activate in cis the indicator gene chloramphenicol acetyl transferase. In addition, two promoters encoding two distinct 5' untranslated regions of the Thy-1 mRNA have been identified. This proposal has the following goals: 1) to determine by deletion mapping and oligonucleotide linker exchange the sequence requirements for T cell specific transcription of the Thy-1 gene, 2) determine the developmental significance of these sequences by transfection into cell lines which can be induced to differentiate, 3) introduce specific constructions containing regulatory regions and indicator genes into transgenic mice to access the developmental significance in vivo, 4) undertake identification and/or isolation of trans-acting gene products required to activate or represss the T cell enhancer elements using a novel indicator genes, the bacterial luciferase genes from the V. fischeri lux operon, and 5) localize cis-regulatory regions in genes for other T cell-specific surface proteins, notably the Tla thymocyte-specific class I MHC gene, L3/T4 and Lyt-2/T8 T-lineage specific genes, and lymphocyte-restricted class I MHC genes from the mouse Qa region. The long term goals of this project are to define intragenic regulatory elements which control T cell specific gene expression at different points in the lymphocyte lineage, and to understand how these elements may contribute normal differentiation to disease states involving developmental abnormalities.
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