. The availability of recombinant colony-stimulating factors (CSFs) for clinical use has presented medical science with its first opportunity to stimulate blood cell formation and host defense in man. Potential benefits of CSF administration include treating primary hematologic diseases, enhancing host defense against infection, and preventing chemotherapy-induced cytopenias. Elucidating the role colony-stimulating factors play in normal hematopoiesis, inflammation and leukemia cell growth requires a thorough understanding of the mechanisms by which cells control their production of CSFs. Studies on the inducible expression of GM-CSF in T cells and the constitutive expression of GM-CSF in HTLV infected T cell lines have identified a critical region of the GM-CSF promoter required for its mitogen-inducible activity and its responsiveness to the HTLV-I and -II tax proteins. In this grant we propose to investigate the DNA-protein interactions in this region to understand the role that expression of sequence-specific DNA-binding proteins play in the normal and abnormal production of hematopoietic growth factors. We will identify the precise nucleotide sequence recognized by GM-CSF promoter DNA-binding proteins by mutating specific nucleotides in the promoter and analyzing their effect on the binding of transcription factors using gel shift and DNase I footprinting assays. The proteins binding specifically to the GM-CSF promoter sequences will be characterized using Southwestern blotting. UV cross-linking and biochemical manipulations designed to identify post-transcriptional protein modifications. We will use these techniques to determine the presence and activity of these factors in nuclear extracts prepared from stimulated and unstimulated mature T cell lines and from a variety of immature or transformed cell lines. T cell DNA-binding proteins will be purified using DNA affinity chromatography and amino acid sequencing will enable us to design oligonucleotides for cloning DNA-binding factors from a T cell cDNA library. We will obtain polyclonal antisera for screening our lambda gtll T cell cDNA expression library and will also use radiolabelled DNA recognition site probes to isolate transcription factor cDNA clones. The studies proposed will provide valuable insights into the transcriptional mechanisms controlling hematopoiesis.
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