Many of the hematopoietic cells that participate in immune and inflammatory responses [including mast cell, basophils, natural killer cells, cytotoxic T lymphocytes, eosinophils, neutrophils, macrophages, and platelets] store a unique family of proteoglycans in an intracellular secretory granule compartment. Because these proteoglycans are present in such a wide range of effector cells of the immune response and because the gene that encodes the peptide core of these proteoglycans is expressed very early in the differentiation of hematopoietic cells, it is now clear that the regulation of this particular gene is extremely important of the development and function of these hematopoietic cells. The objective of the research proposed in this application are to determine the primary sequence of the secretory granule proteoglycan peptide core gene in two evolutionarily distant species, and then to understand how this gene is regulated in hematopoietic cells. Genomic fragments of about 18 kilobases in size have been isolated that contain the gene that encodes the peptide core of this novel family of proteoglycans. Double stranded nucleotide sequencing using chemically synthesized oligonucleotide primers will be used to determine the complete nucleotide sequence of the human and mouse gene. Data have already been obtained that the nucleotide sequences that encode the N- terminus of the peptide core are highly conserved through evolution, as are specific nucleotide sequences within the 5' and 3' untranslated regions of their mRNA transcripts. Likewise, when the 5' flanking region of the human gene was compared to the corresponding 5' flanking region of the analogous mouse gene, a region that immediately precedes the transcription-initiation site was found to be nearly identical. The observation that this latter sequence is even more highly conserved than any corresponding region of the gene that is translated into protein implies that this 5' flanking region contains cis acting regulatory elements that are critical for expression of this gene in hematopoietic cells. Hematopoietic cells and non- hematopoietic cells will be transfected with chimeric constructs to elucidate the regions of the gene which are important for its transcriptional regulation. Antibodies will be raised that recognize the peptide core of the mouse proteoglycan to study the translation and glycosaminoglycan modification of this proteoglycan in transfected cells.
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