Increased knowledge of the molecular basis for gene expression during myelopoiesis would help to clarify the fundamental mechanisms by which cell growth and differentiation occur, and might suggest ways to treat myeloproliferative disorders more effectively. Human myeloid leukemia cells express membrane-associated glycoproteins that are also expressed by normal myeloid progenitors and subsets of differentiated myeloid cells in a lineage-specific manner. The genes encoding such proteins are ideal candidates for studies of lineage-restricted transcriptional regulations during myelopoiesis. We have partially characterized two human genes that encode glycoproteins (gp150 and gp67) expressed by myeloid cells. The genes were isolated from mouse cell lines after DNA-mediated gene transfer, and transformants with highly amplified expression of both genes were isolated with a fluorescence-activated cell sorter, by use of specific monoclonal antibodies. Portions of the genes have been molecularly cloned, and appropriate probes have been used to identify transcripts expressed in human myeloid cell lines. We propose to characterize the 5' regulatory sequences of both genes and define elements that control their lineage-specific expression in vitro and in vivo. Plasmid constructs containing transcriptional regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) gene will be assayed for their ability to be expressed in different hematopoietic cell lines, and will be used to test for the presence of specific DNA binding proteins that coordinate gene expression. The role of defined regulatory sequences from the human genes or their mouse homologues will be tested in vivo by introducing retroviral vectors containing these sequences into mouse hematopoietic stem cells. Different """"""""reporter"""""""" gene sequences (including CAT, oncogenes, and the coding sequences of gp150 and gp67 themselves) will be used to determine the ability of regulatory sequences to program gene expression during myelopoiesis. These experiments will test the concept tha cis-controlling elements adjacent to coding regions of the introduced genes can interact with cellular factors in a lineage-specific manner. The studies will provide insights into the molecular basis of differential gene expression during hematopoiesis, and may provide a conceptual framework for investigations of altered gene expression in human myeloid leukemias.
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