The normal development of mammalian hematocyte stem cells requires proteins and genes which participate in signal transduction pathways. Understanding these pathways is important to diseases such as Chronic Myelogenous Leukemia (CML), in which the c-Abl protein tyrosine kinase functions abnormally. However, it is not possible at this time to systematically characterize an entire hematopoietic regulatory pathway using mammalian experimental systems. The fruit fly Drosophila melanogaster offers an excellent alternative, because of the powerful genetic and molecular techniques available. We propose to use the fly as a model system for studying the pathways that regulate hematocyte stem cell development. The majority of fly blood cells are functionally analogous to human macrophages. One dominant Drosophila mutation, Tumorous-lethal(Tum-l), causes an overproliferation and premature differentiation of blood stem cells, resulting in large melanotic masses, invasiveness into other tissues, and lethality. The Tum-l gene encodes a putative nonreceptor protein tyrosine kinase, with homology to oncogenes such as the vertebrate fes feline retroviral transforming protein. We propose to investigate the molecular mechanisms by which this mutation causes a misregulation of hematocyte stem cells, and to identify other genes whose products interact with Tum-l in the same signal transduction pathway. Ultimately, we will attempt to determine whether a similar pathway regulates human hematopoiesis. Specifically, (i) The functional differences between normal and mutant Tum-l protein will be determined. Polyclonal antibodies to the Tum-l protein will be raised. These antibodies will be used to compare the protein concentration, localization, and in vitro tyrosine kinase activity of the normal and mutant Tum-l proteins. (ii) The amino acid substitution(s) responsible for the mutant Tum-l protein will be determined. Mutant Tum-l DNA will be sequenced and compared to its wild- type counterpart. For each amino acid substitution found, the corresponding nucleic acid alteration will be made in the wild-type gene by in vitro mutagenesis. Transgenic fly stocks will then be made for each construct by P element-transformation, and the resulting effects on blood stem cells and overall development will be determined. (iii) Other genes participating in the Tum-l stem cell signal transduction pathway will be identified. A chemical mutagenesis scheme will be used to identify genes whose products interact with Tum-l. (iv) The most promising mutation identified will be further analyzed. The specific effects of this mutation on blood stem cells will be studied, and the mutated gene will be cloned and characterized molecularly.
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