The biological factors which regulate megakaryocyte differentiation and platelet production in vivo have not been identified. We propose to use a number of innovative molecular biological techniques to isolate and characterize a series of candidate genes whose gene products are involved in megakaryocytopoiesis. Human leukemias associated with rearrangements of band 3g21 (often with 3g26) have characteristic dysmegakaryocytopoiesis and thrombocytosis, indicating that gene(s0 in this region may regulate megakaryocytopoiesis. We shall precisely microdissect band 3g21, employing a high resolution system we have developed, and amplify microdissected chromosomal DNA by PCR using """"""""universal"""""""" primers containing six degenerate bases. The amplified product will be radioisotopically labelled and used to probe, 1) a normal human bone marrow cDNA library, and 2) a cDNA library derived from leukemic myeloblasts carrying the t (3;3) (g21;26.2) translocation, two cell sources predicted to express the gene(s) of interest located at 3g21. Isolated clones will be mapped to metaphase chromosomes using fluorescent in situ hybridization an characterized by insert size, restriction length analysis and by DNA sequencing. An additional set of cDNA clones will be isolated using microdissected band 3g26.2 DNA as probe. The primary structure of novel clones will be subjected to computer homology search and analysis to elucidate structure/function relationships with known genes, particularly hematopoietic growth factors and plasma membrane receptors. This methodology provides a rapid and direct approach for isolating novel expressed genes from specific chromosome regions. The gene products of the isolated genes expressed in bone marrow and specific leukemic cells may have an important role in pathological conditions of megakaryocytes and be useful in the treatment of drug-induced thrombocytopenia.
