This research effort will deal with the expression of specific genes which determine distinctive normal and abnormal phenotypes of human hematopoietic cells. Two separate but related areas will be explored. The first involves functional abnormalities in globin gene expression causing deficient synthesis of Alpha and Beta globin in the thalassemia syndromes. The second area focuses on the regulation of specific genes whose selective expression or nonexpression produces the distinctive phenotypes of erythrocytes (e.g., globin and glycophorin) granulocytes (e.g. lysozyme and lactoferrin) and platelets (e.g., platelet glycoprotein). Studies of thalassemia will be oriented toward delineation of abnormalities of globin mRNA metabolism predicted by mutations already known (in some cases), or to be identified by recombinant DNA cloning and nucleotide sequence analysis of the abnormal genes. Transcription, processing, stability and function of globin mRNAs will be examined by use of molecular genetic methods including S1 nuclease mapping, primer extension analysis, in vitro transcription and translation, and analysis of radioactive mRNA synthesis and turnover in intact erythroid cells and foreign host cells carrying cloned globin genes. Studies of gene regulation during hematopoietic cell maturation will utilize normal and leukemic progenitor cellslfrom patients and human leukemia cell lines which exhibit phenotypes of selected lines of hematopoietic development (e.g., K562 cells (erythroid); HL60 (granulocytic)). Libraries of cloned mRNA sequences prepared from each cell type will be screened by in vitro hybrid selected translation and immunopreiciptation techniques to identify genes expressing proteins known to contribute to each of the above phenotypes. Families of shared and unique mRNAs will be identified by cross-hybridization of cDNA probes derived from each cell type with clones of the other. Clones containing unique or shared sequences will be used as hybridization probes to examine the differential expression of these genes in the different cell types, by nalysis of mRNA synthesis, accumulation, and translation. The goal of these studies will be improved understanding of patterns of gene expression which generate normal, thalassemic, and leukemic blood cells.
| Emanuel, J R; Garetz, S; Schneider, J et al. (1986) Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells. Mol Cell Biol 6:2476-81 |
