The general objective of this research is to delineate the control of the switch from Gamma to Beta globin gene activity in development, differentiation and in erythroleukemia lines using the approach of somatic cell hybridization. Our approach exploits experimental innovations done during the current period of the grant which make the investigation of the switches of the Beta globin genes feasible in spite of the lack of a selective system for chromosome 11. We will first focus on control of Beta and Gamma expression in HEL cells. With studies of HEL X MEL hybrids we will ask whether the evidence, from our current studies, that Gamma expression in HEL cells is controlled trans and by an element that segregates independently of the Beta gene cluster, is supported by cytogenetic analysis. We will examine whether the absence of Beta globin in HEL cells is due to presence of an inhibitor of Beta expression or to lack of elements that activate the Beta genes. Complementation experiments will test whether Beta activation is a multistep process. Analyses of HEL X MEL hybrids will test whether and how phenotypes of globin expression correlate with the methylation status of Beta and Gamma genes. Second, we will investigate the control of globin switching in normal cells. With fusions of MEL cells with erythroid cells from various stages of human development we will ask a central question in the biology of Hb switching, i.e., whether switching is controlled in cis or in trans. We will further examine whether the activation of Hb F in the adult follows the pattern of control that cells of earlier stages of development display and whether adult erythroid progenitors have an active F-program, as hypothesized. Third, we will apply our approach in the analysis of human mutants that are associated with continuation of Hb F in the adult; having the mutant chromosome alone in hybrid cells will allow studies that cannot be done with analyses of heterozygous cells. Fourth, we will examine whether the hybrids express other erythroid human characters, investigate the expression of such characters and attempt their chromosomal assignment. We will also attempt, using panels of monoclonal antibodies, to detect a cell surface character that is a marker of chromosome 11. This will enhance the usefulness of the somatic cell hybridization approach in studies of Gamma to Beta switching, by allowing application of cell sorting for hybrid selection. Overall this research will generate a significant amount of new information on the control of switching and will produce lines of cells that will be of general usefulness in cellular and molecular analysis of the switching phenomena.
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