The overall objective of this proposal is to understand the processes which control hematopoietic differentiation at the molecular level. The following specific aims will be addressed: 1. To develop a more complete understanding of the factors which control globin gene expression by: a.) Evaluating the impact on the expression of the mouse beta globin gene of mutation at sites in the IVS2 region which exhibit specific binding and footprinting for tissue specific binding proteins B1 and B2 b.) Further characterizing and purifying DNA binding proteins B1 and B2 and c.) Extending DNA binding studies to additional sites in the mouse beta globin gene. 2. To extend our initial studies identifying mRNA regions which contribute to mRNA stability in beta globin and c-fos mRNAs by: a.) Developing additional chimeric constructs between human c- fos and beta globin mRNAs to discriminate with greater precision mRNA sequences or structures which contribute to the differences in stability between these mRNA species. b.) Extending the chimeric mRNA approach to additional mRNA species including alpha globin, c-myc and delta globin mRNAs. C.) Identifying an inducible expression system which will allow us to extend the evaluation of mRNA stability to differentiating hematopoietic cells d.) Developing biochemical and genetic methods for identifying cellular mechanisms which respond to signals within mRNA molecules which lead to differences in half life. 3.) To develop somatic cell genetic approaches to hematopoietic differentiation by: a. Applying gene transfer procedures to determine the significance of changes in expression levels of specific polypeptides in the control of the differentiation program. b. Developing the use of retroviral vectors for insertional mutagenesis in the MEL cell system c. Developing multidrug resistance gene system for use as a selection system for bone marrow progenitor cells and pluripotent stem cells.
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