ERYTHROID DIFFERENTIATION IN FRIEND LEUKEMIA CELLS

本提案的总体目标是了解 控制造血分化的过程， 分子水平。 将处理以下具体目标： 1. 为了更全面地了解 其通过以下方式控制球蛋白基因表达：a.）评价 对小鼠β珠蛋白基因表达的影响 IVS 2区域中表现出特异性结合的位点处的突变 和组织特异性结合蛋白B1和B2的足迹法（footprinting）B.） 进一步表征和纯化DNA结合蛋白B1和 B2和c.）将DNA结合研究扩展到 小鼠β珠蛋白基因。 2. 为了扩展我们最初的研究，确定mRNA区域， 通过以下方式促进β珠蛋白和c-fos mRNA的mRNA稳定性： a.）开发人c- fos和β珠蛋白mRNA，以更高的精度区分 mRNA序列或结构，有助于 这些mRNA种类之间的稳定性差异。 B.） 将嵌合mRNA方法扩展到其他mRNA 包括α珠蛋白、c-myc和δ珠蛋白mRNA。C.）的 确定一个诱导表达系统，使我们能够 将mRNA稳定性的评估扩展到分化 造血细胞d.）发展生物化学和遗传学 用于鉴定响应于以下的细胞机制的方法 mRNA分子内的信号，导致一半的差异， 生活 3.）第三章发展体细胞遗传学方法， 造血分化通过：a.应用基因转移 确定表达变化的重要性的程序 特异性多肽在控制分化中的水平 程序. B.开发逆转录病毒载体的用途， MEL细胞系统中的插入诱变c.发展中 用作选择系统的多药耐药基因系统 骨髓祖细胞和多能干细胞。

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.