ERYTHROID DIFFERENTIATION IN FRIEND LEUKEMIA CELLS

这项建议的总体目标是了解 控制造血细胞分化的过程 分子水平。将实现以下具体目标： 1.对这些因素有更全面的了解 它们通过以下方式控制珠蛋白基因的表达：a.)评估 对小鼠β-珠蛋白基因表达的影响 IVS2区显示特异性结合的位点的突变 以及组织特异性结合蛋白B1和B2b的足迹。) DNA结合蛋白B1和DNA结合蛋白的进一步鉴定和纯化 B2和c.)将DNA结合研究扩展到更多的 小鼠β珠蛋白基因。 2.扩大我们的初步研究，确定哪些信使核糖核酸 通过以下方式促进β珠蛋白和c-fos mRNAs的mRNA稳定性： A.)开发更多的嵌合结构在人类c- Fos和β珠蛋白mRNAs能更精确地区分 对基因序列或结构有贡献的mRNA序列 不同种类的信使核糖核酸稳定性差异。B.) 将嵌合信使核糖核酸方法扩展到额外的信使核糖核酸 物种包括α珠蛋白、c-myc和增量珠蛋白mRNAs。(C) 识别一种可诱导的表达系统，使我们能够 将信使核糖核酸稳定性的评价扩展到分化 造血细胞d.发展中的生化和遗传学 识别应答的细胞机制的方法 导致一半差异的mRNA分子内的信号 生活。3.)发展体细胞遗传学方法 通过：a.应用基因转移进行造血分化 确定表达变化的重要性的程序 特异性多肽水平在调控分化中的作用 程序。B.发展逆转录病毒载体用于 MEL细胞系发育中的插入突变 多药耐药基因系统作为多药耐药选择系统的应用 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.