REC-DNA ANALYSIS OF HUMAN HEMATOPOIETIC DIFFERENTIATION

人类造血分化的 REC-DNA 分析

• 批准号: 3170166
• 负责人: WINSTON SALSER
• 金额: $ 20.43万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1987-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3170166
• 关键词: DNA; cell differentiation; electron microscopy; electrophoresis; gene expression; genetic manipulation; genetic transcription; genome; hematopoiesis; histochemistry /cytochemistry; human therapy evaluation; human tissue; messenger RNA; molecular cloning; molecular oncology; myelogenous leukemia; neoplasm /cancer classification /staging; neoplasm /cancer diagnosis; neoplasm /cancer genetics; neoplasm /cancer therapy; nucleic acid sequence; radiotracer; ribosomal RNA; structural genes

The human leukemia cell line HL-60 can be induced to differentiate in vitro, permitting a recombinant DNA analysis of the complex series of differentiation steps leading from promyelocytes to either macrophages or granulocytes. We have isolated many genes which are strongly regulated. Characterization during the past year included discovery of a new human tubulin gene and the first human ferritin L subunit clones among the regulated genes as well as a gamma-actin with an amino acid substitution in a sequence normally highly conserved. Mutant beta-actin genes have been implicated as important in affecting oncogenic or metastatic potential. This is the first indication that gamma-actin alterations may have similar effects. Our main emphasis remains the development of a system for using site-directed mutagenesis to study commitment and terminal differentiation in human cells. We have now succeeded in obtaining efficient transfection of HL-60 cells with the pSV2neo vector. By cotransformation, tagged versions of genomic clones for genes which show strong regulation during HL-60 differentiation have also been inserted. Several tagged genes are expressed at high levels and show appropriate regulation. This suggests that we have succeeded in developing the first system permitting the use of site-directed mutagenesis to study the irreversible gene regulation changes occurring during the terminal differentiation of human cells. At the same time we have obtained evidence suggesting that gene regulation during myeloid terminal differentiation involves a complex series of gene switching events. Instead of a single coordinate turn-on, as has been seen in muscle or reticulocyte differentiation, we see a complex series of changes in the levels of different mRNAs. Translational regulation is also important, and in the case of the alpha-tubulin mRNA we have identified an unusual sequence in the 5' untranslated region which may explain a very strong translational regulation. (M)

人白血病细胞株HL-60可在体外诱导分化 体外，允许对复杂的系列进行重组DNA分析 从早幼粒细胞到巨噬细胞的分化步骤 粒细胞。我们已经分离出许多受强烈调控的基因。 在过去的一年里，人们的特征包括发现了一种新的人类 微管蛋白基因和第一个人铁蛋白L亚基克隆 受调控的基因以及带有氨基酸替代的γ-肌动蛋白 通常高度保守的序列。突变的β-肌动蛋白基因已经被 被认为是影响致癌或转移潜能的重要因素。 这是第一次有迹象表明，伽马-肌动蛋白的改变可能具有类似的 效果。 我们的主要重点仍然是开发一种使用 定点突变研究承诺和末端分化 在人类细胞中。我们现在已经成功地获得了高效的转染剂 将pSV2neo载体与HL-60细胞共培养。通过共转换，标记 在此期间表现出强烈调控的基因的基因组克隆版本 HL-60分化也已被插入。几个标记的基因是 在高水平表达，并表现出适当的监管。这表明 我们已经成功地开发出了第一个允许使用 定点突变研究不可逆性基因调控变化 在人类细胞的终末分化过程中发生的。同时 我们已经获得的证据表明，基因调控在 髓系终末分化涉及一系列复杂的基因 切换事件。而不是像已经看到的那样，打开单个坐标 在肌肉或网织红细胞分化中，我们看到一系列复杂的 不同mRNAs水平的变化。翻译监管也是 重要的是，在α-微管蛋白mRNA的情况下，我们已经识别出一种 5‘端非翻译区的异常序列可能解释了 强有力的转化性监管。(M)