PRCTEIN-DNA INTERACTIONS IN HUMAN CHROMOSOMES

人类染色体中 PRCTEIN-DNA 相互作用

基本信息

批准号：
3283479
负责人：
SAMUEL A. LATT
金额：
$ 24.75万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-01-01 至 1989-01-31
项目状态：
已结题

项目摘要

The research proposed is aimed at understandng the chemical and cytological features of human chromosome structure, replication and the response to DNA damage. Fluorescent dyes, including new dyes with red fluorescence, will be used singly and in pairs, the latter sensing energy transfer, together with flow cytometry, to characterize better the DNA content, DNA replication kinetics, and chromatin condensation of normal and genetically abnormal cells (from patients with diseases (Fanconi's anemia (FA) and ataxia telangiectasia (AT), associated with a predisposition for the development of cancer), before and after DNA damage. Cytochemical detection of BrdU incorporation will contribute to this work and to experiments focusing on sister chromatid exchange (SCE) formation. Cytological and biochemical studies of factors, e.g. DNA methylation, which influence SCE formation, a sensitive index of cellular exposure to environmental mutagens, will be performed, and controlled damage(e.g. with psoralens) of DNA used to test hypotheses for the mechanism of SCE formation and to search for biochemical evidence of DNA interchange associated with SCE formation in defined DNA sequences. Cloned DNA probes complementary to highly amplified DNA sequences, localizable at a cytological level, and employed in Southern blots will be important for this work. Biochemical studies probing the repair defects in FA and AT are planned, as are DNA transformation experiments, using human DNA and rodent cells with DNA repair defects modelling these diseases, with the goal of better diagnosis and understanding of the genetic changes and molecular pathology of FA and AT. Analysis of DNA methylation, chromatin condensation, and DNA replication kinetics will also be extended to a molecular level to study the control of gene expression associated with human X chromosome inactivation. Several cloned genomic and cDNA probes, with homology to the human X, will be employed, together with quantitative Southern blotting, selective photodegradation of BrdU-substituted DNA sequences, northern blotting, digestion of DNA with isoschizomer restriction enzyme pairs, DNA'se digestion of chromatin, and protein analysis on flow sorted active and inactive human X chromosomes to investigate the molecular correlates and mechanism of X inactivation, particularly as they vary between different regions of the X or different cell types. The results should aid diagnosis and understanding of the phenotypic consequences of human X chromosome abnormalities.

拟议的研究旨在了解化学和细胞学人类染色体结构、复制和对 DNA 的反应的特征损害。荧光染料，包括具有红色荧光的新型染料，将可以单独使用，也可以成对使用，后者一起感应能量转移使用流式细胞术，更好地表征 DNA 含量、DNA 正常和遗传的复制动力学和染色质浓缩异常细胞（来自患有疾病（范可尼贫血 (FA) 和毛细血管扩张共济失调 (AT)，与以下倾向相关 DNA 损伤之前和之后。细胞化学 BrdU 掺入的检测将有助于这项工作并重点关注姐妹染色单体交换（SCE）形成的实验。因素的细胞学和生化研究，例如DNA 甲基化，影响 SCE 的形成，这是细胞暴露于环境诱变剂，将被执行，并控制损害（例如与 DNA 补骨脂素 (psoralens) 用于检验 SCE 机制的假设形成并寻找 DNA 交换的生化证据与特定 DNA 序列中 SCE 的形成相关。克隆DNA探针与高度扩增的 DNA 序列互补，可定位于细胞学水平，并用于 Southern 印迹对于这项工作。探讨 FA 和 AT 修复缺陷的生化研究计划使用人类 DNA 和啮齿动物进行 DNA 转化实验具有 DNA 修复缺陷的细胞模拟这些疾病，目的是更好地诊断和理解遗传变化和分子 FA 和 AT 的病理学。 DNA甲基化、染色质分析 DNA复制动力学也将扩展到分子水平研究与相关基因表达的控制人类X染色体失活。几个克隆的基因组和 cDNA 探针，与人类 X 同源，将与定量一起使用 Southern blotting，BrdU 取代 DNA 的选择性光降解序列、RNA印迹、同裂酶消化 DNA 限制性内切酶对、染色质 DNA 消化和蛋白质对流式排序的活性和非活性人类 X 染色体进行分析研究 X 失活的分子相关性和机制，特别是因为它们在 X 的不同区域或不同的区域之间有所不同细胞类型。结果应有助于诊断和理解人类 X 染色体异常的表型后果。