PROTEIN-DNA INTERACTIONS IN HUMAN CHROMOSOMES

提出这项研究的目的是为了了解化学和细胞学 人类染色体结构、复制和对DNA反应的特点 损坏。荧光染料，包括带有红色荧光的新型染料，将 单独使用和成对使用，后者一起感应能量转移 用流式细胞仪更好地表征DNA含量，DNA 正常和遗传的复制动力学和染色质凝集 异常细胞(来自疾病患者)(Fanconi贫血(FA)和 共济失调毛细血管扩张症(AT)，与 癌症的发展)，在DNA损伤前后。细胞化学 BrdU掺入的检测将有助于这项工作和 以姐妹染色单体交换(SCE)形成为重点的实验。 细胞学和生物化学研究的因素，例如DNA甲基化， 影响细胞暴露的敏感指标--姐妹染色单体交换的形成 将执行环境诱变剂，并控制损害(例如，使用 补骨脂素)，用于检验姐妹染色单体交换机制的假说 DNA交换的形成及其生化证据的寻找 在已定义的DNA序列中与姐妹染色单体交换形成相关。克隆的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 blotts将是重要的 这项工作。探讨FA和AT修复缺陷的生化研究是 计划中的，以及使用人类DNA和啮齿动物的DNA转化实验 带有DNA修复缺陷的细胞模拟这些疾病，目标是 更好地诊断和了解遗传变化和分子 FA和AT的病理改变。DNA甲基化、染色质分析 缩合和DNA复制动力学也将扩展到 在分子水平上研究相关基因表达的调控 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，将与量化一起使用 BrdU取代DNA的Southern印迹、选择性光降解 测序，Northern杂交，DNA同裂异构体消化 限制性内切酶对、染色质的DNA消化和蛋白质 人类X染色体活体和非活体染色体流向分析 研究X失活的分子相关因素和机制， 尤其是当它们在X的不同区域或不同 单元类型。这些结果应该有助于诊断和理解 人类X染色体异常的表型后果。

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.