CHROMATIN STRUCTURE AND FUNCTION HISTONE MODIFICATIONS A

这些建议的长期目标旨在 了解活性染色质的结构和功能。证据 已经积累了支持染色质功能状态的观点 结构域与变量有关，例如：1)核心的乙酰化 组蛋白；2)组蛋白H1的可能耗尽或丢失；3)HMG的结合 蛋白质14和17；4)H_2A和H_2B的泛素修饰；5)DNA 甲基化；6)DNA超螺旋和多态。想必这样的变化 允许访问特定蛋白质以识别并与DNA调节结合 站点，并提供结构正确的模板和DNA拓扑 以供抄写。我们的细胞周期研究只将最高 H3和H4与转录的乙酰化状态，也表明 UH_2A和uH_2B在中期之前的前期消失， 后期迅速重现。染色质的这些特定区域被标记 UH_2A和uH_2B似乎最后被包装到中期染色体中。 已有研究表明，uH2A位于潜在的可转录区域 染色质。关于组蛋白乙酰化作用的研究正在进行中， 泛素化与HMG蛋白14和17在结构上的结合 和寡核小体的结构转变以及DNA的结构 闭合环状pBR322的拓扑结构和转录效率 含有基因插入物的微小染色体。这些研究将允许进行一项测试 我们早先提出的组蛋白乙酰化破坏34 nm的稳定性 螺线管。来避免前面由于使用 组蛋白乙酰化的混合状态，我们正在分馏不同的 用于结构/功能清洁研究的乙酰化组蛋白的状态 两性关系。我们有证据证明一种不寻常的相互作用 H4多肽(1-23)和(1-37)与被乙酰化抑制的DNA结合。 这些多肽与已知DNA十二聚体的详细研究 结构应该提供对这种交互的理解，以及它是如何 可能会影响染色质结构。我们正在培养小鼠乳腺TS85细胞 它在G2早期的非允许温度停滞时没有 在允许的温度下，H2A被泛素化，尽管它是 随后在中期之前去泛素。我们建议 含核小体和寡核小体的uH_2A的分离和鉴定 询问它们是否与潜在的活性基因相关 那种细胞类型。为了涉及染色质的体外研究，我们建议 分离和鉴定转录和非转录片段 不同生命周期核仁的核糖体DNA染色质 多头绒泡菌属(Physarum Polycephalum)

The long-term objectives of these proposals are directed to an understanding of the structure and function of active chromatin. Evidence has accumulated to support the view that the functional states of chromatin domains are associated with variables such as: 1) acetylation of core histones; 2) probable depletion or loss of histone Hl; 3) binding of HMG proteins 14 and 17; 4) ubiquitin modifications of H2A and H2B; 5) DNA methylation; 6) DNA supercoiling and polymorphism. Presumably such changes allow access for specific proteins to identify and bind to DNA regulatory sites and also provide the correctly structured template and DNA topology for transcription. Our cell cycle studies associate only the highest states of acetylation of H3 and H4 with transcription and show also that uH2A and uH2B disappear in prophase immediately before metaphase and reappear rapidly in anaphase. These specific regions of chromatin labeled with uH2A and uH2B appear to be packaged last into metaphase chromosomes. It has been suggested that uH2A is located in potentially transcribable chromatin. Studies are in progress of the effects of histone acetylation, ubiquitination and the binding of HMG proteins 14 and 17 on the structures and structural transitions of oligonucleosomes and on the structures, DNA topologies and transcriptional efficiencies of closed circular pBR322 minichromosomes containing gene inserts. These studies will allow a test of our earlier proposal that histone acetylation destabilizes the 34 nm solenoid. To avoid the previous ambiguities resulting from the use of mixed states of histone acetylation, we are fractionating the different states of acetylated histones for clean studies of structure/function relationships. We have evidence for an unusual type of interaction of the H4 peptides (1-23) and (1-37) with DNA which is suppressed by acetylation. Detailed studies of these peptides with the DNA dodecamer of known structure should provide an understanding of this interaction and how it might affect chromatin structure. We are growing mouse mammary ts85 cells which at the non-permissive temperature arrest in early G2 and have no uH2A; at the permissive temperature H2A is ubiquitinated, although it is subsequently deubiquitinated just prior to metaphase. We propose to isolate and characterize uH2A containing nucleosomes and oligonucleosomes to ask whether they are associated with the potentially active genes of that cell type. To relate in vitro studies of chromatin, we propose to isolate and characterize transcribed and non-transcribed pieces of ribosomal DNA chromatin from nucleoli at different states of the life cycle of Physarum polycephalum.