CHROMATIN STRUCTURE AND FUNCTION HISTONE MODIFICATIONS A

这些建议的长期目标是 了解活性染色质的结构和功能。 证据 已经积累到支持这样的观点，即染色质的功能状态 结构域与变量相关，例如：1）核心的乙酰化 组蛋白; 2）组蛋白Hl的可能消耗或损失; 3）HMG的结合 蛋白质14和17; 4）H2 A和H2 B的泛素修饰; 5）DNA 6）DNA超螺旋和多态性。 这样的变化， 允许获得特定的蛋白质，以识别并结合到DNA调节 位点，并提供正确结构的模板和DNA拓扑结构 用于转录。 我们的细胞周期研究只将最高的 H3和H4的乙酰化状态与转录，也表明， uH 2A和uH 2B在中期前的前期消失， 在后期迅速重现。 这些特定的染色质区域 uH 2A和uH 2B似乎最后包装到中期染色体中。 已经表明uH 2A位于潜在的可转录区域， 染色质 组蛋白乙酰化作用的研究正在进行中， 泛素化和HMG蛋白14和17在结构上的结合 和寡核小体的结构转换， 闭环pBR 322的拓扑结构和转录效率 含有基因插入物的微型染色体。 这些研究将允许测试 我们早期的建议，组蛋白乙酰化不稳定的34纳米 螺线管。 为了避免以前由于使用 组蛋白乙酰化的混合状态，我们正在分馏不同的 乙酰化组蛋白的状态，用于结构/功能的清洁研究 关系。 我们有证据表明， H4肽（1-23）和（1-37）与被乙酰化抑制的DNA。 这些肽与已知的DNA十二聚体的详细研究 结构应该提供对这种相互作用的理解，以及它如何 可能会影响染色质结构。 我们正在培养小鼠乳腺TS 85细胞 在非允许温度下，在G2早期停滞， uH 2A;在允许温度下，H2 A是泛素化的，尽管它是 随后在中期之前去泛素化。 我们建议 含有uH 2A的核小体和寡核小体的分离和表征 以了解它们是否与潜在的活性基因有关， 这种细胞类型。 为了与染色质的体外研究相关，我们建议 分离和表征转录和非转录片段 来自生活史不同状态的核仁的核糖体DNA染色质 多头绒泡菌

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.