NUCLEAR & CHROMATIN PACKAGING OF MAMMALIAN X CHROMOSOME

核

• 批准号: 2519045
• 负责人: JEANNE Bentley LAWRENCE
• 金额: $ 21.86万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2519045
• 关键词: cell cycle; chromatin; chromosome translocation; cytogenetics; flow cytometry; immunofluorescence technique; nucleoproteins; sex chromosomes

By coupling the unique model system of the mammalian X chromosome with powerful new ways to probe the organization of specific genes. RNAs, or chromosomes, the work proposed here has potential to advance our understanding of higher-level chromosome and nuclear structure, in general, and its relationship to transcription and X inactivation, in particular. We will systematically investigate and compare specific aspects of the molecular cytology of Xi (inactive X) and Xa (active X), in the normal native state, in naturally occurring chromosomal rearrangements. and in genetically manipulated cells. Experiments proposed largely relate to areas of recent developments in nuclear structure, each of which has fundamental significance and is supported by strong preliminary studies. The first concerns emerging evidence for nuclear compartmentalization. We hypothesize that the genome, as embodied by interphase chromosomes, is non randomly integrated in a gene specific manner with respect to splicing factor/poly A RNA rich "compartments" or "domains", specialized for a function(s) related to RNA metabolism. We further suggest that this spatial arrangement related to gene expression. Specific aim 1 compares euchromatic Xa versus heterochromatic Xi, including genes which escape inactivation, to address aspects of this and related ideas. Analysis of the important Fragile X region is included where chromosome structure is known to be perturbed by a trinucleotide repeat expansion. The second recent breakthrough concerns XIST a gene strongly implicated in X inactivation. We hypothesize that RNA from the XIST gene is itself involved in inactivation. Preliminary studies suggest that this RNA may provide a precedent for RNA involvement chromosome and nuclear structure. The molecular cytology of this RNA becomes key to understanding the structure/function relationship. Aim 2 proposes to examine the RNA distribution relative to Xi DNA sequences on normal and rearranged chromosomes, by both 2-D and detailed 3-D analysis. Analysis of specific genes on Xi will contribute to the formation of a model for the interphase organization of genes which escape inactivation. Aim 3 will investigate the relationship of XlST RNA to Xi and nuclear structure in cells in which the RNA, or portions of it, are expressed from foreign chromosomal or nuclear context. Reciprocal transgenes, consisting of an autosomal gene translocated to Xi, will examine inter-relationships between XIST RNA. the splicing factor rich compartment, and underlying nuclear structure. Aim 4 will extend aspects of the above studies to immunofluorescence differences in protein content on Xi, Xa, an Xi:autosome translocations, for example to explore proteins known to bind XIST RNA, or to explore an innovative means to isolate pure populations of Xi and Xa as a resource for studies of chromatin.

通过将哺乳动物X染色体的独特模型系统 用强大的新方法来探测特定基因的组织。 RNA，或染色体，这里提出的工作有可能取得进展 我们对更高层次的染色体和核结构的理解， 一般而言，及其与转录和X失活关系， 特别是。我们将系统地调查和比较 Xi（非活性X）和Xa的分子细胞学的特定方面 （活性X），在正常的天然状态下，在自然发生的 染色体重排和基因操作的细胞。 所提议的实验主要涉及最近发展的领域 在核结构中，每一个都具有重要意义， 这得到了初步研究的有力支持。第一个问题涉及 新出现的核分区证据 我们假设 由间期染色体所体现的基因组， 以基因特异性方式随机整合， 富含剪接因子/多聚ARNA的“区室”或“结构域”， 专门用于与RNA代谢相关的功能。我们进一步 表明这种空间排列与基因表达有关。 具体目标1比较常染色质Xa与异染色质Xi， 包括逃避失活的基因，以解决这一问题的各个方面， 和相关的想法。重要的脆性X区域的分析是 包括在已知染色体结构被 三核苷酸重复扩增。最近的第二个突破 XIST是一个与X染色体失活密切相关的基因。我们 假设来自XIST基因的RNA本身参与了 失活 初步研究表明，这种RNA可以提供 RNA参与染色体和核结构的先例。 这种RNA的分子细胞学成为理解 结构/功能关系目标2：检测RNA 相对于Xi DNA序列的分布在正常和重排 染色体，通过2-D和详细的3-D分析。分析 Xi上的特定基因将有助于形成一个模型， 逃避失活的基因的间期组织。 目的 3将研究XlST RNA与Xi和核的关系， 细胞中表达RNA或其部分的结构 与外来染色体或细胞核无关。相互的转基因， 由一个常染色体基因易位到Xi，将检查 XIST RNA之间的相互关系。富含剪接因子 细胞核结构和细胞核结构。目标4将扩大 上述研究的方面，以免疫荧光的差异， Xi、Xa、an Xi上的蛋白质含量：常染色体易位，例如 探索已知结合XIST RNA的蛋白质，或探索 创新的方法来分离Xi和Xa的纯群体， 染色质研究的资源。