CHROMATIN DOMAINS IN X INACTIVATION

哺乳动物的染色体被组织成一系列的环，称为染色质 结构域，它们作为独立的扭转单元和DNA单元 复制的 这项研究的长期目标是测试 假设染色质结构域的功能单位 转录能力，因为每个独立的结构域可以是 作为活性或非活性染色质组装， 染色质结构域在S期前半期完成复制， 而不活跃的染色质结构域通常在第二次细胞周期中复制， 半 具体目标是测试这个假设的四个预测：（A） 活性和非活性染色质之间的边界应标记为 结构域端点。 (B)转基因应该自主行为， 它们占据了自己的染色质结构域， 与它们插入的染色体区域一致， 它们并不占据专属领域。 (C)域边界位置 应该独立于转录活性。 (D)活动域 应该在S期的前半部分复制，而非活性的 结构域应在S期后期复制。 这项研究对于 正确理解基因的组织特异性和阶段特异性模式 表达，表达发生变化的基础， 癌症、衰老和遗传疾病，以及如何治疗遗传疾病， 疾病状态（包括某些癌症）。 染色质结构域边界的位置和复制时间将是 确定了六个染色体区域。 复制试验将 对基因的活性和非活性拷贝进行检测。 待研究位点 包括小鼠X染色体上的四个转基因（转铁蛋白， 金属硫蛋白-加压素融合基因，和两个独立的插入， 酪氨酸酶）、Xist（无活性X特异性转录物）基因和H19 和易受基因组印记影响的Igf 2基因座。 研究利用 细胞系和转基因小鼠，使用各种分子技术， 包括核基质附着和拓扑异构酶切割位点 作图，核酸酶敏感性测定，聚合酶链反应（PCR）， 逆转录、北方和南方印迹以及脉冲场 凝胶电泳

Mammalian chromosomes are organized as a series of loops called chromatin domains, which behave as independent torsional units and units of DNA replication. The long-term objective of this research is to test the hypothesis that chromatin domains are the functional units of transcriptional competence in that each independent domain can be assembled either as active or as inactive chromatin, and that active chromatin domains complete replication during the first half of S phase, while inactive chromatin domains are often replicated during the second half. Specific aims are to test four predictions of this hypothesis: (A) The border between active and inactive chromatin should be marked by a structural domain endpoint. (B) Transgenes should behave autonomously if they occupy a chromatin domain of their own, but should behave in a manner consistent with the chromosomal region into which they have inserted if they do not occupy an exclusive domain. (C) Domain border positions should be independent of transcriptional activity. (D) An active domain should replicate during the first half of S phase, while an inactive domain should replicate later in S phase. The research is crucial to understanding correct tissue-specific and stage-specific patterns of gene expression, of the basis for the alterations in expression that occur in cancer, aging, and genetic disease, and of how one might treat genetic disease states (including certain cancers). Position of chromatin domain borders and replication timing will be determined for six chromosomal regions. Replication assays will be performed on both active and inactive copies of genes. Loci to be studied include four transgenes on the mouse X chromosome (transferrin, metallothionein-vasopressin fusion gene, and two independent insertions of tyrosinase), the Xist (inactive X specific transcript) gene, and the H19 and Igf2 loci which are subject to genomic imprinting. Research utilizes cell lines and transgenic mice, using a variety of molecular techniques, including nuclear matrix attachment and topoisomerase cleavage site mapping, nuclease sensitivity assays, polymerase chain reaction (PCR), reverse transcription, Northern and Southern blotting, and pulsed-field gel electrophoresis.