CHROMATIN DOMAIN AS A UNIT OF X CHROMOSOME INACTIVATION AND IMPRINTING

哺乳动物的染色体是由一系列称为染色质的环组成的 结构域，表现为独立的扭转单位和DNA单位 复制。这项研究的长期目标是测试 认为染色质结构域是细胞的功能单位 转录能力，因为每个独立的结构域都可以 被组装为活性或非活性染色质，并且该活性 染色质结构域在S阶段的前半部分完成复制， 而不活跃的染色质结构域通常在第二个 一半。 具体目的是测试这一假设的四个预测：(A) 活性染色质和非活性染色质之间的边界应用 结构域终结点。(B)转基因应在下列情况下自主行为 它们占据着自己的染色质区域，但应该以一种方式 与它们插入的染色体区域一致，如果 它们并不占据专属领域。(C)领域边界位置应 独立于转录活动。(D)活动域名应 在S阶段的上半段复制，而一个不活跃的域 应该在S阶段以后复制。这项研究对 了解正确的组织特异性和阶段特异性基因模式 表达式中出现的表达式更改的基础 癌症、衰老和遗传病，以及如何治疗遗传病 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。复制分析将是 在活跃和非活跃的基因拷贝上执行。有待研究的基因座 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(失活特异转录物)基因和H19 和受基因组印记影响的Igf2基因座。学生们将拥有 在问题和实验设计中的重要投入，执行 这些实验，在解释结果时，在回顾 文献，以及在成果的出版和介绍方面， 目标是鼓励他们考虑从事科学工作。学生 将与细胞系和转基因小鼠合作，使用各种现代 分子技术，包括核基质附着和 拓扑异构酶裂解位点定位，核酸酶敏感性分析， 聚合酶链式反应、逆转录、Northern和 Southern杂交和脉冲场凝胶电泳法。

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- specific transcript) gene, and the H19 and Igf2 loci which are subject to genomic imprinting. Students will have significant input in the design of questions and experiments, carrying out the experiments, in the interpretation of the results, in reviewing the literature, and in the publication and presentation of results, with the objective of encouraging them to consider careers in science. Students will work with cell lines and transgenic mice, using a variety of modern 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.