CHARACTERIZING REGULATORY REGIONS OF CYCLIN D IN STRONGYLOCENTROTUS PURPURATUS

紫荠中 CYCLIN D 调控区的特征

• 批准号: 8360317
• 负责人: CHRISTOPHER MCCARTY
• 金额: $ 4.2万
• 依托单位: MOUNT DESERT ISLAND BIOLOGICAL LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360317
• 关键词: Cell Differentiation process; Cell division; Cyclin D1; DNA; Embryo; Embryonic Development; Funding; Genes; Grant; Green Fluorescent Proteins; Health; Human; Human Biology; Knowledge; Lytechinus variegatus; Maine; Malignant Neoplasms; Methods; Microinjections; Microscopy; Mutate; National Center for Research Resources; Neurodegenerative Disorders; Nucleic Acid Regulatory Sequences; Principal Investigator; Reporter; Research; Research Infrastructure; Resources; Role; Sea Urchins; Source; Strongylocentrotus purpuratus; Techniques; Time; Transgenes; United States National Institutes of Health; Vertebrates; base; comparative; cost; egg; functional genomics; interest; protein expression

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The cyclin D gene is an important regulator of cell division and differentiation. In this project, "cis-regulatory" DNA regions that control when and where cyclin D is activated during embryonic development of the purple sea urchin Strongylocentrotus purpuratus are being analyzed to determine their specific roles. Sea urchins and vertebrates share a common ancestor, so knowledge gained from this study can be used to understand aspects of human biology. In humans, cyclin D genes are mis-regulated in certain cancers and neurodegenerative diseases, so this research has health implications. In the current project, potential cis-regulatory regions have been identified computationally based on their similarity to regions in the cyclin D gene of the sea urchin Lytechinus variegatus, as well as by various additional criteria. These candidate cis-regulatory regions were incorporated individually into a "reporter" transgene encoding green fluorescent protein (GFP), for microinjection into fertilized sea urchin eggs. As the injected embryos develop, GFP expression can be studied by microscopy to determine where in the embryo the cis-regulatory region of interest exerts its action, and quantitatively, by a technique termed Real-Time PCR. From these methods, we have isolated two regions that regulate the activation of the cyclin D gene in the purple sea urchin. These regions are being broken down further, by mutating subregions hypothesized to be required for regulatory function, then re-examining the abilities of the altered regions to induce activation of GFP.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 细胞周期蛋白D基因是细胞分裂和分化的重要调节因子。在这个项目中，控制着紫海胆胚胎发育过程中细胞周期蛋白D何时何地被激活的顺式调控DNA区域正在被分析，以确定它们的具体作用。海胆和脊椎动物有着共同的祖先，因此从这项研究中获得的知识可以用来理解人类生物学的各个方面。在人类中，细胞周期蛋白D基因在某些癌症和神经退行性疾病中被错误调控，因此这项研究具有健康意义。在目前的项目中，基于它们与海胆Lytechinus varegatus的Cyclin D基因区域的相似性以及各种附加标准，已经通过计算确定了潜在的顺式调控区域。这些候选的顺式调控区域被分别整合到编码绿色荧光蛋白(GFP)的报告基因中，用于显微注射到受精的海胆卵中。随着注射胚胎的发育，GFP的表达可以通过显微镜来研究，以确定感兴趣的顺式调控区在胚胎中发挥作用的位置，并通过一种名为Real-time PCR的技术进行定量。从这些方法中，我们分离出了调控紫海胆细胞周期蛋白D基因激活的两个区域。这些区域被进一步分解，通过突变假定为调节功能所需的亚区，然后重新检查改变的区域诱导GFP激活的能力。