BIOCHEMICAL ANALYSIS OF PROMOTERS REGULATING DEVELOPMENT

调节发育的启动子的生化分析

• 批准号: 2392127
• 负责人: ALBERT J COUREY
• 金额: $ 23.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-07 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392127
• 关键词: Drosophilidae; developmental genetics; embryogenesis; gene induction /repression; genetic enhancer element; genetic promoter element; intermolecular interaction; laboratory mouse; laboratory rabbit; mutant; nucleic acid probes; nucleic acid sequence; oncogenes; tissue /cell culture; transcription factor

DESCRIPTION: This proposal addresses one of biology's most basic questions: how is a group of undifferentiated cells transformed into an ordered array of differentiated tissues? The focus is on the initial phase of this transformation in which a field of cells is divided into a few discrete domains. To examine this process, biochemical and genetic approaches will be combined to study the molecular interactions that control Drosophila embryogenesis. The dorsal morphogen is a promoter selective transcription factor that determines cell fate as a function of position along the dorsal/ventral axis of the Drosophila embryo. Early in development, this protein comes to be distributed in an activity gradient, with high activity on the ventral side and low activity on the dorsal side of the embryo. This graded distribution of Dorsal results in the spatially regulated expression of genes that are essential for germ layer establishment. For example, since Dorsal is an activator of the twist (twi) gene, twi is only transcribed in the ventrally- situated presumptive mesoderm where Dorsal activity is high. In contrast, Dorsal is a repressor of the decapentaplegic (dpp) and zerknllt (zen) genes, which are therefore only transcribed in the dorsally-situated presumptive ectoderm where Dorsal activity is low. The goal of this research is to determine how Dorsal interacts with other nuclear factors to activate some promoters and repress others. These factors include certain basic helix-loop- helix (BHLH) transcription factors, which interact with Dorsal to broaden and intensify domains of Dorsal-mediated activation, as well as co-repressor proteins, which assist in Dorsal-mediated transcriptional repression. By contributing to an understanding of animal development, these studies should help to elucidate the basis of human developmental disorders. The specific aims are to 1) Analyze the synergistic interactions between Dorsal and bHLH transcription factors. In vitro transcription and transient transfection assays will be employed to examine the molecular interactions that allow Dorsal to synergize with the bHLH factors encoded by twi, daughterless, and the Achaete Scute complex. In addition, the protein domains responsible for the synergy will be mapped and analyzed to determine if they mediate direct contacts between the factors. Using information gained from this analysis, dominant negative alleles of the factors will be developed and introduced into the embryo to determine the developmental role of Dorsal/BHLH factor synergy. 2) Characterize the interactions between Dorsal and co- repressors to elucidate the mechanisms of ventral repression. First the germline transformation assay will be employed to map the domains in Dorsal that are required for repression and which may, therefore, directly contact co-repressor proteins. Second, through the use of genetic mosaics, NTF-1, a putative co-repressor, will be analyzed to determine if this factor is essential for normal dorsal/ventral pattern formation. Third, additional putative co-repressor proteins will be characterized. Fourth, the role in dorsal/ventral pattern formation of direct interactions between Dorsal and co- repressors will be assessed.

描述：该提案涉及生物学最基本的一种 问题：一组未分化的细胞如何转化为一个 有序的分化组织阵列？ 重点是最初 这种转换的阶段，其中一个细胞场被分为 一些离散域。 要检查这个过程，生化和 遗传方法将合并以研究分子相互作用 控制果蝇的胚胎发生。 背面形态学是启动子选择性转录因子， 确定细胞命运是沿背/腹侧的位置的函数 果蝇胚胎的轴。 在开发的早期，这种蛋白质来了 分布在活动梯度中，对 腹侧和低活性在胚胎的背侧。 这 背面的分级分布在空间调节中 表达对于生殖层建立至关重要的基因。 例如，由于背侧是扭曲（TWI）基因的激活因子TWI 仅在腹侧定位的中胚层中转录 背活动很高。 相比之下，背是 因此 在背侧的假定外胚层中转录在背侧 活动很低。 这项研究的目的是确定背侧 与其他核因素相互作用，以激活某些启动子和 压抑别人。 这些因素包括某些基本的螺旋环螺旋 （BHLH）转录因子，与背侧相互作用以扩大和 强化背介导的激活的结构域以及共抑制剂 蛋白质，有助于背介导的转录抑制。 通过对动物发展的理解做出贡献，这些研究 应该有助于阐明人类发育障碍的基础。 具体目的是1）分析之间的协同相互作用 背和BHLH转录因子。 体外转录和 瞬态转染测定法将用于检查分子 允许背侧与BHLH因子协同作用的相互作用 由Twi，Daughsterless和Achaete Scute Complex编码。 在 此外，将映射负责协同作用的蛋白质域 并分析以确定它们是否介导 因素。 利用从此分析中获得的信息，主要负面 这些因素的等位基因将被开发并引入胚胎 确定背或BHLH因子协同作用的发育作用。 2） 表征背侧和联合压迫者之间的相互作用 阐明腹侧抑制的机制。 首先是种系 转换测定将用于绘制背侧的域 镇压需要，因此可能直接接触 共抑制蛋白。 其次，通过使用遗传镶嵌物， NTF-1是一种推定的共抑制器，将分析以确定是否 因子对于正常的背/腹图形成至关重要。 第三， 将表征其他推定的共抑制蛋白。 第四，在直接的背/腹图形成中的作用 将评估背侧和联合压迫者之间的相互作用。