DEVELOPMENTALLY-REGULATED DNA-BINDING PROTEINS

发育调控的 DNA 结合蛋白

• 批准号: 3305007
• 负责人: ERIC LAM
• 金额: $ 12.66万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305007
• 关键词: DNA binding protein; Leguminoseae; cell type; complementary DNA; developmental genetics; gene expression; genetic library; genetic promoter element; genetic transcription; genetically modified plants; hormone therapy; immunofluorescence technique; in situ hybridization; messenger RNA; molecular cloning; northern blottings; nucleic acid probes; nucleic acid sequence; plant growth /development; protein biosynthesis; site directed mutagenesis; southern blotting; stress proteins; tobacco; tobacco mosaic virus; transcription factor; transfection

The objective of this proposal is to elucidate the role of two DNA-binding proteins in the development of a multicenter organism. ASF-1 and HSBF are tobacco factors which bind to a hexameric motif present in the upstream region of plant histone genes. This motif, TGACGT, is related to the cAMP-responsive element in mammalian systems. ASF-1 and HSBF have different sequence specificities as demonstrated by crosscompetition studies with different binding site probes. In transgenic tobacco, recent data suggest that the activity of a binding site specific for ASF-1 is induced by a plant hormone and is also developmentally-regulated. Tobacco cDNA clones that encode these two DNA-binding proteins have homologies to transcription factors containing the leucine-zipper motif. Northern analyses show that the expression of these genes is organspecific and developmentally-regulated. Both proteins are encoded by small multigene families. We want to characterize the regulation of the various members of these two gene families. Since each individual member may be regulated differently, we propose to isolate gene-specific probes from either the 5' or 3' non-coding region of each member of these two gene families. These probes will be used for nuclear run-on transcription assays to determine whether the genes are regulated at the transcriptional level, and to localize the mRNA of the particular gene to specific cell types by in situ hybridization. The distribution of the gene products will also be examined at the protein level by immune-detection techniques. The role of these gene products in plant development will be studied by alterations of their concentration in vivo through ectopic expression of the corresponding cDNA in transgenic tobacco.

该提案的目的是阐明两种 DNA 结合的作用 多中心生物体发育中的蛋白质。 ASF-1 和 HSBF 是 与上游存在的六聚基序结合的烟草因子 植物组蛋白基因区域。这个主题，TGACGT，与 哺乳动物系统中的 cAMP 响应元件。 ASF-1和HSBF有不同 交叉竞争研究证明了序列特异性 不同的结合位点探针。在转基因烟草中，最近的数据表明 ASF-1 特异性结合位点的活性是由 植物激素，也是发育调节的。烟草 cDNA 克隆 编码这两种 DNA 结合蛋白的蛋白与转录具有同源性 含有亮氨酸拉链基序的因子。北方分析表明 这些基因的表达是器官特异性的 发育调节。两种蛋白质均由小型多基因编码 家庭。我们想要描述各个成员的监管特征 这两个基因家族。由于每个会员都可能受到监管 不同的是，我们建议从 5' 端分离基因特异性探针 或这两个基因家族每个成员的3'非编码区。这些 探针将用于核连续转录测定，以确定 基因是否在转录水平受到调控，以及 通过原位将特定基因的 mRNA 定位到特定细胞类型 杂交。基因产物的分布也将被检查 通过免疫检测技术在蛋白质水平上进行检测。这些基因的作用 植物开发中的产品将通过改变其 通过相应 cDNA 的异位表达来确定体内浓度 在转基因烟草中。