TFIIIA GENE EXPRESSION IN OOCYTES AND SOMATIC CELLS

卵母细胞和体细胞中的 TFIIIA 基因表达

• 批准号: 3296022
• 负责人: WILLIAM L TAYLOR
• 金额: $ 16.21万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3296022
• 关键词: DNA; DNA binding protein; Xenopus; cell transformation; developmental genetics; electroporation; fusion gene; genetic manipulation; genetic transcription; genome; messenger RNA; microinjections; mutagens; mutant; nucleic acid sequence; oogenesis; radiotracer; sea urchins; transfection

The levels of the 5S RNA gene specific positive transcription factor, TFIIIA, appear to play a critical role in the developmental regulation of 5S RNA genes in Xenopus. The expression of the TFIIIA gene is known to be developmentally regulated at the level of mRNA accumulation. It is the objective of this proposal to identify the factors which control TFIIIA gene expression in oocytes and in somatic cells. This work is specifically directed at examining the molecular interactions involved in expression of-the TFIIIA gene in oocytes and somatic cells. In addition, differences between the expression of the TFIIIA gene in oocytes and somatic cells will be examined. Experiments to define cis-acting sequences of the TFIIIA gene and surrounding genomic DNA sequences, which affect the transcription of the gene, will be performed using standard gene fusion and transfection techniques. Deletion mutations of the TFIIIA gene and surrounding genomic DNA sequences will be prepared by in vitro mutagenesis protocols. The mutated sequences will then be assayed for the ability to direct specific transcription of the TFIIIA gene in oocytes and somatic cells. In this manner, it should be possible to define DNA sequences from the TFIIIA gene and surrounding genomic regions which act in a positive and/or negative fashion to control the expression of the TFIIIA gene in oocytes and somatic cells. Differences in the expression of the deletion mutants in oocytes and somatic cells will be examined carefully. These differences are likely to define regions of the TFIIIA gene and surrounding genomic sequences which are important in the tissue specificity of the TFIIIA gene. Finally, the interaction of cis-acting sequences which affect the expression of the TFIIIA gene with proteins (trans-acting factors) from oocytes and somatic cells will be examined using various DNA- protein binding assays. The presence or absence in oocytes or somatic cells or cell type specific modification of the DNA binding proteins may play a crucial role in the expression of the TFIIIA gene. Purification of the proteins which interact with cis-acting sequences which control the expression of the TFIIIA gene will be attempted. The purification of these proteins should allow the detailed examination of their role in the transcription of the TFIIIA gene in oocytes and somatic cells, including any modifications and tissue specificity.

5S RNA基因特异性正转录水平 TFIIIA 因子似乎在发育中发挥着关键作用 非洲爪蟾 5S RNA 基因的调控。 TFIIIA的表达 已知基因在 mRNA 水平上受到发育调控 积累。本提案的目的是确定 控制卵母细胞和细胞中 TFIIIA 基因表达的因素 体细胞。 这项工作专门针对检查分子 卵母细胞中 TFIIIA 基因表达涉及的相互作用 和体细胞。另外，表达式之间的差异 将检查卵母细胞和体细胞中 TFIIIA 基因的变化。 定义 TFIIIA 基因顺式作用序列的实验 周围基因组 DNA 序列，影响转录 的基因，将使用标准基因融合进行和 转染技术。 TFIIIA 基因的缺失突变和 周围的基因组DNA序列将通过体外制备 诱变协议。 然后将检测突变序列 具有指导 TFIIIA 基因特异性转录的能力 在卵母细胞和体细胞中。 按照这种方式，应该是 可以定义 TFIIIA 基因的 DNA 序列，并且 周围基因组区域发挥积极和/或消极作用 控制卵母细胞中 TFIIIA 基因表达的时尚 体细胞。 删除表达的差异 卵母细胞和体细胞中的突变体将被仔细检查。 这些差异可能定义了 TFIIIA 基因的区域 以及在组织中重要的周围基因组序列 TFIIIA 基因的特异性。 最后，顺式作用序列的相互作用影响 TFIIIA 基因与蛋白质（反式作用因子）的表达 来自卵母细胞和体细胞的检查将使用各种DNA- 蛋白质结合测定。卵母细胞中存在或不存在或 DNA 结合的体细胞或细胞类型特异性修饰 蛋白质可能在 TFIIIA 的表达中发挥至关重要的作用 基因。 与顺式作用相互作用的蛋白质的纯化 控制 TFIIIA 基因表达的序列将是 尝试过。这些蛋白质的纯化应该允许 详细检查它们在转录中的作用 卵母细胞和体细胞中的 TFIIIA 基因，包括任何 修饰和组织特异性。