TFIIIA GENE EXPRESSION IN OOCYTES AND SOMATIC CELLS

5S RNA基因特异性正转录水平 因子，TFIIIA，似乎在发展中发挥关键作用， 非洲爪蟾5S RNA基因的调控。TFIIIA的表达 已知该基因在mRNA水平上受到发育调控 积累本建议的目的是确定 控制卵母细胞中TFIIIA基因表达的因子， 体细胞 这项工作是专门针对检查分子 卵母细胞中TFIIIA基因表达的相互作用 和体细胞。此外，表达方式之间的差异 将检测卵母细胞和体细胞中TFIIIA基因的表达。 确定TFIIIA基因的顺式作用序列的实验， 周围的基因组DNA序列，影响转录 将使用标准基因融合进行， 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， 诱变方案。 然后将分析突变的序列 用于指导TFIIIA基因的特异性转录的能力 在卵母细胞和体细胞中。 这样，应该 可以从TFIIIA基因定义DNA序列， 围绕基因组区域，所述基因组区域以正向和/或负向作用， 控制卵母细胞中TFIIIA基因表达的方式， 体细胞 删除的表达差异 将仔细检查卵母细胞和体细胞中的突变体。 这些差异很可能定义TFIIIA基因的区域 以及周围的基因组序列，这些基因组序列在组织中很重要， TFIIIA基因的特异性。 最后，研究了顺式作用序列之间的相互作用， TFIIIA基因与蛋白质（反式作用因子）的表达 卵母细胞和体细胞将使用各种DNA进行检查， 蛋白质结合测定。卵母细胞中是否存在或 体细胞或细胞类型特异性修饰的DNA结合 蛋白质可能在TFIIIA的表达中起关键作用， 基因 与顺式作用蛋白相互作用的蛋白的纯化 控制TFIIIA基因表达的序列将被 企图。这些蛋白质的纯化应该允许 详细审查他们在转录《圣经》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。

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.