TFIIIA GENE EXPRESSION IN OOCYTES AND SOMATIC CELLS

5S RNA基因特异性正转录水平 因子，TFIIIA，似乎在发育中发挥关键作用 非洲爪哇5S RNA基因的调控TFIIIA基因的表达 已知基因在mRNA水平上受发育调节 积累。这项提案的目的是确定 控制TFIIIA基因在卵母细胞和卵巢中表达的因素 体细胞。 这项工作专门针对研究分子 TFIIIA基因在卵母细胞中表达的相互作用 和体细胞。此外，表达之间的差异 将检测TFIIIA基因在卵母细胞和体细胞中的表达情况。 确定TFIIIA基因顺式作用序列的实验和 周围的基因组DNA序列，影响转录 将使用标准的基因融合和 转基因技术。TFIIIA基因缺失突变和 周围的基因组DNA序列将通过体外制备 诱变方案。然后将对突变的序列进行分析 对于引导TFIIIA基因的特定转录的能力 在卵母细胞和体细胞中。在这种情况下，应该是 从TFIIIA基因和 以正向和/或负向作用的周围基因组区域 如何控制TFIIIA基因在卵母细胞中的表达 体细胞。缺失表达的差异 将仔细检查卵母细胞和体细胞中的突变。 这些差异可能定义了TFIIIA基因的区域 以及周围在组织中很重要的基因组序列 TFIIIA基因的特异性。 最后，顺式作用序列的相互作用影响 TFIIIA基因在蛋白质(反式作用因子)中的表达 从卵母细胞和体细胞中分离出来的DNA 蛋白质结合分析。卵母细胞的存在或不存在 体细胞或细胞类型特异的DNA结合修饰 蛋白质可能在TFIIIA的表达中起关键作用 吉恩。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 已尝试。这些蛋白质的纯化应该允许 详细考察它们在转录中的作用 TFIIIA基因在卵母细胞和体细胞中的表达 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.