MECHANISM OF SELECTIVE EXPRESSION OF TRNA GENES

拟议中的实验的目标是确定 TRNA转录的组织特异性调控。我们将重点关注两个方面 家蚕的tRNA/A1a基因，其中(C)是转录的 另一种(SG)仅存在于蚕丝腺中。我们将使用这两种方法 体外和体内启动子功能的检测，并将分析在 详细的蛋白质-DNA复合体，包括C和C的上游片段 SG发起人。此数据段很有趣，因为它控制 两个启动子的体外差异转录，同源 转录系统。这两个基因的上游启动子都是 包含在其上游的-35个碱基对DNA片段内 C基因的转录起始点和启动子功能是 由这一细分市场中的两个AT丰富的盒子授予。特定的序列 或者是造成差异的结构性差异 C和SG启动子的转录情况尚不清楚。 我们计划进行以下实验，以了解这一关键 启动子片段功能： 我们将使用体外和体内试验(在培养的细胞和在 完整的丝腺)以鉴定序列和/或结构特征 在功能上区分C和SG基因的上游启动子， 这赋予了SG启动子丝腺特异性。 我们将比较TFIIIB与上游的C和SG的相互作用 使用从丝腺或组织分离的TFIIIB的启动子 (卵巢)中SG启动子不活跃。根据这个分析， 我们将鉴定和纯化可能是 C和SG启动子之间的关键区别因素。 在体外发现的调节SG转录的多肽将 通过确定它们的调节能力在体内进行功能测试 SG在培养的果蝇中异位表达时的转录 家蚕细胞和完整的、分化的家蚕组织中。

The goal of the proposed experiments is to determine the mechanism of tissue-specific regulation of tRNA transcription. We will focus on two tRNA/A1a genes of silkworms, one of which (C) is transcribed constitutively, and the other (SG) only in silkglands. We will use both in vitro and in vivo assays of promoter function, and will analyze in detail protein-DNA complexes that include the upstream segment of C and SG promoters. This segment is interesting because it controls differential transcription from the two promoters in vitro, in homologous transcription systems. The upstream promoters for both genes are contained within -35 bp DNA segments immediately upstream of their transcription initiation sites, and promoter function for the C gene is conferred by two AT-rich boxes within this segment. The specific sequence or structural distinction that is responsible for differential transcription from C and SG promoters is not known. We plan the following experiments to understand how this critical promoter segment functions: We will use both in vitro and in vivo assays (in cultured cells and in intact silkglands) to identify the sequence and/or structural features that functionally distinguish the upstream promoters of C and SG genes, and that confer silkgland specificity on the SG promoter. We will compare the interaction of TFIIIB with C and SG upstream promoters, using TFIIIB isolated either from silkglands or from a tissue (ovaries) in which the SG promoter is inactive. Based on this analysis, we will identify and purify candidate polypeptides that are likely to be key discriminators between C and SG promoters. Polypeptides that are found to regulate SG transcription in vitro will be functionally tested in vivo by determining their ability to regulate SG transcription when ectopically expressed in cultured Drosophila and silkworm cells and in intact, differentiated silkworm tissues.