MOLECULAR BASIS FOR SELECTIVE EXPRESSION OF TRNA GENES

TRNA 基因选择性表达的分子基础

• 批准号: 3282016
• 负责人: KAREN U SPRAGUE
• 金额: $ 8.79万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1986-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282016
• 关键词: DNA directed RNA polymerase; alanine; cell differentiation; cell free system; developmental genetics; eukaryote; gene expression; genetic manipulation; genetic transcription; genome; microinjections; molecular cloning; natural gene amplification; nucleic acid sequence; radiotracer; transfer RNA

We wish to analyze the mechanisms responsible for the tissue-specific accumulation of alanine tRNAs in the silkworm, Bombyx mori. An understanding of the molecular basis of this phenomenon should provide insight into the mechanisms involved in eukaryotic cellular differentiation. Recent work in our laboratory has established that the constitutive and silkgland-specific types of alanine tRNA (which are nearly identical in sequence) are in fact encoded by distinct genes. This observation makes it unlikely that selective post-transcriptional modification accounts for the tissue-specific pattern of alanine tRNA production in vivo. In this proposal, we have focused on other levels at which tRNA synthesis might be regulated. Tissue-specific amplification or rearrangement of alanine tRNA genes, or, differential transcription of the two callses of tRNA genes are most likely to explain the selective appearance of the silkgland-specific type of alanine tRNA. The number and structure of alanine tRNA genes in genomic DNA from different silkworm tissues will be examined with hybridization probes that discriminate sharply between the two classes of genes. Tissue-specific changes associated with one or the other gene class should be easily detected by this method. To learn whether these genes are regulated at the level of transcription, we will compare the transcriptional properties of cloned representatives from each gene class in cell-free extracts from Bombyx ovaries and silkglands, as well as by microinjection of isolated genes into intact ovary and silkglands cells. By fractionating the Bombyx RNA polymerase III transcription apparatus, we will attempt to identify the component(s) responsible for any tissue-specific transcriptional differences we observe. We will also construct hybrid alanine tRNA genes in order to identify the nucleotide sequence responsible for functional differences between the constitutive and silkgland-specific type genes.

我们希望分析组织特异性的机制 家蚕 (Bombyx mori) 体内丙氨酸 tRNA 的积累。 一个 了解这种现象的分子基础应该提供 深入了解真核细胞的机制 差异化。 我们实验室最近的工作表明 丙氨酸 tRNA 的组成型和丝腺特异性类型（几乎是 序列相同）实际上是由不同的基因编码的。 这 观察使得选择性转录后不太可能 修饰解释了丙氨酸 tRNA 的组织特异性模式 体内生产。 在本提案中，我们重点关注了其他层面 tRNA 合成可能受到调节。 组织特异性扩增或 丙氨酸 tRNA 基因的重排，或差异转录 tRNA 基因的两次调用最有可能解释选择性 丝腺特异性类型的丙氨酸 tRNA 的出现。 基因组 DNA 中丙氨酸 tRNA 基因的数量和结构 不同的蚕组织将用杂交探针进行检查 严格地区分两类基因。 组织特异性 与一种或另一种基因类别相关的变化应该很容易 通过该方法检测到。 了解这些基因是否受到调控 转录水平，我们将比较转录特性 从无细胞提取物中克隆出每个基因类别的代表 家蚕卵巢和丝腺，以及通过显微注射分离的 基因进入完整的卵巢和丝腺细胞。 通过分馏家蚕 RNA 聚合酶 III 转录装置，我们将尝试识别 负责任何组织特异性转录的成分 我们观察到的差异。 我们还将构建杂合丙氨酸 tRNA 基因 为了鉴定负责功能的核苷酸序列 组成型基因和丝腺特异性基因之间的差异。