MOLECULAR BASIS FOR SELECTIVE EXPRESSION OF TRNA GENES

TRNA 基因选择性表达的分子基础

• 批准号: 3282015
• 负责人: KAREN U SPRAGUE
• 金额: $ 9.22万
• 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/3282015
• 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.

我们希望分析导致组织特异性的机制。 家蚕体内丙氨酸tRNAs的积累一个 对这种现象的分子基础的理解应该提供 真核细胞参与的机制研究进展 差异化。我们实验室最近的研究表明， 构成和丝腺特异类型的丙氨酸tRNA(几乎是 序列相同)实际上由不同的基因编码。这 观察表明，选择性转录后转录 修饰解释了丙氨酸tRNA的组织特异性模式 活体生产。在这项建议中，我们将重点放在其他层面上 其中tRNA的合成可能受到调控。组织特异性扩增或 丙氨酸tRNA基因重排，或差异转录 TRNA基因的两个调用最有可能解释选择性 丝腺特异型丙氨酸tRNA的出现。 日本血吸虫基因组DNA中丙氨酸tRNA基因的数量和结构 将用杂交探针检测不同的家蚕组织 对这两类基因进行了明显的区分。组织特异性 与一个或另一个基因类别相关的变化应该很容易 用这种方法检测到的。为了了解这些基因是否在 转录水平，我们将比较 在无细胞提取液中克隆了每个基因类别的代表 家蚕卵巢和丝腺，以及微量注射分离的 基因进入完整的卵巢和丝腺细胞。通过对家蚕进行分级 RNA聚合酶III转录装置，我们将尝试鉴定 负责任何组织特定转录的组件(S) 我们观察到的差异。我们还将构建杂交的丙氨酸tRNA基因 为了确定负责功能的核苷酸序列 丝腺特异型基因与构成型基因的差异。