MOLECULAR BASIS FOR SELECTIVE EXPRESSION OF TRNA GENES

我们希望分析负责组织特异性 家蚕中丙氨酸tRNA的积累。 一个 对这种现象的分子基础的理解应该提供 深入了解真核细胞 分化 我们实验室最近的工作已经证实， 组成型和丝腺特异性类型的丙氨酸tRNA（其几乎是 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 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，我们侧重于其他层面， 哪些tRNA合成可能受到调控。 组织特异性扩增或 丙氨酸tRNA基因的重排，或者， tRNA基因的两个愈伤组织最有可能解释选择性的 丝腺特异性丙氨酸tRNA的出现。 中国对虾基因组DNA中丙氨酸tRNA基因的数量和结构 将用杂交探针检查不同的蚕组织， 在这两类基因之间有明显的区别。 组织特异 与一个或另一个基因类相关的变化应该很容易 用这种方法检测。 为了了解这些基因是否在细胞内受到调控， 转录水平，我们将比较转录特性， 在无细胞提取物中克隆来自每个基因类的代表， 卵巢和丝腺，以及通过显微注射分离的 基因注入完整的卵巢和丝腺细胞。 通过分离100%的 RNA聚合酶III转录装置，我们将试图确定 负责任何组织特异性转录的组分 我们观察到的差异。 我们还将构建杂合丙氨酸tRNA基因 为了确定负责功能性的核苷酸序列， 组成型和丝腺特异型基因之间的差异。

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.