ORGANIZATION AND EXPRESSION OF CHLOROPLAST TRNA GENES

叶绿体基因组通常被认为编码所有的转移 核糖核酸（tRNA）参与叶绿体蛋白质合成。 由于叶绿体 基因组体积小，容易以完整的形式分离， 将其克隆到所需大小的片段中作为重组DNA，这是一种可行的方法。 目的是定位和表征每个叶绿体tRNA基因， 确定每个的反密码子和同源氨基酸。 我们一直 参与了一套完整的tRNA基因的表征， 纤细裸藻叶绿体中的侧翼序列。 这个项目是 重点是完成了tRNA编码基因座的DNA序列分析， 裸藻叶绿体基因组，决定tRNA基因是如何组织成 转录单位并转录成前体tRNA， 了解tRNA基因表达调控机制 在叶绿体生物发生过程中。 tRNA编码基因座的DNA序列将被 用化学裂解或引发的双脱氧方法进行。 初级tRNA转录和加工反应的表征 将基于同源体外转录系统的研究 来自纤细裸藻和菠菜叶绿体。 DNA 模板修饰实验将用于确定DNA的作用 转录的调控、起始和终止序列。 最后，合成的脱氧寡核苷酸将用作杂交 研究体内tRNA的合成和成熟的探针， 叶绿体发育

Chloroplast genomes are generally believed to encode all of the transfer RNAs (tRNAs) involved in chloroplast protein synthesis. Since chloroplast genomes are of small size, readily isolated in intact form, and easily cloned in fragments of desired size as recombinant DNAs, it is a feasible goal to locate and characterize every chloroplast tRNA gene, and to determine the anticodon and cognate amino acid for each. We have been involved in a characterization of the complete set of tRNA genes and flanking sequences in chloroplasts of Euglena gracilis. This project is focused on completing the DNA sequence analysis of tRNA coding loci of the Euglena chloroplast genome, determining how tRNA genes are organized into transcription units and transcribed into precursor tRNAs, and on understanding mechanisms involved in regulation of tRNA gene expression during chloroplast biogenesis. DNA sequence of tRNA coding loci will be carried out with either the chemical cleavage or primed, dideoxy method. Characterization of primary tRNA transcription and processing reactions will be based on studies with homologous in vitro transcription systems from both Euglena gracilis and Spinacia oleracea choloroplasts. DNA template modification experiments will be used to determine the role of DNA sequences in regulation, initiation and termination of transcription. Finally, synthetic deoxyoligonucleotides will be used as hybridization probes to study the synthesis and maturation of tRNAs in vivo during chloroplast development.