ORGANIZATION AND EXPRESSION OF CHLOROPLAST TRNA GENES

叶绿体基因组通常被认为编码了所有的转移 参与叶绿体蛋白质合成的RNA(TRNAs)。因为叶绿体 基因组大小小，很容易以完整的形式分离出来，而且很容易 克隆成所需大小的片段作为重组DNA，是一种可行的 目的定位和鉴定每一个叶绿体tRNA基因，并 确定每一个的反密码子和同源氨基酸。我们一直在 参与了完整的tRNA基因的表征和 纤细裸藻叶绿体的侧翼序列。这个项目是 重点完成了猪瘟病毒tRNA编码区的DNA序列分析 Euglena叶绿体基因组，决定tRNA基因如何组织成 转录单位并转录成前体tRNAs，以及 了解tRNA基因表达的调控机制 在叶绿体生物发生过程中。TRNA编码区的DNA序列将是 用化学裂解或底物双脱氧方法进行。 初级tRNA转录和加工反应的特征 将基于对同源体外转录系统的研究 从纤细裸藻和菠菜胆绿体中分离得到。脱氧核糖核酸 模板修饰实验将用于确定DNA的作用 转录调控、启动和终止的序列。 最后，合成的脱氧寡核苷酸将被用作杂交 TRNAs在体内合成和成熟的研究进展 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.