MOLECULAR BIOLOGY OF CHLOROPLAST RIBOSOMES

我们建议研究1)编码基因的翻译位置 叶绿体核糖体的蛋白质，2)这些蛋白质的细胞内定位 基因；3)这些基因在叶绿体DNA上的物理位置。这个 叶绿体核糖体蛋白的合成地点将通过In 体内误译试验和翻译抑制物研究。孤立 将测试叶绿体和细胞质部分的内源合成 叶绿体核糖体蛋白的体外研究。细胞中的信使RNA组分 细胞质和叶绿体将被检测在体外编程的能力 1)大肠杆菌翻译合成叶绿体核糖体蛋白 核糖体，2)重组烟草叶绿体系统，3)小麦胚芽 核糖体和4)网织红细胞裂解物系统。含信使核糖核酸的分离组分 特定叶绿体核糖体蛋白的活性将允许物理作图 叶绿体核糖体蛋白的基因。然后，叶绿体DNA基因将被 物理定位由DNA-RNA杂交、限制性内切酶 作图程序，核糖体与叶绿体DNA的结合，以及偶联 使用单个限制性片段的转录-翻译研究 叶绿体DNA。

We propose to study 1) the site of translation of the genes which code for the proteins of chloroplast ribosomes, 2) the intracellular location of these genes, and 3) the physical location of these genes on chloroplast DNA. The site of synthesis of chloroplast ribosomal proteins will be examined by an in vivo mistranslation assay and by translation inhibitor studies. Isolated chloroplast and cytoplasmic fractions will be tested for endogenous synthesis of chloroplast ribosomal proteins in vitro. Messenger RNA fractions from cell cytoplasm and chloroplasts will be examined for ability to program in vitro synthesis of chloroplast ribosomal proteins by translation with 1) E. Coli ribosomes, 2) a reconstituted Nicotiana chloroplast system, 3) wheat germ ribosomes and 4) a reticulocyte lysate system. Isolated fractions with mRNA activity for specific chloroplast ribosomal proteins will allow physical mapping of genes for chloroplast ribosomal proteins. Chloroplast DNA genes will then be physically located by a combination of DNA-RNA hybridization, restriction enzyme mapping procedures, ribosome binding to chloroplast DNA, and coupled transcription-translation studies using individual restriction fragments of chloroplast DNA.