CHLOROPLAST GENE STRUCTURE AND REGULATION

叶绿体(Ct)中的大多数(如果不是全部)多聚体酶是由 结构基因分散在两个不同细胞中的亚基 隔室(核和ct)。我们的长期目标是了解 这些基因的表达如何协调和调控。作为一名 迈向这一目标的第一步，我们希望收集更多关于罚款的数据 Ct基因本身的结构。尽管ct DNA有可能 编码了100多个多肽，其中只有两个已被识别 到目前为止。这项建议涉及到识别和表征 编码光系统(PS)多肽亚单位的黄瓜ct基因 II情结。在ct内形成的多肽亚基将是 在没有或存在特定信号的情况下由脉冲标记确定 抑制剂。将产生针对ct合成的亚单位的抗体。 很可能是ct基因产物。黄瓜的限制性片段 CT dna将用于从总ct rna中选择特定的mrna。 混合物。选定的信使核糖核酸将被翻译成网织红细胞裂解物 抗体免疫沉淀法鉴定系统及其产物 转化为PS II多肽。这些实验将识别编码ct基因。 PS II多肽，并将其定位于特定的限制性片段 已知的地图位置。编码PS II的每个基因的5‘端起始位置 多肽将通过对S1技术的修改来绘制。每个人 基因将根据其转录方向进行表征 以及它是否包含内含子。该基因的3‘和5’区 将通过Maxam-Gilbert技术进行测序，以查看是否有 在基因调控中可能很重要的共识序列。学习ct 基因在体外的表达和调控我们将建立一种DNA依赖的 利用叶绿体裂解物的可溶性转录系统。截断的片段 羧基酶大亚基(LS)基因和32kd多肽基因将 用作模板。我们预测这两个基因在ct中的转录。 但在黄体裂解物中只有LS基因。我们希望利用 这两个叶绿体裂解产物的差异转录反应 隔离特定需要的因子的类型 32kd多肽基因在ct中的转录。

Most, if not all, multimeric enzymes in the chloroplast(ct) are made up of subunits whose structural genes are dispersed in two different cellular compartments (nucleus and ct). Our long term objective is to understand how the expression of these genes are coordinated and regulated. As a first step toward this goal, we wish to gather more data on the fine structures of ct genes themselves. Although ct DNA has the potential to code for more than 100 polypeptides, only two of them have been identified so far. This proposal deals with the identification and characterization of cucumber ct genes encoding polypeptide subunits of the photosystem (PS) II complex. Polypeptide subunits that are made inside the ct will be determined by pulse-labeling in the absence or presence of specific inhibitors. Antibodies will be raised against the ct-synthesized subunits which are likely to be ct gene products. Restriction fragments of cucumber ct DNA will be used to select for specific mRNA from a total ct RNA mixture. The selected mRNA will be translated in a reticulocyte lysate system and the products identified by immunoprecipitation with antibodies to PS II polypeptides. These experiments will identify ct genes encoding PS II polypeptides and localize them to specific restriction fragments of known map positions. The 5' initiation site of each mRNA encoding PS II polypeptide will be mapped by a modification of the S1 technique. Each gene will be characterized with respect to its direction of transcription and whether it contains any introns. The 3' and 5' regions of the gene will be sequenced by the Maxam-Gilbert technique to see if there are consensus sequences that may be important in gene regulation. To study ct gene expression and regulation in vitro we will establish a DNA-dependent soluble transcription system using plastid lysates. Truncated fragments of the carboxylase large subunit (LS) gene and the 32 kd polypeptide gene will be used as templates. We predict transcription of both genes in the ct lysate but only the LS gene in the etioplast lysate. We hope to exploit this differential transcriptional response of lysates from the two plastid types of isolate a factor that is specifically required for the transcription of the 32 kd polypeptide gene in ct.