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CHLOROPLAST GENE STRUCTURE AND REGULATION

叶绿体基因结构和调控

基本信息

批准号：
3278557
负责人：
NAM-HAI CHUA
金额：
$ 17万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-05-01 至 1987-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3278557
关键词：
antibody formation autoradiography chloroplasts density gradient ultracentrifugation electron microscopy endonuclease gel electrophoresis gene expression genetic mapping genetic regulation genetic transcription immunoprecipitation messenger RNA nucleic acid sequence structural genes

项目摘要

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.

大多数，如果不是全部的话，叶绿体（ct）中的多聚体酶是由其结构基因分散在两个不同的细胞中，隔室（核和CT）。我们的长期目标是了解这些基因的表达是如何协调和调节的。作为为了实现这一目标，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有可能编码超过100种多肽，其中只有两种已被确定迄今本提案涉及对编码光系统（PS）多肽亚基的黄瓜ct基因 II复合体。在ct内产生的多肽亚基在不存在或存在特异性抑制剂的抗体会针对ct合成的亚基其可能是CT基因产物。黄瓜的限制性片段 ct DNA将用于从总ct RNA中选择特异性mRNA 混合物. 选择的mRNA将在网织红细胞裂解物中翻译系统和通过抗体免疫沉淀鉴定的产物至PS II多肽。这些实验将确定ct基因编码 PS II多肽，并将其定位于PS II多肽的特异性限制性片段。已知的地图位置编码PS II的每个mRNA的5 '起始位点多肽将通过S1技术的修改来作图。每个基因的特征在于其转录方向以及它是否含有内含子。基因的3 '和5'区将通过Maxam-Gilbert技术进行测序，看看是否有在基因调控中可能很重要的共有序列。学习ct 体外基因表达和调节我们将建立一个依赖DNA的使用质体裂解物的可溶性转录系统。截短的羧化酶大亚基（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中的转录裂解物中，但只有LS基因的原生质体裂解物。我们希望利用两种质体裂解物的不同转录反应隔离类型是一个特定要求的因素， CT中32kD多肽基因的转录。