GENE EXPRESSION IN CELL ORGANELLES

拟议研究的主要目标是：(1)确定 DNA依赖的rpoA、rpoB、rpoC之间的关系 水稻叶绿体DNA中编码的RNA聚合酶亚基基因 纤细裸藻和高等植物与RNA聚合酶 叶绿体的活性，(2)定义rpoB复合体如何- RpoC转录单位被转录，产生的Pre- 对mRNAs进行加工和翻译以产生多肽 亚基，(3)鉴定、分离和鉴定任何其他 叶绿体RNA的叶绿体或核定位基因 聚合酶，以及(4)确定如何表达 叶绿体RNA聚合酶亚单位基因在 光诱导细胞器发育。重点将放在 转录与转录后事件在脑内的重要性 转录与转录后事件的调控 调节发育中的质体的转录状态。 叶绿体包含两种类型的转录活性。按顺序 为了确定rpoA、B和C基因是否编码任何一个亚单位 或同时具有聚合酶活性、多肽编码区 叶绿体rpoA、B和C基因将在大肠杆菌中表达 用于产生单特异性多肽的产率融合多肽 针对腐烂RNA聚合酶的抗体 亚单位。抗体将被用来免疫纯化酶， 通过Western blotting鉴定亚基，并特异性地抑制 不同的转录反应。均一制剂 这些酶中的一种应该通过免疫亲和力获得 层析和FPLC，除了目前可用的 净化程序。RNA聚合酶亚基的基因 不是由rpoA、B和C基因座编码的基因也将被研究。在……里面 以确定如何处理rpoC抄本，以及 这一过程有一些不同寻常的特点，如交替拼接 将制备rpoC mRNA的位点、cDNA克隆和 已排序。通过上述类型的实验和 RNA聚合酶表达调控的研究 亚单位基因，以及由此产生的酶活性，它将是 有可能定义RNA的许多重要的基本方面 在细胞器中合成。

The main objectives of the proposed research are (1) to determine the relationship between the rpoA, rpoB, rpoC DNA-dependent RNA polymerase subunit genes encoded in chloroplast DNA of Euglena gracilis and higher plants and the RNA polymerase activities of chloroplasts, (2) to define how the complex rpoB- rpoC transcription units are transcribed, and the resulting pre- mRNAs are processed and translated to yield polypeptide subunits, (3) to identify, isolate and characterize any additional chloroplast or nuclear localized genes for chloroplast RNA polymerases, and (4) to determine how the expression of the chloroplast RNA polymerase subunit genes is regulated during light induced organelle development. Emphasis will be placed on the importance of transcriptional vs post-transcriptional events in regulating the transcriptional vs post-transcriptional events in regulating the transcriptional status of the developig plastid. Chloroplast contain two types of transcriptional activity. In order to determine if the rpoA, B, and C genes encode subunits of either or both polymerase activities, polypeptide coding domains of the chloroplast rpoA, B, and C genes will being expressed in E. coli to yield fusion polypeptides for use in generating monospecific antibodies directed against the putatative RNA polymerase subunits. Antibodies will be used to immunopurify the enzymes, to identify subunits by Western blotting, and to specifically inhibit the different transcriptional reactions. Homogenous preparations of these enzymes should be obtained via immunoaffinity chromatography and FPLC, in addition to currently available purification procedures. Genes for RNA polymerase subunits that are not encoded by the rpoA, B, and C loci will also be studied. In order to determine how rpoC transcripts are processed, and if there are unusual features to this process such as alternat splice sites, cDNA clones of rpoC mRNA will be prepared and sequenced. Through the types of experiments described above and studies on the regulation of expression of the RNA polymerase subunit genes, and of the resulting enzyme activities, it will be possible to define many important fundamental aspects of RNA synthesis in cell organelles.