GENE EXPRESSION IN CELL ORGANELLES

本研究的主要目的是：（1）确定 rpoA、rpoB、rpoC DNA依赖性 叶绿体DNA中编码的RNA聚合酶亚基基因 纤细裸藻与高等植物及RNA聚合酶 活动的叶绿体，（2）以确定如何复杂的rpoB- rpoC转录单位被转录，并且所得的前- mRNA被加工和翻译以产生多肽 （3）识别、分离和表征任何额外的亚单位， 叶绿体RNA或核定位基因 聚合酶，和（4）以确定如何表达的 叶绿体RNA聚合酶亚基基因在 光诱导细胞器发育。 重点将放在 转录与转录后事件的重要性 调节转录与转录后事件， 调节发育质体的转录状态。 叶绿体具有两种类型的转录活性。 为了 以确定rpoA、B和C基因是否编码以下任一亚基： 或两者的聚合酶活性，所述多肽的多肽编码结构域 叶绿体rpoA、B和C基因将在E. 杆菌以 产生用于产生单特异性的融合多肽 针对腐败RNA聚合酶的抗体 亚单位。 抗体将用于免疫纯化酶， 通过Western印迹鉴定亚基，并特异性抑制 不同的转录反应。 均质制剂 这些酶应该通过免疫亲和 色谱和FPLC，除了目前可用的 纯化程序。 RNA聚合酶亚基的基因， 不是由rpoA、B和C基因座编码的。 在 以确定如何处理rpoC成绩单，如果 这种方法有一些不寻常的特点， 位点，将制备rpoC mRNA的cDNA克隆， 测序 通过上述类型的实验， RNA聚合酶表达调控的研究 亚基基因，以及由此产生的酶活性，它将是 可以定义RNA的许多重要的基本方面 细胞器中的合成。

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.