Genetic analysis of pleiotropic mutation independent of A factor derived from Streptomyces griseus

为了揭示次级代谢中基因表达的调控机制，对灰色链霉菌多效性突变体的RNA聚合酶活性和σ因子进行了测定。获得的结果如下1.在亲本和多效性突变体中纯化RNA聚合酶。以主代谢基因和次级代谢基因为模板，测定体外转录活性。突变体的RNA聚合酶不具有转录次级代谢基因的活性，表明突变体中不存在或缺少sigma因子.筛选出与链霉素生物合成相关的strB基因启动子区结合的蛋白。在启动子基因上发现了一个结合蛋白，但该蛋白对多效性突变体是非特异性的. S.筛选了灰蝶。S.以coelicolorA3（2）为探针进行菌落和Southern杂交实验。含有pCE8的克隆与探针DNA强烈杂交。测定pCE8的核苷酸序列。序列中发现一个开放阅读框（ORF），长度为1335bp。该ORF含有36bp的rpoB box，该片段保存了几种sigma因子基因的核苷酸序列。hrdB与pCE8的同源性为86.3%。用北方印迹法测定了链霉菌菌丝生长过程中几个阶段的sigma样蛋白。该蛋白在菌丝生长的各个阶段都被识别。这些结果表明，pCE8中的蛋白质不是次级代谢因子，而是主要代谢因子，并暗示链霉菌中存在另一个与次级代谢有关的σ因子。

In order to clue a control mechanism of gene expression in secondary metabolism, RNA polymerase activity and sigma factor were determined in pleiotropic mutant derived from Streptomyces griseus. Results obtained are as follow1. RNA polymerase were purified in both parent and pleiotropic mutant. In vitro transcriptional activities using both principal and secondary metabolic gene as template were determined. RNA polymerase from mutant possessed no activity transcribing secondary metabolic gene, suggesting that a sigma factor was unusual or lack in the mutant.2. A protein binding to promoter region on strB gene which contributed to streptomycin biosynthesis was screened. A binding protein attached to promoter gene was foud, but the protein was non specific for pleiotropic mutant.3. A sigma factor in S. griseus was screened. Gene hrdB of S. coelicolor A3(2) was used as probe for both colony and Southern hybridization experiment. A clone houvouring pCE8 was hybridized strongly to the probe DNA.4. Nucleotide sequences of pCE8 was determined. A ORF(1335bp) was found in the sequence. The ORF contained 36bp rpoB box, which was preserved nucleotide sequence in gene of several sigma factor. DNA homology of hrdB to pCE8 was 86.3%.5. The sigma like protein derived from pCE8 were determined in several phase of growth in streptomyces mycelium, using Northern bloting method. The protein was recognized in all phases in myceliul growth. These result revealed that the protein from pCE8 was not secondary metabolic factor but principal one and suggested the presence of another sigma factor concerning secondary metabolism in Streptomyces.