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

为了揭示基因在次生代谢中的表达调控机制，对灰链霉菌多效突变体的RNA聚合酶活性和sigma因子进行了测定。所得结果如下：1。在亲本和多效突变体中均纯化了RNA聚合酶。以主代谢基因和次代谢基因为模板，测定其体外转录活性。来自突变体的RNA聚合酶不具有转录次级代谢基因的活性，这表明突变体中不寻常或缺乏sigma因子。筛选了一种与strB基因启动子区结合的链霉素生物合成蛋白。在启动子基因上发现了一个结合蛋白，但该蛋白对多效突变体无特异性。筛选了griseus的sigma因子。以S. coelicolor A3(2) hrdB基因为探针进行群体杂交和南方杂交实验。一个包含pCE8的克隆与探针dna进行了强杂交。测定pCE8的核苷酸序列。在该序列中发现一个ORF（1335bp）。ORF含有36bp的rpoB box，保存了多个sigma因子基因的核苷酸序列。hrdB与pCE8的DNA同源性为86.3%。用Northern blotting法测定了链霉菌菌丝生长的不同阶段中pCE8衍生的sigma样蛋白。该蛋白在菌丝生长的各个阶段均被识别。这些结果表明pCE8蛋白不是链霉菌次生代谢因子，而是主要代谢因子，提示链霉菌中存在另一个与次生代谢有关的sigma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.