Cloning and Analysis of Tautomycin Biosynthetic Genes

Tautomycin （TM）从一种培养培养基中分离的Streptomyces spiroverticillatus是蛋白磷酸酶1和2A的抑制剂。自从TM及其模拟被期望成为有用的探测器，以促进哺乳动物细胞的信号转化，我们研究了一个绝对的结构、调整性分析、结构活动关系和多酮化合物的生物合成， TM。在目前的研究中，我们研究了TM生物合成基因的克隆和分析。在此，我报告了1996、1997和1998年的实验结果。转换系统的发展：TM生产者利用报告的其他链霉菌的程序实现了原型形成和再生。在添加到实验上，该转换系统已被确定为通过热休克治疗治愈的生产者的加密质粒。2x 10型D15型D1转化者/μgDNA使用pIJ 702质粒获得的满意度，结果第三次 ... More 在我们最优化的转换系统的各种条件下。可移植TM生物合成基因的克隆：KS和AT引物，是从PKS I型出版的序列设计的，用于PCR策略以获得产品。初级设计策略是成功的，可以识别出来自S的碎片。spiroverticillatus基因组DNA和cosmid pwe 15上的PKS。PCR产品的序列分析，我们强烈建议在TM生产商中克隆了PKS基因标志。Gene disruption of the putative TM biosynthetic gene： To determine whether the PKS gene fragment obtained from PCR strategy indeed encodes PKS involving in TM biosynthesis， the fragments were then used for gene disruption by recombinational insertion of the cloned 1.8 kb region into the corresponding region on the chromosome。结果15条直线是增强的，并被确认为TM的存在。没有一个是由strains生产的，另一个抗生素xanthostatin是由全strains生产的。研究人员展示了TM生物合成基因在这些菌株中被基因组DNA删除的基因组南方杂交分析。Less（低）

Tautomycin (TM) isolated from a culture broth of Streptomyces spiroverticillatus is an inhibitor of protein phosphatases 1 and 2A. Since TM and its analogues are expected to be useful probes for elucidating the signal trasduction of mammalian cells, we studied on the absolute structure, conformational analysis, structure-activity relationship, and biosynthesis of the polyketide compound, TM. In the present study, we have studied on the cloning and analysis of TM biosynthetic genes. Here I report the results of experiments done in 1996, 1997 and 1998.1. Development of transformation system: Protoplast formation and regeneration of the TM producer were achieved by using the reported procedure for other Streptomyces sp. In addition to above experiments, the transformation system was established by curing the producer of the cryptic plasmid by a heat shock treatment. The satisfactory efficiency of 2 x 10ィイD15ィエD1 transformants/μgDNA was obtained using the pIJ702 plasmid, with the result th … More at we optimized various conditions of the transformation system.2. Cloning of the putative TM biosynthetic gene: The KS and AT primers, which were designed from the published sequences in PKS Type I, were used in PCR strategy to obtain products. The primer design strategy was successful in identifying a fragment from S. spiroverticillatus genomic DNA and a PKS on cosmid pWE15. Sequence analysis of the PCR product strongly suggests that we have cloned a PKS gene flagment in the TM producer.3. Gene disruption of the putative TM biosynthetic gene: To determine whether the PKS gene fragment obtained from PCR strategy indeed encodes PKS involving in TM biosynthesis, the fragments were then used for gene disruption by recombinational insertion of the cloned 1.8 kb region into the corresponding region on the chromosome. The resulting 15 strains were fermented and assayed for the presence of TM. Although none of the strains produced TM, an another antibiotic xanthostatin was produced by all strains. Genomic Southern hybridization analyses showed that TM biosynthetic genes in these strains were deleted from the genomic DNA. Less