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Functional Analysis of Plasmid pSAl and Its Derivatives in Streptomyces azureus

天蓝色链霉菌质粒pSAl及其衍生物的功能分析

基本信息

批准号：
60560120
负责人：
OGATA Seiya
金额：
$ 1.15万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-60560120/
关键词：
Pock formation Pock plasmid Function of pock plasmid Defective plasmid Restriction enzyme cleavage map of plasmid Inhibition of spore formation Integrated state of plasmid 胞子形成抑制機能

项目摘要

Streptomyces azureus ATCC 14921 (PK0) harbored one copy or less of plasmid pSA1 (8.8kb), also its primal sequences in an integrated state, and elicited pocks at 0.1 to 1% frequency on the plasmid-free strains. Strain PK100 carried 20 to 30 copies of pSA1.1 (8.8kb), a derivative of pSA1, and elicited pocks at 100% frequency. Stain PK10 carried two plasmids (pSA1.1, pSA1.2), and elicited pocks at 10 to 50% frequency. Plasmid pSA1.2 (7.6kb; copy size, 5 to 10) had two deleted portions (ca. 1.2kb and 30b long) of pSA1.1 together with no pock-forming ability. Either one or both of the deletion segments seem to be essential to the pock formation. The production of spores and thiostrepton was very low in PK100, normal in PK0 and PK10, and abundant in plasmid-free strains. These results show that the inhibition on the secondary products and the high pock formation are due to pSA1.1 in high copies. Plasmid pSA1.1 from PK10 amplified 20 to 30 copies in transformants as well as from PK100 and inhibited their spore and thiostrepton production, whereas, in PK10, copy size was lower (10 to 20) and no inhibition was observed. These results show that the defective pSA1.2 appears to depress the ability of pSA1.1 on the host cells. Cysteine also depressed the ability of pSA1.1. A specific protein (MW ca. 70,000) was found in the strains having pSA1.1.The hybrid plasmids were easily obtained from pSA1.2 and E. coli plasmid pBR322 or pUC13 using all the unique sites of endonucleases. But, only one hybrid was obtained from pSA1.1 and pBR322 using their BamHI sites. These results indicate that the sequence of defective pSA1.2 is easily amplified in the host-vector system of E. coli but not that of pock-forming pSA1.1. These different features between pSA1.1 and pSA1.2 may be due to the 30b-deletion segments in pSA1.2 near by BamHI site.

蓝色链霉菌ATCC 14921（PK 0）含有一个拷贝或更少的质粒pSA 1（8.8kb），其原始序列也处于整合状态，并且在无质粒菌株上以0.1%至1%的频率引起麻点。菌株PK100携带20 - 30个拷贝的pSA 1.1（8.8kb），pSA 1.1是pSA 1的衍生物，并且以100%的频率引发麻点。PK10菌株携带两种质粒（pSA1.1，pSA1.2），并以10%至50%的频率引发麻点。质粒pSA1.2（7.6kb;拷贝大小，5至10）具有两个缺失部分（约100 kb）。1.2kb，30 kb长），但无形成痘的能力。一个或两个缺失片段似乎是必要的麻点的形成。孢子和硫链丝菌素的产生在PK100中非常低，在PK 0和PK10中正常，并且在无质粒菌株中丰富。这些结果表明，对次级产物的抑制和高麻点形成是由于高拷贝的pSA1.1。来自PK10的质粒pSA1.1在转化体中以及从PK100扩增20至30个拷贝，并抑制它们的孢子和硫链丝菌素产生，而在PK10中，拷贝大小较低（10至20），未观察到抑制。这些结果表明，缺陷型pSA1.2似乎抑制了pSA1.1对宿主细胞的能力。半胱氨酸还抑制pSA 1.1的能力。特定蛋白质（MW ca. 70，000）。从pSA 1.2和E.大肠杆菌质粒pBR 322或pUC 13使用所有独特的内切酶位点。但是，利用pSA 1.1和pBR 322的BamHI位点，仅获得一个杂交体。这些结果表明，缺陷型pSA 1.2的序列在大肠杆菌宿主-载体系统中容易扩增。而pSA1.1则没有。pSA 1.2与pSA 1.1的这些差异可能是由于pSA 1.2中靠近BamHI位点的30 b缺失片段所致。