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基于放线菌质粒pSB24.1和质粒prBR325的杂交质粒设计及其在链霉菌-大肠杆菌系统中的稳定性研究

[Design of hybrid plasmids based on the actinomycete plasmid pSB24.1 and plasmid prBR325 and a study of their stability in the Streptomyces-E. coli system].

作者信息

Podgornova G P, Iarulin V R, Danilenko V N

出版信息

Antibiot Med Biotekhnol. 1987 Oct;32(10):723-30.

PMID:3322185
Abstract

Actinomycete plasmid pSB24.1 was cloned on vector of the E. coli pBR325 system. The following bireplicon plasmids were obtained: pSU501 and pSU502 (by XhoI site of pSB24.1 and SalGI site of pBR325), pSU503 (by Bg1II (c) site of pSB24.1 and BamHI site of pBR325) and pSU504 (by Bg1II(b) site of pSB24.1 and BamHI site of pBR325). In the cells of E. coli C600 plasmids pSU501-504 determined phenotype AprCmrTcs and were stable. In the cells of Str. lividans the initial structure pSU501 selected by Ltz+ phenotype was maintained at a frequency of 12.5 per cent. Analysis of the deletion variants of pSU501 isolated from Str. lividans showed that the deletions were induced by both the pBR325 region and the pSB24.1 DNA fragment near XhoI site. The region from SacII(a) site to Bg1II(b) site clockwise in the map of plasmid pSB24.1 was not significant for its replication and maintenance in Str. lividans. There were detected unique sites of pSB24.1 and its derivatives useful for cloning. Possible shortening of plasmid pSB24.1 by 567 kb (the length between the terminator of the open frame reading translation and XhoI site) was revealed.

摘要

放线菌质粒pSB24.1被克隆到大肠杆菌pBR325系统的载体上。获得了以下双复制子质粒:pSU501和pSU502(通过pSB24.1的XhoI位点和pBR325的SalGI位点)、pSU503(通过pSB24.1的Bg1II (c)位点和pBR325的BamHI位点)以及pSU504(通过pSB24.1的Bg1II(b)位点和pBR325的BamHI位点)。在大肠杆菌C600细胞中,质粒pSU501 - 504决定了AprCmrTcs表型且是稳定的。在变铅青链霉菌细胞中,通过Ltz +表型选择的初始结构pSU501以12.5%的频率得以维持。对从变铅青链霉菌中分离出的pSU501缺失变体的分析表明,缺失是由pBR325区域和XhoI位点附近的pSB24.1 DNA片段诱导的。在质粒pSB24.1图谱中,从SacII(a)位点到Bg1II(b)位点顺时针方向的区域对其在变铅青链霉菌中的复制和维持并不重要。检测到了pSB24.1及其衍生物中可用于克隆的独特位点。揭示了质粒pSB24.1可能缩短567 kb(开放阅读框翻译终止子与XhoI位点之间的长度)。

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