A VECTOR SYSTEM WITH TEMPERATURE-SENSITIVE REPLICATION FOR GENE DISRUPTION AND MUTATIONAL CLONING IN STREPTOMYCETES
A VECTOR SYSTEM WITH TEMPERATURE-SENSITIVE REPLICATION FOR GENE DISRUPTION AND MUTATIONAL CLONING IN STREPTOMYCETES
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DOI：
10.1007/bf00259605
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发表时间：
1989-11-01
期刊：
影响因子：
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通讯作者：
PUHLER, A
中科院分区：
文献类型：
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作者：
MUTH, G;NUSSBAUMER, B;PUHLER, A
加纳链霉菌质粒pSG 5的复制显示出温度敏感性。pSG 5复制子在低于34 ℃的温度下稳定遗传。C，但在高于此的孵育温度下会丢失。使用pSG 5最小复制子和不同标记基因构建了一个克隆载体家族。所获得的载体尺寸小，具有中等拷贝数，具有广泛的宿主范围，并且与一些其他链霉菌载体系统相容。通过提高孵育温度，这些载体可以非常有效地从其宿主细胞中消除。证明了pSG 5载体家族通过基因破坏突变染色体基因的适用性：pBN 10，一种含有膦丝丙酰丙氨酸（PTT）抗性基因pat的内部片段的pSG 5衍生物，被整合到PTT生产者绿产色链霉菌的染色体pat基因中，从而使PTT抗性失活。整合的pBN10质粒与携带PTT生物合成簇DNA的相邻片段一起从染色体中被拯救出来。
Replication of the Streptomyces ghanaensis plasmid pSG5 was shown to be temperature sensitive. The pSG5 replicon is stably inherited at temperatures below 34.degree. C, but is lost at incubation temperatures above this. A family of cloning vectors was constructed using the pSG5 minimal replicon and different marker genes. The vectors obtained are small in size, have an intermediate copy number, possess a broad host range and are compatible with some other streptomycete vector systems. By increasing the incubation temperature, these vectors can be eliminated from their host cells very efficiently. The suitability of the pSG5 vector family for mutating chromosomal genes by gene disruption was demonstrated: pBN10, a pSG5 derivative containing an internal fragment of the phosphinothricyl-alanyl-alanine (PTT) resistance gene pat, was integrated into the chromosomal pat gene of the PTT-producer Streptomyces viridochromogenes thus inactivating PTT resistance. The integrated pBN10 plasmid was rescued from the chromosome, together with an adjacent fragment carrying DNA of the PTT biosynthetic cluster.