Danilenko V N, Potekhin Ia A, Biriukova I V, Navashin S M
Antibiotiki. 1984 Aug;29(8):563-72.
A new vector type was constructed on the basis of SLP 1.2 plasmid and the Kanr determinant of S. rimosus P3. It was shown that the determinant was capable of amplifying in the chromosomes of S. rimosus during its improvement for increasing the level of resistance to kanamycin. Cloning of the Kanr determinant was performed on the Pst I-A fragment of the SLP 1.2 plasmid in S. lividans 66. The Kanr determinant was expressed in the resulting hybrid plasmids, e. g. pSU 3, thus providing resistance of S. lividans to 20 micrograms/ml of kanamycin. As a result of repeated passages variants capable of growing in the presence of 50 000 micrograms/ml of the antibiotic were selected. The electrophoretic analysis of the total DNA fragments obtained after exposure to different endonucleases showed that they were identical to the respective fragments of the hybrid pSU 3 plasmid and amounted to approximately 40 per cent of the total DNA. The presence of the unique sites for the Bam HI, ClaI and SacI restriction endonucleases on the pSU 3 plasmid in an insignificant area and the relative stability (30-100 per cent) of the amplified variants allowed using it for cloning and amplification of DNA in Streptomyces. Plasmids were identified with the genetic and physical methods in 13 strains of the blue systematic group among the 72 strains studied. A multicopy plasmid with a molecular weight of 5.6 MD designated as pSB 24.1 was identified in the family of the plasmids of S. cyanogenus. The deletion and insertion variants of the pSB 24.1 plasmid were obtained. The comparative study of the properties of these plasmids revealed the areas insignificant for replication and maintenance of the pSB 24.1 plasmid and the area determining the Ltz+-phenotype. It was suggested that formation of the deletion and insertion variants of the pSB 24.1 plasmid was provided by the site specific recombination mechanisms. The presence of the unique sites for a number of the restriction endonucleases located in the insignificant area, the presence of the selective marker and the high transformation frequency allowed one to consider the multicopy pSB 24.1 plasmid an acceptable vector for cloning of DNA in Streptomyces.
基于SLP 1.2质粒和龟裂链霉菌P3的卡那霉素抗性决定簇构建了一种新型载体。结果表明，在改良龟裂链霉菌以提高其对卡那霉素的抗性水平的过程中，该抗性决定簇能够在龟裂链霉菌的染色体中扩增。卡那霉素抗性决定簇的克隆是在变铅青链霉菌66中对SLP 1.2质粒的Pst I - A片段进行的。卡那霉素抗性决定簇在所得的杂合质粒（如pSU 3）中表达，从而赋予变铅青链霉菌对20微克/毫升卡那霉素的抗性。经过多次传代，筛选出了能够在50000微克/毫升抗生素存在下生长的变体。对用不同核酸内切酶处理后获得的总DNA片段进行的电泳分析表明，它们与杂合pSU 3质粒的相应片段相同，约占总DNA的40%。pSU 3质粒上在一个不显著区域存在Bam HI、ClaI和SacI限制性核酸内切酶的独特位点，以及扩增变体的相对稳定性（30% - 100%），使得它可用于链霉菌中DNA的克隆和扩增。在所研究的72株菌株中，用遗传和物理方法在13株蓝色系统群菌株中鉴定出了质粒。在产蓝链霉菌的质粒家族中鉴定出了一种分子量为5.6 MD的多拷贝质粒，命名为pSB 24.1。获得了pSB 24.1质粒的缺失和插入变体。对这些质粒特性的比较研究揭示了对pSB 24.1质粒复制和维持不重要的区域以及决定Ltz + 表型的区域。有人认为，pSB 24.1质粒缺失和插入变体的形成是由位点特异性重组机制导致的。位于不显著区域的多种限制性核酸内切酶的独特位点的存在、选择标记的存在以及高转化频率使得人们可以将多拷贝pSB 24.1质粒视为链霉菌中DNA克隆的一种可接受的载体。
