转座子Tn4560在吸水链霉菌应城变种中的转座

TRANSPOSITION OF Tn4560 IN STREPTOMYCES HYGROSCOPICUS VAR. YINGCHENGENSIS

Tn4556是在弗氏链霉菌UC8592中发现的一个类似于Tn3的转座子,在它转坐的非必需区中间插入紫霉素抗性基因vph所组成的复合体(Tn4556::vph),称为Tn4560.把Tn4560克隆到一个温敏性质粒pMT660(由PIJ702衍生而来)构建的重组质粒(pMT660::Tn4560)称为pUC1169.pUC1169不能直接转化吸水链霉菌应城变种1O-22,但可以以一定的频率转化其衍生菌株Q105(至少丧失了一个内源质粒的受体菌株.)从转化子中检测DNA,可以观察到完整的PUC1169的存在(仍为高拷贝).将携带了pUC1169的Q105菌株的孢子悬液以一定的稀释度涂布到无抗生素的平板上获得单个分散的菌株,把平板放在39℃下培养,待菌落长出丰满的孢子后,再分别影印到含有紫霉素和硫链丝菌素的培养基平板上,分离紫霉素抗性(vio^r)和硫链丝菌素敏感(thio^s)的菌落,即为pMT660“自杀”,而Tn4560在细胞中发生了转座的个体.从18个这类菌落中提取总DNA,经PvuⅡ酶解后,在琼脂糖凝胶上电泳,把电泳后的DNA转移到硝酸纤维膜上,与非放射性方法标记的pUC1169探针杂交揭示,每一个衍生菌株都丢失了对应于载体PMT660的区域,却在染色体或内源质粒的不同位点上保留了Tn4560.从vio^r thio^s的突变体中已获得了5102-Ⅲ抗生素生物合成的阻断突变株.

Tn4556, a transposon of the Tn3 family, was discovered in Streptamyces fradiae UC8592. Incorporation of a viomycin resistant gene (vph) into Tn4556 gave Tn4560 (Tn4556: : vph). The Tn4560 was cloned into pMT660, a temperature sensitive derivative of the wide host range plasmid pIJ702, to give pUCH69 (pMT660:: Tn4560). pUC1169 can not be introduced by transforming directly into S. hygroscopicus 10-22, but can be transformed into one of its derivatives, Q105 (at least one of the five indigeous plasmid present in 10-22 was cured). Intact pUC1169 can be visualized at high copy number in Q105. Spores of Q105 carrying pUC1169 were harvested and plated on antibiotic-free medium for single colonies. The plates were incubated at 30 C and then shifted up to 39℃. After sporulation, the colonies are replica plated to agar containing viomycin or thiostrepton. Viomycin resistant (vior) and thiostrepton sensitive (thios) colonies must have lost pUC1169 but retained Tn4560 by transposition to the chromosome (or other replicons). The total DNA of 18 vio1 thios colonies were digested with Pvu Ⅱ respactively. fractionated on agarose gel and transferred to nitrocellulose filter. After hybridization with nonradioactively (DIG) labelled pUC1169, all the colonies were found to have lost the region corresponding to pMT660, but retained Tn4560 at chromosome or plasmids. Blocked mutants for 5102-Ⅲ antibiotics biosynthesis have been obtained among vior thios mutants.