摘要
在分离和定位链霉菌高拷贝质粒 pIJ101上启动子活性区域的过程中,我们将 Bcll-E 片段经 MboI 进一步酶解后,克隆到由 pIJ101基本复制功能区所衍生的载体 pIJ425的 BgIII位点上,从转化变铅青链霉菌 TK64的原生质体所获得的硫链丝菌素抗性转化子中,发现了两个拷贝数极其异常的衍生质粒,命名为 pIJ2743和 pIJ2744。与 pIJ425相比,pIJ2743的拷贝数增加约10倍,而 pIJ2744的拷贝数则降低约10倍。拷贝数的剧增和剧减不是由于质粒宿主的突变,因为它们在重新转化的宿主中仍显示了突变型拷贝数特征。拷贝数的变化也不是由于载体的突变,因为切除插入片段后,载体区域再连接后的质粒仍与原始 pIJ425的拷贝数相同。将高拷贝突变子中0.63kb 的插入片段以两种不同的取向克隆到另一个类似于pIJ425的载体 pIJ486的 BamHI 位点,在一种取向插入时,新衍生的质粒仍显示出极高的拷贝数,而在另一种取向时,质粒的拷贝数不发生变化,说明拷贝数的剧增不仅只与插入片段有关,而且其功用具有明显的方向性,即为一顺式作用因子。
In the process of isolating and localizing the promoter-active regions,the Bcl-E fragmentof pIJICl digested with Mobl was ligated with BgllI-drgested pIJ425,a vector derived fromthe basic replicon of plJ101.Among the thiostrepton-resistant transformants obtained after trans-formation of S.liuidans TK64,two plasmids were found to have unusural copy-number.Theywere designated as PIJ2743 and pIJ2744.While pIJ2743 has an extremely high copy-number,about ten times of that of the vector pIJ425,pIJ2744 has low copy-number,about ten times lowerthan that of the vector pIJ425 Two extreme copy number were maintained when the two pla-smids were introduced into fresh TK64 protoplasts and were thus not caused by host muta-tions.Removal of the 0.63 kb insert from pIJ2743 and 0.68 kb insert from pIJ2744 gave plas-raids with the same copy-number as the original vector pIJ425.This suggested that it is notthe mutation(s)in the vector which caused the changes of the copy-number.For pIJ2743,proof that the cloned piece of DNA was acturally alone responsible for the increased copy-num-ber came from subcloning the 0.63 kb Bglll insert into the BamHI site of pIJ486,a vector si-milar to pIJ425.The extremely high copy-number was observed when the fragment was in-serted in one orientation.With the same insert in the other orientation,a similar copy-num-her as for the vector without insert,pIJ425,was observed,suggesting that the effect of the insertmight be cis-acting.
出处
《微生物学报》
CAS
CSCD
北大核心
1993年第2期104-107,共4页
Acta Microbiologica Sinica
关键词
链霉菌属
拷贝
质粒
Copy-number mutants
pIJ101
Streptomyces
