Ogura M, Tanaka T, Seto H, Otake N
Institute of Applied Microbiology, University of Tokyo, Japan.
J Antibiot (Tokyo). 1990 Jul;43(7):873-82. doi: 10.7164/antibiotics.43.873.
We cloned six different DNA fragments from a curromycin producing strain, Streptomyces hygroscopicus 358AV2, which confer curromycin-resistance on a curromycin non-producing and sensitive strain, S. hygroscopicus Rgll, a protoplast regenerant of the strain 358AV2. We studied the plasmid pSHR2 carrying one of the DNA fragments. By Southern blot analysis, the cloned DNA sequence in pSHR2 was found to be deleted in the Rgll genome. From the Rgll strain, a curromycin producing revertant A-4 was obtained, indicating that the structural genes for the curromycin biosynthesis and resistance are retained in the Rgll genome. Based on the existence of A-4 and the deletion of the DNA region corresponding to the cloned DNA sequence in the Rgll genome, we conclude that the cloned DNA sequence carries a regulatory gene governing curromycin-resistance but not the resistance gene itself. The smallest DNA region in pSHR2 conferring curromycin-resistance was sequenced, and it was found that there were two small open reading frames (ORF) on each strand of the cloned DNA. In-frame fusion of ORFs to the reporter gene lacZ revealed that one ORF designated cre was indeed translated in vivo. The putative gene product deduced from the cre ORF is a basic and hydrophilic protein having a calculated molecular weight of 6 kdaltons.
我们从产curromycin的菌株吸水链霉菌358AV2中克隆了6个不同的DNA片段，这些片段可使不产curromycin且对其敏感的菌株——358AV2的原生质体再生菌株吸水链霉菌Rgll产生curromycin抗性。我们研究了携带其中一个DNA片段的质粒pSHR2。通过Southern印迹分析，发现pSHR2中的克隆DNA序列在Rgll基因组中缺失。从Rgll菌株中获得了一个产curromycin的回复突变体A-4，这表明curromycin生物合成和抗性的结构基因保留在Rgll基因组中。基于A-4的存在以及Rgll基因组中与克隆DNA序列相对应的DNA区域的缺失，我们得出结论，克隆的DNA序列携带一个调控curromycin抗性的基因，而不是抗性基因本身。对pSHR2中赋予curromycin抗性的最小DNA区域进行了测序，发现在克隆DNA的每条链上有两个小的开放阅读框（ORF）。将ORF与报告基因lacZ进行框内融合，结果显示一个命名为cre的ORF确实在体内被翻译。从cre ORF推导的推定基因产物是一种碱性亲水蛋白，计算分子量为6千道尔顿。
