Sheldon P J, Johnson D A, August P R, Liu H W, Sherman D H
Department of Microbiology and Biological Process Technology Institute, Gortner Laboratory, University of Minnesota, St. Paul 55108, USA.
J Bacteriol. 1997 Mar;179(5):1796-804. doi: 10.1128/jb.179.5.1796-1804.1997.
In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an Mr of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.
为了表征细胞针对抗肿瘤抗生素丝裂霉素C（MC）进行自我保护所涉及机制的多样性，将来自产生该抗生素的生物体（淡紫色链霉菌）的DNA片段导入变铅青链霉菌，并筛选出对该药物具有抗性的转化体。对一个4.0 kb的BclI片段进行亚克隆，发现了一个MC抗性决定簇mrd。核苷酸序列分析确定了一个由130个氨基酸组成的开放阅读框，预测分子量为14364。转录分析表明，mrd组成型表达，在MC存在时转录增加。mrd在大肠杆菌中的表达导致合成了一种Mr为14400的可溶性蛋白，该蛋白赋予细胞对MC和一系列结构相关天然产物的高水平抗性。纯化的MRD被证明作为一种药物结合蛋白发挥作用，通过阻止MC的还原激活来防止DNA交联。
