Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae.
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.
Sheldon
P J
PJ
Department of Microbiology and Biological Process Technology Institute, Gortner Laboratory, University of Minnesota, St. Paul 55108, USA.
Johnson
D A
DA
August
P R
PR
Liu
H W
HW
Sherman
D H
DH
D000595:Amino Acid Sequence; D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins; D016685:Mitomycin / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D000903:Antibiotics, Antineoplastic; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; C104641:MRD protein, Streptomyces lavendulae; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016685:Mitomycin