Stable amplification of a linear extrachromosomal DNA in mycophenolic acid-resistant Leishmania donovani.
Pulsed field gel electrophoretic analysis of chromosomes of MPA100 cells, a strain of Leishmania donovani that possesses an approx. 15-fold amplified IMP dehydrogenase (IMPDH) gene copy number, revealed a new 280-kb extrachromosomal DNA, IMPDH-280, that was not present in wild type parental cells. Southern blots of these pulsed field gels revealed that the vast majority of the amplified impdh genes were localized on IMPDH-280. In addition to the 700-kb wild type chromosome, the impdh probe also recognized a 740-kb chromosome in the MPA100 genome. The pulse time-dependent relative mobility of IMPDH-280 in pulsed field gels, the failure of limited gamma-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to lambda-exonuclease digestion, demonstrated that IMPDH-280 was a linear molecule. IMPDH-280 was also recognized by a telomere probe but not by fragments derived from amplified DNAs found in other drug-resistant Leishmania. IMPDH-280 and the drug resistance phenotype remained stable when MPA100 cells were propagated in the absence of drug for 2 years. The appearance of IMPDH-280 in MPA100 cells represents one of the first examples of an amplification of a linear extrachromosomal DNA element mediating drug resistance in Leishmania and the first instance of a linear DNA amplification that is stable in the absence of selective pressure.
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098.
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K
Wilson
S M
SM
Beverley
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B
Ullman
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D015342:DNA Probes; D004270:DNA, Circular / Q000032:analysis; D016054:DNA, Protozoan / Q000032:analysis*; D004351:Drug Resistance; D016521:Electrophoresis, Gel, Pulsed-Field; D005784:Gene Amplification; D017125:Genes, Protozoan; D007168:IMP Dehydrogenase / Q000235:genetics*; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D009173:Mycophenolic Acid / Q000494:pharmacology*; D010957:Plasmids; D016615:Telomere / Q000737:chemistry
D015342:DNA Probes; D004270:DNA, Circular; D016054:DNA, Protozoan; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid