TY - JOUR
T1 - Stable amplification of a linear extrachromosomal DNA in mycophenolic acid-resistant Leishmania donovani
AU - Wilson, Keith
AU - Beverley, Stephen M.
AU - Ullman, Buddy
N1 - Funding Information:
This work was supported by Grants AI23682 and AI32036 (B.U.) and Grant AI21903 (S.B) from the National Institute of Allergy and Infectious Diseases. K.W.'s salary was supported in part by the N. L. Tartar Trust Fellowship Fund. We thank C. M. Coburn, D. D. Rogers and L. Tolmasky for technical assistance and Dr. Kenneth Stuart and Dr. Lex van der Ploeg for providing the pCD-1-B8 and pTT6 probes, respectively. We also thank Dr. Sheri Hanson for critical reading of the manuscript and many helpful discussions.
PY - 1992/10
Y1 - 1992/10
N2 - Pulsed field gel electrophoretic analysis of chromosomes of MPA 100 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 γ-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to λ-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 MPA 100 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.
AB - Pulsed field gel electrophoretic analysis of chromosomes of MPA 100 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 γ-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to λ-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 MPA 100 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.
KW - Drug resistance
KW - Extrachromosomal element
KW - Gene amplification
KW - IMP dehydrogenase
KW - Leishmania donovani
KW - Pulsed field gel electrophoresis
UR - http://www.scopus.com/inward/record.url?scp=0026657269&partnerID=8YFLogxK
U2 - 10.1016/0166-6851(92)90140-F
DO - 10.1016/0166-6851(92)90140-F
M3 - Article
C2 - 1359409
AN - SCOPUS:0026657269
SN - 0166-6851
VL - 55
SP - 197
EP - 206
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1-2
ER -