DNA amplification in tunicamycin-resistant Leishmania mexicana. Multicopies of a single 63-kilobase supercoiled molecule and their expression.
Tunicamycin-resistant variants of Leishmania mexicana were found to contain elevated activity of N-acetylglucosamine-1-phosphate transferase and amplified DNA (Kink, J. A., and Chang, K.-P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1253-1257). Complete digestion of their DNA with restriction endonucleases produced discrete ethidium bromide-staining bands after agarose gel electrophoresis. All four BamHI fragments of the amplified DNA were cloned separately into pBR322 and found to share no substantial sequence homology. DNA complementary to each of the cloned fragments is 64-128-fold more abundant in the variants than in the wild type cells. The amplified DNA appears to originate from a single chromosomal region of 63 kilobases. Individual copies of the 63 kilobases are each circularized at the newly formed junction site producing multiple extrachromosomal supercoiled molecules in the drug-resistant cells. There is overproduction of RNA ranging in size from 1.9 to 6.6 kilobases complementary to the amplified DNA in these cells.
D000818:Animals; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004278:DNA, Superhelical / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D006863:Hydrogen-Ion Concentration; D007894:Leishmania mexicana / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010770:Phosphotransferases / Q000378:metabolism; D012313:RNA / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D017855:Transferases (Other Substituted Phosphate Groups); D014415:Tunicamycin
D004274:DNA, Recombinant; D004278:DNA, Superhelical; D014415:Tunicamycin; D012313:RNA; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C023222:UDPacetylglucosamine-dolichyl-phosphate acetylglucosamine-1-phosphate transferase; D004262:DNA Restriction Enzymes