Expression and cellular localization of ribonucleotide reductase small subunit M2 protein in hydroxyurea-resistant Leishmania mexicana amazonensis.
We raised a specific antiserum against the recombinant M2 subunit protein of ribonucleotide reductase of Leishmania mexicana amazonensis in rabbit. This antiserum was used to study the expression and cellular location of the M2 protein in wildtype as well as hydroxyurea-resistant variants (HuR) of the parasite. The protein increased with increasing dose of the drug used for selection of resistance. The increase in protein level was accompanied by an increase in the copy numbers of mRNA of the M2 gene in the variants. In contrast to mammalian cells, the M2 protein of Leishmania is located in the nucleus rather than in the cytoplasm. The number of cells expressing M2 protein is also different in mammalian cells versus Leishmania. In mammalian cells, expression of M2 protein is a strictly S-phase-correlated event and in exponentially growing cells only approximately 50% of the cells are in S-phase and only these cells synthesize M2 protein. In L. m. amazonensis, however, almost all exponentially growing cells are positive for M2 protein. This makes it unlikely that M2 protein expression in Leishmania is S-phase dependent. In view of these findings, a fresh look in the future into the regulatory mechanisms of synthesis and the site of action of RNR in L. m. amazonensis is warranted.
Lye
L F
LF
Division of Infectious Diseases, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC.
Chiang
S C
SC
Hsu
J Y
JY
Lee
S T
ST
D000818:Animals; D000913:Antibodies, Protozoan / Q000096:biosynthesis / Q000276:immunology; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004351:Drug Resistance; D005455:Fluorescent Antibody Technique; D006918:Hydroxyurea / Q000494:pharmacology*; D007894:Leishmania mexicana / Q000187:drug effects* / Q000201:enzymology* / Q000254:growth & development; D016253:Microscopy, Immunoelectron; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D012262:Ribonucleoside Diphosphate Reductase; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*
D000913:Antibodies, Protozoan; D000981:Antiprotozoal Agents; D011993:Recombinant Fusion Proteins; D012264:Ribonucleotide Reductases; C093713:ribonucleotide reductase M2; D012262:Ribonucleoside Diphosphate Reductase; D006918:Hydroxyurea