Expression and cellular localization of ribonucleotide reductase small subunit M-2 protein in hydroxyurea-resistant Leishmania mexicana amazonensis

We raised a specific antiserum against the recombinant M-2 subunit protein of ribonucleotide reductase of Leishmania mexicana amazonensis in rabbit. T his antiserum was used to study the expression and cellular location of the M-2 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 M-2 gene in the variants. In contrast to mammalian cells, the M-2 protein of Leishmania is located i n the nucleus rather than in the cytoplasm. The number of cells expressing M-2 protein is also different in mammalian cells Versus Leishmania. In mamm alian cells, expression of M-2 protein is a strictly S-phase-correlated eve nt and in exponentially growing cells only similar to 50% of the cells are in S-phase and only these cells synthesize M-2 protein. In L. m. amazonensi s, however, almost all exponentially growing cells are positive for M-2 pro tein. This makes it unlikely that M-2 protein expression in Leishmania is S -phase dependent. In view of these findings, a fresh look in the future int o the regulatory mechanisms of synthesis and the site of action of RNR in L . In. amazonensis is warranted. (C) 1999 Elsevier Science B.V. All rights r eserved.