Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major.

Abstract

We have determined the nucleotide sequence of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) gene of the protozoan parasite Leishmania major (dihydrofolate reductase, EC 1.5.1.3 and thymidylate synthase, EC 2.1.1.45). The DHFR-TS protein is encoded by a single 1560-base-pair open reading frame within genomic DNA, in contrast to vertebrate DHFRs or mouse and phage T4 TSs, which contain intervening sequences. Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that (i) the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, (ii) the Leishmania bifunctional DHFR-TS evolved independently and not through a phage T4-related intermediate, and (iii) the rate ofevolution of both the DHFR and TS domains has not detectably changed despite the acquisition of new functional properties by the bifunctional enzyme. The Leishmania gene is 86% G+C in the third codon position, in contrast to genes of the parasite Plasmodium falciparum, which exhibit an opposite bias toward A+T. The DHFR-TS locus is encoded within a region of DNA amplified in methotrexate-resistant lines, as previously proposed.

The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection

“…are protozoa parasites among which are species that cause leishmaniasis, a disease that occurs in different clinical forms (cutaneous, mucocutaneous and visceral) and is endemic in many countries around the globe. Disease control and treatment are still inefficient and parasite drug resistance is a challenge since it may involve DNA amplification (Beverley et al 1986;Grogl et al 1989;Beverley, 1991;Ouellette et al 2004;Berman, 2005). Therefore, efforts for the establishment of intensive research to better understand the molecular biology of these parasites are encouraged.…”

Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.

“…DISCUSSION Our initial attempts to isolate the TS gene from P. carinii by screening the genomic library using a variety of heterologous hybridization probes failed. As probes, we tried the TS genes from Leishmania major (26,27), Plasmodium falciparum (28), and Herpesvirus saimiri (29), as well as two degenerate oligonucleotides based on conserved sequences of TS; the hybridization conditions were also varied to such low stringency that numerous false positives were uncovered. To obtain a suitable hybridization probe, we decided to use 256-fold degenerate oligonucleotides as PCR primers to amplify a fragment of the TS gene from P. carinii genomic DNA.…”

<i>IN SILICO</i> AND <i>IN VITRO</i> STUDIES: TRYPAREDOXIN PEROXIDASE INHIBITOR ACTIVITY OF METHOTREXATE FOR ANTILEISHMANIAL ACTIVITY

“…In most organisms DHFR and TS exist as separate molecular entities. Whereas, in Leishmania, these enzymes are a part of a bi-functional DHFR-TS complex (Beverley et al, 1986). Studies have shown that it is possible to achieve inhibition selectivity of the parasite against the human by targeting the DHFR-TS complex of the Leishmania major.…”