TY - JOUR
T1 - Identification of surface-membrane P-type ATPases resembling fungal K+- and Na+-ATPases, in Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani
AU - Stiles, J. K.
AU - Kucerova, Z.
AU - Sarfo, B.
AU - Meade, C. A.
AU - Thompson, W.
AU - Shah, P.
AU - Xue, L.
AU - Meade, J. C.
PY - 2003/6
Y1 - 2003/6
N2 - Genomic DNA fragments encoding nine, novel, P-type ATPases in trypanosomatid organisms were amplified in PCR, using degenerate oligonucleotide primers that recognize the ATP-binding and -phosphorylation sites present in all P-type ATPases. Subsequent phylogenetic analysis, based on the presence of conserved motifs in predicted peptide sequences for six Trypanosoma brucei, T. cruzi or Leishmania donovani PCR fragments, identified calcium-,proton- and phospholipid-translocating ATPases. DNA fragments that predict proteins homologous to the fungal, type-IID, P-type, ATPase pumps that transport Na+ or K+ ions were also present in T. brucei (TBCA1; 1022 nucleotides representing 340 amino acids), T. cruzi (TCNA1; 1022 nucleotides representing 340 amino acids) and L. donovani (LDCA1; 1031 nucleotides representing 343 amino acids). Southern blots showed that the Na+-ATPases were each present as a single-copy gene. The LDCA1 fragment was used to clone the complete LDCA1 gene from an L. donovani genomic-DNA library. The LDCA1 gene encodes a protein, of 1047 amino acids, with a predicted molecular mass of 115,501 Da. The results of analyses based on northern blots and the rapid amplification of cDNA ends (RACE) indicated that LDCA1 was expressed in promastigotes and amastigotes from axenic cultures and in animal-derived amastigotes. TBCA1 was expressed, as a 5.0-kb transcript, in procyclic culture stages and bloodstream trypomastigotes, with the 5.0-kb message up-regulated six-fold in the trypomastigote stage. Western blots probed with an antibody to the partial TBCA1 peptide identified a 150-kDa protein that was detected, by immunofluorescence, on the surface membrane of procyclic T. brucei..
AB - Genomic DNA fragments encoding nine, novel, P-type ATPases in trypanosomatid organisms were amplified in PCR, using degenerate oligonucleotide primers that recognize the ATP-binding and -phosphorylation sites present in all P-type ATPases. Subsequent phylogenetic analysis, based on the presence of conserved motifs in predicted peptide sequences for six Trypanosoma brucei, T. cruzi or Leishmania donovani PCR fragments, identified calcium-,proton- and phospholipid-translocating ATPases. DNA fragments that predict proteins homologous to the fungal, type-IID, P-type, ATPase pumps that transport Na+ or K+ ions were also present in T. brucei (TBCA1; 1022 nucleotides representing 340 amino acids), T. cruzi (TCNA1; 1022 nucleotides representing 340 amino acids) and L. donovani (LDCA1; 1031 nucleotides representing 343 amino acids). Southern blots showed that the Na+-ATPases were each present as a single-copy gene. The LDCA1 fragment was used to clone the complete LDCA1 gene from an L. donovani genomic-DNA library. The LDCA1 gene encodes a protein, of 1047 amino acids, with a predicted molecular mass of 115,501 Da. The results of analyses based on northern blots and the rapid amplification of cDNA ends (RACE) indicated that LDCA1 was expressed in promastigotes and amastigotes from axenic cultures and in animal-derived amastigotes. TBCA1 was expressed, as a 5.0-kb transcript, in procyclic culture stages and bloodstream trypomastigotes, with the 5.0-kb message up-regulated six-fold in the trypomastigote stage. Western blots probed with an antibody to the partial TBCA1 peptide identified a 150-kDa protein that was detected, by immunofluorescence, on the surface membrane of procyclic T. brucei..
UR - http://www.scopus.com/inward/record.url?scp=0038575686&partnerID=8YFLogxK
U2 - 10.1179/000349803235002362
DO - 10.1179/000349803235002362
M3 - Article
C2 - 12831521
AN - SCOPUS:0038575686
SN - 0003-4983
VL - 97
SP - 351
EP - 366
JO - Annals of Tropical Medicine and Parasitology
JF - Annals of Tropical Medicine and Parasitology
IS - 4
ER -