| Adenine nucleotide transport has been demonstrated in Plasmodium falciparum (P.f.) via an adenine nucleotide translocator (ANT). A DNA product obtained by genomic PCR using two primers lying within conserved ANT domains, was applied as a probe to screen a P.f. cDNA library. A single hybridizing cDNA clone was isolated and sequenced, consisting of an ant open reading frame of 903 base pairs which encodes a basic polypeptide of 33.5 kilodaltons(kD). Using various ant -specific probes designed from the ant cDNA sequence, a single P.f. ant gene was implicated by genomic Southern analysis. Ant transcription was detected throughout the P.f. asexual cycle by Northern blot analysis, yielding transcripts of 4.1, 3.4 and 2.3 kilobases, all larger than the ant cDNA sequence. To determine the 5$spprime$ upstream ant sequence, several cDNA and gDNA clones were obtained by PCR and genomic library screening. One of the clones contains the 5$spprime$ upstream gDNA continuous to the ant coding sequence and its sequence is under determination. In order to detect and localize P.f. ANT(s), polyclonal antisera were raised against a glutathione S-transferase (GST)-P.f. ANT fusion protein. After immunoadsorption and affinity purification, anti-PfANT antibodies were isolated and they recognized P.f. proteins of 30 kD and 33 kD by Western blot analysis, similar in size to that predicted from the ant cDNA sequence. This suggests that the ant cDNA clone may have the full-length ant coding sequence. These two products were not detected by anti-GST antibodies, indicating that they are ANT-specific. ANT expression was shown throughout the P.f. asexual cycle, giving the two products at early stages but only the 30 kD protein at late stages of the cycle. By Western blot analysis and immunoflorescence using anti-PfANT antibodies, P.f. ANT was localized in P.f. membrane(s) but not P.f. mitochondrion. |
