Athrombospondin-related anonymous protein (TRAP) of the human malaria parasite Plasmodium falciparum shares highly conserved amino acid sequence motifs with the circumsporozoite protein of all plasmodia sequenced so far, as well as with unrelated proteins like thrombospondin and properdin. Although it was first described as an asexual blood stages protein, there has been some controversy about its expression in these stages. Pursuant to our interest in the conserved sequences within the malaria antigens, we synthesized an 18-residue peptide (18-mer) representing a conserved motif of TRAP and raised polyclonal antibodies against it. In an immunoblot assay in which we probed proteins from the asexual blood stages of the parasite, we found that this antibody recognized predominantly a 78-kDa protein in the whole parasite lysate. Furthermore, in another immunoblot, the recombinant TRAP constructs containing the conserved-motif sequence were distinctly recognized by the antipeptide antibodies, whereas a construct lacking the motif sequence was not, suggesting that the antibodies specifically cross-reacted with a protein which might be a TRAP-like protein present in the asexual blood stages of the parasite. Also, in an immunofluorescence assay, this antibody brightly stained the acetone-fixed trophozoites of the parasite. Most significantly, anti-18-mer immunoglobulin G, as well as antipeptide antibody against a smaller (nonamer) construct representing the most conserved motif within the 18-mer, inhibited the merozoite invasion of erythrocytes in a dose-dependent manner. These results provide evidence of the expression of TRAP or a TRAP-like protein in the asexual blood stages of the parasite and of a possible role of the conserved motifs in the parasite-host cell interaction during the process of invasion.
The complete nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica DSM 43387 was determined. Sequence analysis revealed that pA387 is 30,157 bp long and has a G+C content of 71.74%. To obtain a minimal transferable replicon capable of self-replication, a 2,176 bp fragment of pA387 was cloned, and we demonstrated that this fragment is sufficient for autonomous replication. The replication region of pA387 exhibited no significant homology to any known replication proteins available in databases. Putative maintenance and transfer functions were identified on pA387. The predicted products of open reading frames, ORF 2 and ORF 12, resembled the plasmid stabilizing proteins, a DNA resolvase and a ParA protein, respectively. The putative translational products of ORF 15 and ORF 16 showed similarity to known bacterial conjugation proteins, TraG and TraA, respectively. A conjugative Escherichia coli -Amycolatopsis shuttle-cloning vector was constructed by using the pA387 replicon and designated pSETRL1. Shuttle vector pSETRL1 successfully transformed Amycolatopsis mediterranei DSM 40773 and Amycolatopsis orientalis NBRC 12806 by conjugation and electroporation, and is likely to be a useful vector in Amycolatopsis research.
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