Molecular Cloning Of A Putative Abstrakt Gene From Plasmodium Flaciparum And CDNA Cloning, Expression And ATPase Assay Of A Putative EIF-4A Of Plasmodium Cynomolgi

The ATP-dependent RNA helicase of the DEAD-box family, involved in almost every celluar process of RNA metabolism, play many essential roles during cell development and growth in almost all organisms. Malaria still remains a major health problem in many parts of the world. One of the possible ways to control malaria could be to discover inhibitors specific for the essential enzymes of malaria parasite like DNA/RNA helicases. In order to follow this approach we have initiated a systematic study of helicase from malaria parasite. We present here the research work on 2 fields in this study: 1. The cloning of a putative abstract gene from Plasmodum flaciparum and sequence analysis of different DEAD-box proteins. 2. The cDNA cloning, expression in Eschrichia coli, purification and ATPase assay of an eIF-4A homologue from Plasmodium cymologi.In this study , FH1F, the full-length gene of a putative abstrakt , was isolated by screening the genomic library of Plasmodium falciparum .FH1F was inferred located in the chromosome 5 in the whole genome. FH1F is composed of 2804 bp and encodes a predicted protein of 386 aa. Alignment analysis of the predicted protein sequence with many typical DEAD-box proteins suggested FH1F protein was an abstrakt of DEAD-box family. By phylogenetic analysis we realized that DEAD-box proteins assembled into different subgroups. Then we listed different organization of conserved motifs in abstrakt, eIF-4A , Vasa , P68 proteins as compared to the general DEAD-box protein consensus , and pointed out the novel residue substitutions in abstrakt proteins in the highly conserved positions. Our analysis will be important for providing general insights into the structural characteristics of different DEAD-box proteinsLikewise, CH1F, the full-length cDNA of a putative eIF-4A , was isolated by screening the cDNA library of Plasmodium cynomolgi , and then expressed in E. coli DH5 α . CH1F is composed of 1753 bp including the ORF of 1197 bp encoding a protein of 398 amino acid residues. Alignment analyses indicated that the deduced protein sequence was much more similar to other eIF-4As than other DEAD-box proteins. The recombinant protein of about 45 kD was purified , refolded and characterized. The ATPase assay showed that ATPase activity of the fusion protein was very low and seemed independent of nucleic acids. Here are three possible explanations for the results , and the results have no contradictions with the assumption that CH1F is an eIF-4A , derived from alignment analyses.