Studies of schistosome DNA: Species and strain differences from melting and drug resistance DNA analysis and cloning, sequencing and expression analysis of a putative praziquantel binding Ca(++)-ATPase gene

This thesis addresses the investigation of schistosome DNA with melting, drug-resistance and cloning, sequencing and characterization of a putative Ca++-ATPase gene. The study of experimental and computed high resolution melting curves for S. mansoni, S. japonicum and S. haematobium presented in Chapter 2 show the %(G+C) in S. mansoni, S. japonicum and S. haematobium genomes are 29.4, 26.1 and 31.5, respectively. Moreover, they have their own distribution of repetitive DNAs in their experimental melts. Despite the limited size of the datasets available in GENBANK, those tables presented in Chapter 2 show that coding sequences of S. mansoni, S. japonicum and S. haematobium melt over a higher temperature range than flanking and intron sequences. So MELTSIM could potentially be used as a useful tool to distinguish these DNA functional classes in the genome.;The genetic differences between Schistosoma mansoni strains from different geographic areas, which were reportedly resistant or sensitive to antischistosomal drugs were studied with randomly amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) PCR and representational difference analysis (RDA) assays in Chapter 3. Out of the 20 RAPD primers we chose, 19 of them showed the capacity to produce a medium to high level of amplification and 6 revealed difference PCR bands between drug-resistant and drug-sensitive strains. One particular primer, 5'-CAGCGACAAG-3 ', showed 2 major difference bands between Praziquantel (PZQ)-resistant and PZQ-sensitive strains from one endemic area of Egypt. Those RAPD and RDA results demonstrate that defined sequence primers could be applied as a useful tool for differentiating drug-resistant and sensitive schistosome parasites in the field; genomic differences between drug-resistant and drug-sensitive strains could also be isolated by representational difference analysis.;Chapter 4 has been cloned, isolated, sequenced and characterized a novel Ca++-ATPase homologue, SM3, from S. mansoni. This clone has 3900 bp in length, including a 3030 bp open reading frame (ORF) and a 870 bp 3'-untranslated sequence. The %(G+C) in ORF is 37%, while the 3'-untranslated sequence is 27% in agreement with our MELTSIM results. An initiator methionine has not yet been unambiguously identified. Therefore, all of the sequence analysis is currently based on the near-complete sequence. From the analysis of the sequence and amino acid alignment compared to those Ca++-ATPases from other organisms in the public database, SM3 has approximately 51% identity and 64% similarity to the third class of Ca++-ATPase from rat (RS10-31) and 40% identity and 53% similarity to yeast (PMR1). SM3 only has 24-27% identity and 41--43% similarity to the PMCAs and SERCAs from rat, human and C. elegans. SM3 is predicted to have a Mr 110,794.5, pI of 6.48 and 10 hydrophobic, potentially transmembrane domains. The deduced amino acid sequence shows the presence in SM3 of all the conserved domains commonly found in P-type ATPases.