Sequencing and expression analysis of a Schistosoma mansoni gene homologue to a Drosophila gene involved in germ plasm assembly.

Abstract

Genes coding for proteins involved in generegulation and/or development are of great inter-est in the study of the biology of Schistosomamansoni. This trematode is the etiologic agent ofschistosomiasis and presents a complex life cyclewith drastic morphologic changes between stages.Recently, some strains have become resistant tothe drugs currently in use to eradicate the disease(D Cioli et al. 1995 Pharmac Ther 68: 35-85). Thestrategy of gene discovery program in S. mansoniby using the EST (expressed sequence tag) ap-proach (GR Franco et al. 1995 Gene 152 : 141-147)has been very efficient in the discovery of new S.mansoni genes, which were unlikely to be identi-fied using classical procedures based on pheno-type. A class of genes that interested us particu-larly were those that in other organisms wereknown to be involved in the regulation of embryo-genesis. Among these, we selected one that pre-sented a high homology to a Drosophila genenamed mago nashi. In diptera this gene is involvedin the process of germ plasm assembly and itsmutation results in sterility of F1 progeny and alsoin the formation of the perpendicular axes (REBoswell et al. 1991 Development 113: 373-384).We reasoned that this gene might conceivably playa role in the morphogenetic changes seen in thelife cycle of the parasite . We thus decided to char-acterize S. mansoni mago nashi further by obtain-ing its full length cDNA and genomic sequences,as well as studying its expression pattern at thedifferent life stages of the worm.The whole cDNA was sequenced in both di-rections yielding 485 nucleotides (nt) that codedfor a protein of 146 amino acids with 84% of ho-mology to the Drosophila homologue (Fig. 1). Pro-cedures for plasmidial DNA preparation, sequenc-ing and analysis of sequences have been previouslydescribed (Franco loc. cit.). From the cDNA se-quence, primers were designed and the genomicgene amplified from S. mansoni total DNA. Thegenomic amplified product was bigger than thecorrespondent cDNA amplified product when twodifferent pairs of primers were used (Fig. 2). ThePCR amplification products were cloned in a plas-mid vector (pUC18) by using the Pharmacia Sureclone kit and sequenced in both directions usingfluorescent primers in an automated DNA se-quencer. Consistent with the results of Fig. 2, threeintrons were identified in the genomic sequence,two of 34 nt and one with 33 nt producing a totalof a 101 nt of intron sequences (Fig. 3).Our next step was to investigate the gene ex-pression pattern at the different life cycle stages.Through the RT-PCR technique using cDNA ob-tained from different stages and specific primers,it was shown that mago nashi is expressed in allstages studied i.e. egg, schistosomula and adultworm (data not shown). This was not unexpectedalthough this gene was first identified in droso-phila embryos, it was also identified in adult flies(PA Newmark & RE Boswell 1994 Development120: 1303-1313). The gene had also been shownto be expressed in a large variety of human adulttissues such as lung, kidney, liver, heart, pancreas,brain and placenta (X- Zhao et al. 1998 Genomics47: 319-322).Since this work was started, the mago nashigene has been identified in a variety of differentorganisms. These include mouse, human,Caenorhabditis elegans, Brugia malayi,Arabidopsis thaliana and Oryza sativa . Alignmentof the conceptual translations of these mago genes