Schistosoma mansoni is the main causative agent of schistosomiasis, an endemic disease in many countries, being on the second rank behind malaria in terms of socio-economic and public health importance. The study of its genome is very important to understand its biology, the mechanisms of drug resistance and their antigenic variation. According to some authors half of the whole genome is composed of repetitive sequences and more than 20% of these might be comprised of mobile genes retrotransposons. By bioinformatic analysis we were able to find sequence of retrotransposons, Boudicca and Perere previously described, respectively, by Copeland and Verjovski-Almeida in two of the BAC clones from Le Paslier library and available in our laboratory. In order to validate and localize these genes on the S. mansoni chromosomes we used FISH (Fluorescence in situ hybridization) and PRINS (Primed in situ labeling). The FISH technique consists of biotinylating (14-dCTP) BAC clones in a randomly primers amplification labeling system, using Klenow polymerase. Slides containing metaphase cells spreads of S. mansoni sporocysts were fixed with ethanol:acetic acid and used to hybridize with the labeled BAC DNA. The hybridization signals were detected with an Avidin-FITC conjugate. After counter stained of the chromosomes with Propidium Iodide (PI) in the anti fade solution, the slides were covered and observed in a confocal microscopy. The PRINS technique involves annealing of specific oligonucleotides primers of retrotransposons, followed by in situ elongation using Taq DNA polymerase in slides containing metaphase chromosomes. The chromosomes were also counter stained with PI and observed in a confocal microscopy. A signal of hybridization for the Retrotransposon Perere, non LTR, was observed in the heterochromatin region of chromosome 2 and the Retrotransposon Boudicca, LTR, was observed preliminarily on chromosomes 2 and Z. The most interesting finding is that two different families of retrotransposons are localized on the same chromosomes.
Supported by WHO, CPqRR, PIBIC-CNPq, UFOP.