Cluster d'inserzione di retroelementi roo in Drosophila melanogaster: validazione sperimentale e ruolo nella trascrizione alternativa.

Transposable elements (TEs) are DNA fragments that have the ability to mobilize and integrate in various positions within a host genome, exerting a wide range of effects. Previous works showed that a family of TIR DNA transposons, P-elements, preferentially insert into the promoter region of heat shock protein (hsp) genes than into other positions, determining a so called insertion cluster. A more recent work demonstrated that a family of LTR Retrotransposons named roo elements produce, within the proximal promoter of a cold-stress gene, CG18446, an insertion cluster of nine different solo-LTR elements with different molecular and functional consequences. Our interest focused on finding out other contexts in which de novo roo-elements produce insertional clusters. By performing PCR screening and sequence analysis in 55 strains from a North American population of D. melanogaster, we analyzed 19 promoter regions predicted by Transposon Insertion and Deletion AnaLyzer (TIDAL) database to be putative clusters regions harboring three roo insertions each. We found that 16 promoter regions harbored just one single insertion instead of the predicted three, while three promoters, relative to CG11459, CG15879 and CR44657 genes, have shown to harbor two independent roo insertions each, confirming the presence of a putative insertion cluster. Further screenings of these putative cluster regions in different strains of D. melanogaster will be necessary to assess the extent of these insertion clusters. Moreover, phenotypic and molecular assays will be useful to understand the functional consequences of these roo insertions. The validation of the TIDAL dataset we used for screening allowed us also to measure the reliability of the predictions of this bioinformatics tool. We could confirm TIDAL as an accurate tool only for the de novo prediction of TE presence and insertion site, with an error rate of 10% and 0% respectively. Another issue considered in this work is that in natural habitats of D. melanogaster, flies heterozygous for two different roo insertions in the same genomic region have never been reported. However, laboratory experiments showed that flies homozygous for two different roo insertions are viable. Thus, we hypothesized that crosses of flies containing a different roo insertion each have lower fecundity compared with crosses of flies containing the same insertion. We observed that crosses between two strains homozygous for two different roo solo-LTR insertions (FBti0019885 and roo-90) in CG18446 promoter region didn't show any reduced fecundity compared to crosses between two strains homozygous for the same TE insertion. This result limits the possible explanations to the allelic frequencies at which FBti0019885 and roo-90 have been found, in the North Carolina Population, which are low enough to make the discovery of heterozygous individuals in nature an extremely rare event. The nine solo-LTR insertion cluster of CG18446 number in particular a roo insertion, FBti0019985, which, in control conditions, was proven to upregulate gene expression and provide an alternative transcription start site with the production of a longer alternative transcript. Our interest was to test whether these effects are exerted also under cold-stress conditions. Through PCR screening of CG18446 cDNAs, we confirmed the production of a longer alternative transcript in flies carrying FBti0019985 element under stress conditions.