Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae)

Short tandem DNA repeats and telomerase compose the telomere structure in the vast majority of eukaryotic organisms. However, such a conserved organisation has not been found in dipterans. While telomeric DNA in Drosophila is composed of specific retrotransposons, complex terminal tandem repeats are present in chromosomes of Anopheles and chironomid species. In the sciarid Rhynchosciara americana, short repeats (16 and 22 bp long) tandemly arrayed seem to reach chromosome ends. Moreover, in situ hybridisation data using homopolymeric RNA probes suggested in this species the existence of a third putative chromosome end repeat enriched with (dA).(dT) homopolymers. In this work, chromosome micro-dissection and PCR primed by homopolymeric primers were employed to clone these repeats. Named T-14 and 93 % AT-rich, the repetitive unit is 14 bp long and appears organised in tandem arrays. It is localised in five non-centromeric ends and in four interstitial bands of R. americana chromosomes. To date, T-14 is the shortest repeat that has been characterised in chromosome ends of dipterans. As observed for short tandem repeats identified previously in chromosome ends of R. americana, the T-14 probe hybridised to bridges connecting non-homologous polytene chromosome ends, indicative of close association of T-14 repeats with the very end of the chromosomes. The results of this work suggest that R. americana represents an additional example of organism provided with more than one DNA sequence that is able to reach chromosome termini.

Rananos expression pattern during oogenesis and early embryonic development in Rhynchosciara americana

The Dipteran a native Brazilian insect that has become a valuable model system for developmental biology research because it provides an interesting opportunity to study a different type of insect oogenesis. Sequences from a cDNA library that was constructed with poly A + RNA from the ovaries of larvae at different ages were analyzed. Molecular characterization confirmed interesting findings, such as the presence of . The gene encodes a conserved RNA-binding protein that is required during early development for the maintenance and division of the primordial germ cells of Diptera. plays an important role in specifying the posterior regions of insect embryos and is important for abdomen formation. In the present work, we showed the spatial and temporal expression profiles of this important gene, which is involved in oogenesis and early development. Data mining techniques were used to obtain the complete sequence of . Bioinformatic tools were used to determine the following: (1) the secondary structure of the 3'-untranslated region of the mRNA, (2) the encoded protein of the isolated gene, (3) the conserved zinc-finger domains of the Nanos protein, and (4) phylogenetic analyses. Furthermore, RNA in situ hybridization and immunolocalization were used to determine mRNA and protein expression in the tissues that were studied and to define as a germ cell molecular marker.