Project description:Small self-cleaving RNAs, such as the paradigmatic Hammerhead ribozyme (HHR), have been recently found widespread in DNA genomes across all kingdoms of life. In this work, we found that new HHR variants are preserved in the ancient family of Penelope-like elements (PLEs), a group of eukaryotic retrotransposons regarded as exceptional for encoding telomerase-like retrotranscriptases and spliceosomal introns. Our bioinformatic analysis revealed not only the presence of minimalist HHRs in the two flanking repeats of PLEs but also their massive and widespread occurrence in metazoan genomes. The architecture of these ribozymes indicates that they may work as dimers, although their low self-cleavage activity in vitro suggests the requirement of other factors in vivo. In plants, however, PLEs show canonical HHRs, whereas fungi and protist PLEs encode ribozyme variants with a stable active conformation as monomers. Overall, our data confirm the connection of self-cleaving RNAs with eukaryotic retroelements and unveil these motifs as a significant fraction of the encoded information in eukaryotic genomes.
Project description:Penelope-like elements (PLEs) represent a new class of retroelements identified in more than 80 species belonging to at least 10 animal phyla. Penelope isolated from Drosophila virilis is the only known transpositionally active representative of this class. Although the size and structure of the Penelope major transcript has been previously described in both D. virilis and D. melanogaster transgenic strains, the architecture of the Penelope regulatory region remains unknown. In order to determine the localization of presumptive Penelope promoter and enhancer-like elements, segments of the putative Penelope regulatory region were linked to a CAT reporter gene and introduced into D. melanogaster by P-element-mediated transformation. The results obtained using ELISA to measure CAT expression levels and RNA studies, including RT-PCR, suggest that the active Penelope transposon contains an internal promoter similar to the TATA-less promoters of LINEs. The results also suggest that some of the Penelope regulatory sequences control the preferential expression in the ovaries of the adult flies by enhancing expression in the ovary and reducing expression in the carcass. The possible significance of the intron within Penelope for the function and evolution of PLEs, and the effect of Penelope insertions on adjacent genes, are discussed.
Project description:BackgroundThe availability of photon and electron spectra in digital form from current accelerators and Monte Carlo (MC) systems is scarce, and one of the packages widely used refers to linacs with a reduced clinical use nowadays. Such spectra are mainly intended for the MC calculation of detector-related quantities in conventional broad beams, where the use of detailed phase-space files (PSFs) is less critical than for MC-based treatment planning applications, but unlike PSFs, spectra can easily be transferred to other computer systems and users.MethodsA set of spectra for a range of Varian linacs has been calculated using the PENELOPE/PRIMO MC system. They have been extracted from PSFs tallied for field sizes of 10 cm × 10 cm and 15 cm × 15 cm for photon and electron beams, respectively. The influence of the spectral bin width and of the beam central axis region used to extract the spectra have been analyzed.ResultsSpectra have been compared to those by other authors showing good agreement with those obtained using the, now superseded, EGS4/BEAM MC code, but significant differences with the most widely used photon data set. Other spectra, particularly for electron beams, have not been published previously for the machines simulated in this work. The influence of the bin width on the spectrum mean energy for 6 and 10 MV beams has been found to be negligible. The size of the region used to extract the spectra yields differences of up to 40% for the mean energies in 10 MV beams, but the maximum difference for TPR 20,10 values derived from depth-dose distributions does not exceed 2% relative to those obtained using the PSFs. This corresponds to kQ differences below 0.2% for a typical Farmer-type chamber, considered to be negligible for reference dosimetry. Different configurations for using electron spectra have been compared for 6 MeV beams, concluding that the geometry used for tallying the PSFs used to extract the spectra must be accounted for in subsequent calculations using the spectra as a source.ConclusionsAn up-to-date set of consistent spectra for Varian accelerators suitable for the calculation of detector-related quantities in conventional broad beams has been developed and made available in digital form.
Project description:The evolutionary origin of telomerases, enzymes that maintain the ends of linear chromosomes in most eukaryotes, is a subject of debate. Penelope-like elements (PLEs) are a recently described class of eukaryotic retroelements characterized by a GIY-YIG endonuclease domain and by a reverse transcriptase domain with similarity to telomerases and group II introns. Here we report that a subset of PLEs found in bdelloid rotifers, basidiomycete fungi, stramenopiles, and plants, representing four different eukaryotic kingdoms, lack the endonuclease domain and are located at telomeres. The 5' truncated ends of these elements are telomere-oriented and typically capped by species-specific telomeric repeats. Most of them also carry several shorter stretches of telomeric repeats at or near their 3' ends, which could facilitate utilization of the telomeric G-rich 3' overhangs to prime reverse transcription. Many of these telomere-associated PLEs occupy a basal phylogenetic position close to the point of divergence from the telomerase-PLE common ancestor and may descend from the missing link between early eukaryotic retroelements and present-day telomerases.
Project description:Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations (Mn2+ and Mg2+). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon.