The physical and genetic map of mtDNA from Neurospora crassa strain 74-OR23-1A.

The physical and genetic map of mtDNA from Neurospora crassa strain 74-ORS23-1A

Map of Plasmid pRAL1.

Map of Plasmid pRAL1

The physical and genetic map of mtDNA from Neurospora crassa strain 74-OR23-1A.

The physical and genetic map of mtDNA from Neurospora crassa strain 74-ORS23-1A

Requirement of a Membrane Potential for the Posttranslational Transfer of Proteins into Mitochondsria

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocatio

Database for bacterial group II introns

The Database for Bacterial Group II Introns (http://webapps2.ucalgary.ca/~groupii/index.html#) provides a catalogue of full-length, non-redundant group II introns present in bacterial DNA sequences in GenBank. The website is divided into three sections. The first section provides general information on group II intron properties, structures and classification. The second and main section lists information for individual introns, including insertion sites, DNA sequences, intron-encoded protein sequences and RNA secondary structure models. The final section provides tools for identification and analysis of intron sequences. These include a step-by-step guide to identify introns in genomic sequences, a local BLAST tool to identify closest intron relatives to a query sequence, and a boundary-finding tool that predicts 5′ and 3′ intron–exon junctions in an input DNA sequence. Finally, selected intron data can be downloaded in FASTA format. It is hoped that this database will be a useful resource not only to group II intron and RNA researchers, but also to microbiologists who encounter these unexpected introns in genomic sequences

Group II Introns Break New Boundaries: Presence in a Bilaterian's Genome

Group II introns are ribozymes, removing themselves from their primary transcripts, as well as mobile genetic elements, transposing via an RNA intermediate, and are thought to be the ancestors of spliceosomal introns. Although common in bacteria and most eukaryotic organelles, they have never been reported in any bilaterian animal genome, organellar or nuclear. Here we report the first group II intron found in the mitochondrial genome of a bilaterian worm. This location is especially surprising, since animal mitochondrial genomes are generally distinct from those of plants, fungi, and protists by being small and compact, and so are viewed as being highly streamlined, perhaps as a result of strong selective pressures for fast replication while establishing germ plasm during early development. This intron is found in the mtDNA of an annelid worm, (an undescribed species of Nephtys), where the complete sequence revealed a 1819 bp group II intron inside the cox1 gene. We infer that this intron is the result of a recent horizontal gene transfer event from a viral or bacterial vector into the mitochondrial genome of Nephtys sp. Our findings hold implications for understanding mechanisms, constraints, and selective pressures that account for patterns of animal mitochondrial genome evolutio

Localization of a bacterial group II intron-encoded protein in human cells

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.This work was supported by research grants CSD 2009–0006 from the Consolider-Ingenio, BIO2011-24401 and BIO2014-51953-P from the Spanish Ministerio de Economía y Competitividad all including ERDF (European Regional Development Funds). We thank Dr. Antonio Barrientos Durán for technical advice. MRC was supported by an FPI Ph.D grant. J.L.G.P´s laboratory is supported by CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+I 2008–2011 and 2013–2016 (FIS-FEDER-PI11/01489 and FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764) and by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420).Peer Reviewe

Mechanisms Used for Genomic Proliferation by Thermophilic Group II Introns

Studies of mobile group II introns from a thermophilic cyanobacterium reveal how these introns proliferate within genomes and might explain the origin of introns and retroelements in higher organisms

The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4–Dependent and –Independent Mechanisms

Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3′ end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications