Carla Quagliariello

The complete nucleotide sequence of theRattus norvegicus mitochondrial genome: Cryptic signals revealed by comparative analysis between vertebrates

SummaryThis paper reports the nucleotide sequence of rat mitochondrial DNA, only the fourth mammalian mitochondrial genome to be completely sequenced. Extensive comparative studies performed with similar genomes from other organisms revealed a number of interesting features.1)Messenger RNA genes: the codon strategy is mainly dictated by the base compositional constraints of the corresponding codegenic DNA strand. The usage of the initiation and termination codons follows well-established rules. In general the canonical initiator, ATG, and terminators, TAA and TAG (in rat, only TAA), are always present when there is gene overlapping or when the mRNAs possess untranslated nucleotides at the 5′ or 3′ ends.2)Transfer RNA genes: a number of features suggest the peculiar evolutionary behavior of this class of genes and confirm their role in the duplication and rearrangement processes that took place in the evolution of the animal mitochondrial genome.3)Ribosomal RNA genes: accurate sequence analysis revealed a number of significant examples of complementarity between ribosomal and messenger RNAs. This suggests that they might uplay an important role in the regulation of mitochondrial translation and transcription mechanisms. The properties revealed by our work shed new light on the organization and evolution of the vertebrate mitochondrial genome and more importantly open up the way to clearly aimed experimental studies of the regulatory mechanisms in mitochondria

REDIdb: the RNA editing database.

The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at .

Conservation of the organization of the mitochondrial nad3 and rps12 genes in evolutionarily distant angiosperms

The organization of the genesnad3 andrps12 has been investigated in the mitochondrial genome of two dicotyledonous plants —Helianthus andMagnolia — and one monocotyledonous plant (Allium). These plants all contain a completerps12 gene downstream of thenad3 gene. This arrangement is thus highly conserved within angiosperms. The two genes are co-transcribed and the transcript is modified at several positions by RNA editing of the C to U-type, thus confirming that both genes encode functional proteins. Some 26, 35 and 27 editing events have been identified in the PCR-derivednad3-rps12 cDNA population from sunflower,Magnolia and onion, respectively. Editing of thenad3-rps12 transcript is thus more extensive inMagnolia than in the other angiosperms so far investigated and radically changes the genomically encoded polypeptide sequence. A novel species-specific codon modification was observed inMagnolia. Several homologous sites show differences in editing pattern among plant species. A C-to-U alteration is also found in the non-coding region separating thenad3 andrps12 genes in sunflower. The PCR-derived cDNA populations from thenad3-rps12 loci analysed were found to be differently edited. In addition the plant species show marked variations in the completeness of RNA editing, with only theMagnolia nad3 mRNA being edited fully.

REDIdb: An upgraded bioinformatics resource for organellar RNA editing sites

RNA editing is a post-transcriptional molecular process whereby the information in a genetic message is modified from that in the corresponding DNA template by means of nucleotide substitutions, insertions and/or deletions. It occurs mostly in organelles by clade-specific diverse and unrelated biochemical mechanisms. RNA editing events have been annotated in primary databases as GenBank and at more sophisticated level in the specialized databases REDIdb, dbRES and EdRNA. At present, REDIdb is the only freely available database that focuses on the organellar RNA editing process and annotates each editing modification in its biological context. Here we present an updated and upgraded release of REDIdb with a web-interface refurbished with graphical and computational facilities that improve RNA editing investigations. Details of the REDIdb features and novelties are illustrated and compared to other RNA editing databases. REDIdb is freely queried at http://biologia.unical.it/py_script/REDIdb/.

A Novel Additional Group II Intron Distinguishes the Mitochondrial rps3 Gene in Gymnosperms

Comparative analysis of the ribosomal protein S3 gene (rps3) in the mitochondrial genome of Cycas with newly sequenced counterparts from Magnolia and Helianthus and available sequences from higher plants revealed that the positional clustering with the genes for ribosomal protein S19 (rps19) and L16 (rpl16) is preserved in gymnosperms. However, in contrast to the other land plant species, the rps3 gene in Cycas mitochondria is unique in possessing a second intron: rps3i2. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of the transcripts generated from the rps19–rps3–rpl16 cluster in Cycas mitochondria demonstrated that the genes are cotranscribed and extensively modified by RNA editing and that both introns are efficiently spliced. Despite remarkable size heterogeneity, the Cycas rps3i1 can be shown to be homologous to the group IIA introns present within the rps3 gene of algae and land plants, including Magnolia and Helianthus. Conversely, sequences similar to the rps3i2 have not been reported previously. On the basis of conserved primary and secondary structure the second intervening sequence interrupting the Cycasrps3 gene has been classified as a group II intron. The close relationship of the rps3i2 to a group of different plant mitochondrial introns is intriguing and suggestive of a mitochondrial derivation for this novel intervening sequence. Interestingly, the rps3i2 appears to be conserved at the same gene location in other gymnosperms. Furthermore, the pattern of the rps3i2 distribution among algae and land plants provides evidence for the evolutionary acquisition of this novel intron in gymnosperms via intragenomic transposition or retrotransposition.

Lineage-specific group II intron gains and losses of the mitochondrial rps3 gene in gymnosperms

According to PCR assays and sequencing, we now report the shared presence of two rps3 introns, namely the rps3i74 and the rps3i249, in the mitochondria of all the classes representing the surviving lineages of gymnosperms, and unveil several lineages experiencing intron loss. Interestingly, the rps3 intron gains and losses within the four groups of gymnosperms let us sort out the Pinaceae and the non-Pinaceae into intron (+)- and intron (-)-lineages, respectively. Worthy of mention is also the finding that only Gnetum within the Gnetales harbours both the rps3 introns. This intron distribution pattern is consistent with the hypothesis that the two rps3 introns were likely present in the common ancestor of the seed plants and, then, independently lost in the non-Pinaceae during gymnosperm evolution. The derived secondary structural model of the novel group IIA intron improves our understanding of the significance and origin of the extraordinary length polymorphisms observed among rps3i249 orthologs. Despite the remarkable structural plasticity to adopt and reject introns, the rps3 mRNAs undergo accurate processing by splicing and extensive editing in gymnosperm mitochondria. This study provides additional insights into the evolutionarily high dynamics of mitochondrial introns which may come and go in closely related plant species. The turnover of the mitochondrial rps3 group II introns seen among lineages of seed plants further suggests that these introns might be an additional signature to discriminate between particularly cryptical taxonomic groups for which there is a need of a further evaluation of their evolutionary affiliation.

Evolution of the nad3-rps12 Gene Cluster in Angiosperm Mitochondria: Comparison of Edited and Unedited Sequences

We have analyzed thenad3-rps12 locus for eight angiosperms in order to compare the utility of mitochondrial DNA and edited mRNA sequences in phylogenetic reconstruction. The two coding regions, containing from 25 to 35 editing sites in the various plants, have been concatenated in order to increase the significance of the analysis. Differing from the corresponding chloroplast sequences, unedited mitochondrial DNA sequences seem to evolve under a quasi-neutral substitution process which undifferentiates the nucleotide substitution rates for the three codon positions. By using complete gene sequences (all codon positions) we found that genomic sequences provide a classical angiosperm phylogenetic tree with a clear-cut grouping of monocotyledons and dicotyledons with Magnoliidae at the basal branch of the tree. Conversely, owing to their low nucleotide substitution rates, edited mRNA sequences were found not to be suitable for studying phylogenetic relationships among angiosperms.

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNAVal (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNAVal gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNAVal is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNAVal species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.

New sources of cytoplasmic diversity in the Italian population of Olea europaea L. as revealed by RFLP analysis of mitochondrial DNA: characterization of the cox3 locus and possible relationship with cytoplasmic male sterility

Abstract Restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) were investigated to detect intraspecific variation among the Italian cultivated and wild Olea europaea L. populations. RFLPs were visualized by Southern hybridization of EcoRI and HindIII restricted total cellular DNA using two mitochondrial genes (cox3 and atpA) as probes. The mtDNA RFLP data gathered in our analysis allow the identification of three distinct olive tree mitotypes; each characterized by specific hybridization patterns. The result on genetic variability is also confirmed by the unweighted pair group method with arithmetic averages dendrogram (UPGMA) that combines the Italian thirty-seven different sources of Olea europaea L. into three major clusters of cytoplasmic relatedness. Therefore, at least as many maternal lineages are involved in the evolution and origin of the Italian olive tree mitochondrial genomes, with the cultivar Cerasola showing the greatest diversity with the cox3 probe and two restriction enzymes. We, thus, report the identification of three novel diagnostic probe/enzyme combinations that can be used as markers of cytoplasmic diversity in the Euromediterranean O. europaea L. population. Further characterization of the polymorphic cox3 locus in the mtDNA of the cultivar Cerasola by sequencing and expression analysis at the level of transcription and RNA editing pattern suggests a correlation between a duplication event of the cox3 locus and the male sterile phenotype of this cultivar. The mtDNA RFLPs provide, thus, a sensitive and reliable method to assess intraspecific cytoplasmic diversity within the economically important olive tree cultivars in Italy. Our results also provide new insights into the maternal lineages involved in the evolution of the Euromediterranean olive trees.

Software note: EdiPy: A resource to simulate the evolution of plant mitochondrial genes under the RNA editing

EdiPy is an online resource appropriately designed to simulate the evolution of plant mitochondrial genes in a biologically realistic fashion. EdiPy takes into account the presence of sites subjected to RNA editing and provides multiple artificial alignments corresponding to both genomic and cDNA sequences. Each artificial data set can successively be submitted to main and widespread evolutionary and phylogenetic software packages such as PAUP, Phyml, PAML and Phylip. As an online bioinformatic resource, EdiPy is available at the following web page: http://biologia.unical.it/py_script/index.html.