David S. Horner

The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla

The analysis of the first plant genomes provided unexpected evidence for genome duplication events in species that had previously been considered as true diploids on the basis of their genetics. These polyploidization events may have had important consequences in plant evolution, in particular for species radiation and adaptation and for the modulation of functional capacities. Here we report a high-quality draft of the genome sequence of grapevine (Vitis vinifera) obtained from a highly homozygous genotype. The draft sequence of the grapevine genome is the fourth one produced so far for flowering plants, the second for a woody species and the first for a fruit crop (cultivated for both fruit and beverage). Grapevine was selected because of its important place in the cultural heritage of humanity beginning during the Neolithic period. Several large expansions of gene families with roles in aromatic features are observed. The grapevine genome has not undergone recent genome duplication, thus enabling the discovery of ancestral traits and features of the genetic organization of flowering plants. This analysis reveals the contribution of three ancestral genomes to the grapevine haploid content. This ancestral arrangement is common to many dicotyledonous plants but is absent from the genome of rice, which is a monocotyledon. Furthermore, we explain the chronology of previously described whole-genome duplication events in the evolution of flowering plants.

Molecular and Phylogenetic Analyses of the Complete MADS-Box Transcription Factor Family in Arabidopsis: New Openings to the MADS World

MADS-box transcription factors are key regulators of several plant development processes. Analysis of the complete Arabidopsis genome sequence revealed 107 genes encoding MADS-box proteins, of which 84% are of unknown function. Here, we provide a complete overview of this family, describing the gene structure, gene expression, genome localization, protein motif organization, and phylogenetic relationship of each member. We have divided this transcription factor family into five groups (named MIKC, Mα, Mβ, Mγ, and Mδ) based on the phylogenetic relationships of the conserved MADS-box domain. This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins. MADS-box genes also constitute an excellent system with which to study the evolution of complex gene families in higher plants.

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

UTRdb and UTRsite (RELEASE 2010): a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs.

The 5′ and 3′ untranslated regions of eukaryotic mRNAs (UTRs) play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and message stability. UTRdb is a curated database of 5′ and 3′ untranslated sequences of eukaryotic mRNAs, derived from several sources of primary data. Experimentally validated functional motifs are annotated and also collated as the UTRsite database where more specific information on the functional motifs and cross-links to interacting regulatory protein are provided. In the current update, the UTR entries have been organized in a gene-centric structure to better visualize and retrieve 5′ and 3′UTR variants generated by alternative initiation and termination of transcription and alternative splicing. Experimentally validated miRNA targets and conserved sequence elements are also annotated. The integration of UTRdb with genomic data has allowed the implementation of an efficient annotation system and a powerful retrieval resource for the selection and extraction of specific UTR subsets. All internet resources implemented for retrieval and functional analysis of 5′ and 3′ untranslated regions of eukaryotic mRNAs are accessible at http://utrdb.ba.itb.cnr.it/.

Molecular Data Suggest an Early Acquisition of the Mitochondrion Endosymbiont

The three deepest branching eucaryotic lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microspora, Metamonada and Parabasala. They are followed by either the Euglenozoa (e.g. Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eucaryotes. To investigate the hypothesis of an even earlier timing of the mitochondrion endosymbiosis we have amplified a partial cpn-60 coding region from the parabasalid Trichomonas vaginalis and the first such sequence from a percolozoan, Naegleria fowleri. Analysis of predicted protein sequences reveals a high degree of sequence similarity (≥ 40%) with a selection of published bacterial and mitochondrial cpn-60s for both taxa. Both sequences were recovered within a strongly supported monophyletic group, otherwise defined by mitochondrial sequences, which systematically clustered with alpha-proteobacteria. These results provide compelling evidence that the ancestor of T. vaginalis once contained the endosymbiont which gave rise to mitochondria, and suggest that this symbiosis probably occurred before the Trichomonas lineage diverged from the main eukaryote trunk. It also makes feasible the published hypothesis that the Trichomonas hydrogenosome might represent a biochemically modified mitochondrion. Analysis of the N. fowleri cpn-60 did not support the hypothesis that the mitochondrion-containing Percolozoa represent an earlier branch in the cpn-60 tree than Trichomonas or Trypanosoma.

Iron hydrogenases--ancient enzymes in modern eukaryotes.

The distribution of [Fe]-hydrogenases was once thought to be limited to a small number of bacteria and a few peculiar hydrogen-producing anaerobic eukaryotes. However, it is now clear that [Fe]-hydrogenases are more widely distributed among eukaryotes than reports of hydrogen production have suggested. Indeed, genes bearing the hallmark signatures of [Fe]-hydrogenases are found both in our own genome and in the genomes of other higher eukaryotes. At present, the functions of most of these new proteins remain unknown; it is not even known whether they can all make hydrogen. Radical new hypotheses have suggested that hydrogenases played a key role in the formation of the eukaryotic cell. These unique enzymes have thus moved from the margins of eukaryotic biology to become the focus of intense speculation and interest. This article summarizes current knowledge of their distribution, evolution and biochemistry.

Hydrogenosomes, Mitochondria and Early Eukaryotic Evolution

Available data suggest that unusual organelles called hydrogenosomes, that make ATP and hydrogen, and which are found in diverse anaerobic eukaryotes, were once mitochondria. The evolutionary origins of the enzymes used to make hydrogen, pyruvate:ferredoxin oxidoreductase (PFO) and hydrogenase, are unresolved, but it seems likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobes, these proteins are found in diverse eukaryotic cells, including our own, where they are targeted to different cell compartments. Organelles related to mitochondria and hydrogenosomes have now been found in species of anaerobic and parasitic protozoa that were previously thought to have separated from other eukaryotes before the mitochondrial endosymbiosis. Thus it is possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, bearing testimony to the important role that the mitochondrial endosymbiosis has played in eukaryotic evolution. It remains to be seen if members of this family of organelles share a common function essential to the eukaryotic cell, that provides the underlying selection pressure for organelle retention under different living conditions.

Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

In chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant BCR-ABL mutants. We used an ultra-deep sequencing (UDS) approach to resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs and to investigate their clonal structure and evolution over time in relation to therapeutic intervention. To this purpose, we performed a longitudinal analysis of 106 samples from 33 patients who had received sequential treatment with multiple TKIs and had experienced sequential relapses accompanied by selection of 1 or more TKI-resistant mutations. We found that conventional Sanger sequencing had misclassified or underestimated BCR-ABL mutation status in 55% of the samples, where mutations with 1% to 15% abundance were detected. A complex clonal texture was uncovered by clonal analysis of samples harboring multiple mutations and up to 13 different mutated populations were identified. The landscape of these mutated populations was found to be highly dynamic. The high degree of complexity uncovered by UDS indicates that conventional Sanger sequencing might be an inadequate tool to assess BCR-ABL kinase domain mutation status, which currently represents an important component of the therapeutic decision algorithms. Further evaluation of the clinical usefulness of UDS-based approaches is warranted.

Large-scale detection and analysis of RNA editing in grape mtDNA by RNA deep-sequencing

RNA editing is a widespread post-transcriptional molecular phenomenon that can increase proteomic diversity, by modifying the sequence of completely or partially non-functional primary transcripts, through a variety of mechanistically and evolutionarily unrelated pathways. Editing by base substitution has been investigated in both animals and plants. However, conventional strategies based on directed Sanger sequencing are time-consuming and effectively preclude genome wide identification of RNA editing and assessment of partial and tissue-specific editing sites. In contrast, the high-throughput RNA-Seq approach allows the generation of a comprehensive landscape of RNA editing at the genome level. Short reads from Solexa/Illumina GA and ABI SOLiD platforms have been used to investigate the editing pattern in mitochondria of Vitis vinifera providing significant support for 401 C-to-U conversions in coding regions and an additional 44 modifications in non-coding RNAs. Moreover, 76% of all C-to-U conversions in coding genes represent partial RNA editing events and 28% of them were shown to be significantly tissue specific. Solexa/Illumina and SOLiD platforms showed different characteristics with respect to the specific issue of large-scale editing analysis, and the combined approach presented here reduces the false positive rate of discovery of editing events.

Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome‐containing fungus Neocallimastix that have three‐fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC‐ deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.