1,107 research outputs found
Transcriptional responses and AMP transcript diversity in M. galloprovincialis
No full textThe present dissertation contains the work done during my PhD in Biotechnology (2009-2011) and, through three published articles, shows how the research group which I worked in has contributed to the development of functional genomics in the mollusc bivalve Mytilus galloprovincialis.
Using different approaches, we aimed to better understand the peculiarities of a transcriptome only partially investigated, focusing our attention on molecules possibly explaining the mussel innate immunity, defense system that allows the survival of this organism in potentially lethal habitats.
Starting from a publicly available collection of expressed sequence tags (ESTs) generated from various tissues of native mussels or from mussels subjected to various stress conditions, we have analyzed and described relevant transcript groups and singletons involved in the mussel immune responses. A selection of these sequences has been used to design a new DNA-microarray platform (Immunochip 1.0), a new tool for the experimental validation of the many gene transcripts possibly playing a role in the sensing, signaling and effector functions of the mussel innate immunity (Venier et al., 2011).
DNA microarray platforms are widely used to investigate the expression of thousands of transcripts and, in general, they provide reliable and dynamic measures of transcriptional trends. As far as the genus Mytilus, species-specific platforms and also platforms including probes of evolutionary close species are available (Domeneghetti et al., 2011).
Using an advanced sequencing approach, we have finally explored and mapped the transcriptional diversity of nine typical antimicrobial peptides expressed in mussels (defensins, mytilins, myticins and mytimycin): although different one from the other, a high frequency of single nucleotide changes was globally evident (Rosani et al., 2011). These results pave the way to study genetic and epigenetic mechanisms able to explain the observed transcript diversity, yet undetermined in mussel, and to characterize, from a functional point of view, sequence variants expressed in response to different antigenic stimuliLa presente tesi raccoglie il lavoro svolto durante il mio dottorato in biotecnologie (2009-2011) e, tramite tre articoli pubblicati, mostra come il gruppo di ricerca entro il quale mi sono inserito ha contribuito ad ampliare le conoscenze nel campo della genomica funzionale del mollusco bivalve Mytilus galloprovincialis. Utilizzando differenti approcci abbiamo cercato di comprendere aspetti di un trascrittoma solo parzialmente conosciuto, soffermandoci sulle molecole dell'immunità innata, sistema difensivo grazie al quale questo organismo invertebrato sopravvive in ambienti ricchi di patogeni.
Avendo a disposizione una collezione di sequenze espresse (EST) rappresentativa di vari tessuti di mitili nativi e di mitili sottoposti a varie condizioni di stress, abbiamo analizzato e descritto alcuni importanti singole sequenze e gruppi di trascritti coinvolti nelle risposte immuni del comune mitilo mediterraneo. Una selezione di questi è stata utilizzata per disegnare una piattaforma DNA-microarray (Immunochip 1.0) utile a validare sperimentalmente la grande varietà di trascritti genici potenzialmente implicati nel sensing, nel signalling e in funzioni effettrici dell’immunità innata di M. galloprovincialis (Venier et al., 2011).
Le piattaforme DNA microarray sono ormai largamente utilizzate per lo studio in parallello dell'espressione di migliaia di trascritti e, in generale, forniscono misure affidabili e dinamiche degli andamenti trascrizionali. Riguardo al genere Mytilus, sono state sviluppate piattaforme specie-specifiche e piattaforme ibride che includono sonde nucleotidiche di specie evolutivamente affini (Domeneghetti et al., 2011).
Utilizzando un approccio di sequenziamento avanzato, abbiamo infine esplorato e mappato la diversità trascrizionale di nove tipici peptidi antimicrobici espressi in mitilo (varie defensine, mitiline, miticine e mitimicina) scoprendo così, pur con differenze da caso a caso, una elevata frequenza di cambiamenti a singolo nucleotide (Rosani et al., 2011). Sulla base di questi risultati sarà interessante studiare meccanismi genetici ed epigenetici che possano spiegare tale variabilità di sequenza, ancora indeterminati in mitilo, e caratterizzare da un punto di vista funzionale le varianti espresse in risposta a diversi stimoli antigenic
Tracing RNA viruses associated with Nudibranchia gastropods
No full textBACKGROUND: Nudibranchia is an under-studied taxonomic group of gastropods, including more than 3,000 species with colourful and extravagant body shapes and peculiar predatory and defensive strategies. Although symbiosis with bacteria has been reported, no data are available for the nudibranch microbiome nor regarding viruses possibly associated with these geographically widespread species. METHODS: Based on 47 available RNA sequencing datasets including more than two billion reads of 35 nudibranch species, a meta-transcriptome assembly was constructed. Taxonomic searches with DIAMOND, RNA-dependent-RNA-polymerase identification with palmscan and viral hallmark genes identification by VirSorter2 in combination with CheckV were applied to identify genuine viral genomes, which were then annotated using CAT. RESULTS: A total of 20 viral genomes were identified as bona fide viruses, among 552 putative viral contigs resembling both RNA viruses of the Negarnaviricota, Pisuviricota, Kitrinoviricota phyla and actively transcribing DNA viruses of the Cossaviricota and Nucleocytoviricota phyla. The 20 commonly identified viruses showed similarity with RNA viruses identified in other RNA-seq experiments and can be putatively associated with bacteria, plant and arthropod hosts by co-occurence analysis. The RNA samples having the highest viral abundances showed a heterogenous and mostly sample-specific distribution of the identified viruses, suggesting that nudibranchs possess diversified and mostly unknown viral communities
Oyster RNA-seq Data Support the Development of Malacoherpesviridae Genomics
No full textThe family of double-stranded DNA (dsDNA) Malacoherpesviridae includes viruses able to infect marine mollusks and detrimental for worldwide aquaculture production. Due to fast-occurring mortality and a lack of permissive cell lines, the available data on the few known Malacoherpesviridae provide only partial support for the study of molecular virus features, life cycle, and evolutionary history. Following thorough data mining of bivalve and gastropod RNA-seq experiments, we used more than five million Malacoherpesviridae reads to improve the annotation of viral genomes and to characterize viral InDels, nucleotide stretches, and SNPs. Both genome and protein domain analyses confirmed the evolutionary diversification and gene uniqueness of known Malacoherpesviridae. However, the presence of Malacoherpesviridae-like sequences integrated within genomes of phylogenetically distant invertebrates indicates broad diffusion of these viruses and indicates the need for confirmatory investigations. The manifest co-occurrence of OsHV-1 genotype variants in single RNA-seq samples of Crassostrea gigas provide further support for the Malacoherpesviridae diversification. In addition to simple sequence motifs inter-punctuating viral ORFs, recombination-inducing sequences were found to be enriched in the OsHV-1 and AbHV1-AUS genomes. Finally, the highly correlated expression of most viral ORFs in multiple oyster samples is consistent with the burst of viral proteins during the lytic phase
Bioinformatics for Inosine: Tools and Approaches to Trace This Elusive RNA Modification
No full textInosine is a nucleotide resulting from the deamination of adenosine in RNA. This chemical modification process, known as RNA editing, is typically mediated by a family of double-stranded RNA binding proteins named Adenosine Deaminase Acting on dsRNA (ADAR). While the presence of ADAR orthologs has been traced throughout the evolution of metazoans, the existence and extension of RNA editing have been characterized in a more limited number of animals so far. Undoubtedly, ADAR-mediated RNA editing plays a vital role in physiology, organismal development and disease, making the understanding of the evolutionary conservation of this phenomenon pivotal to a deep characterization of relevant biological processes. However, the lack of direct high-throughput methods to reveal RNA modifications at single nucleotide resolution limited an extended investigation of RNA editing. Nowadays, these methods have been developed, and appropriate bioinformatic pipelines are required to fully exploit this data, which can complement existing approaches to detect ADAR editing. Here, we review the current literature on the “bioinformatics for inosine” subject and we discuss future research avenues in the field
A bioinformatics approach reveals seven nearly-complete RNA-virus genomes in bivalve RNA-seq data
No full textViral metagenomics (viromics) can provide a great contribution in expanding the knowledge of viruses and the relationship with their hosts. Viromic studies on marine organisms are still at a very early stage and only little efforts have been spent in the identification of viruses associated to marine invertebrates to date, leaving the complexity of marine viromes associated to bivalve hosts almost completely unexplored. However, the potential use of viromic approaches in the management of viral diseases affecting aquacultured species has been recently evidenced by the flourishing of studies on the Ostreid herpesvirus type-1, which has been associated with bivalve mortality events. Herein we discuss an effective pipeline to retrieve and reconstruct nearly complete and previously unreported viral genomes from existing host RNA-seq data. As a case study, we report the identification of seven RNA-virus genomes within the frame of a highly diversified viral community that characterizes both Crassostrea gigas and Mytilus galloprovincialis samples collected from the lagoon of Goro (Italy)
Comparative analysis of Presence-Absence gene Variations in five hard tick species: impact and functional considerations
No full textTick species are vectors of harmful human and animal diseases, and their expansion is raising concerns under the global environmental changes' scenario. Ticks host and transmit bacteria, protozoa and viruses, making the understanding of host-pathogen molecular pathways critical to development of effective disease control strategies. Despite the considerable sizes and repeat contents of tick genomes, individual tick genomics is perhaps the most effective approach to reveal genotypic traits of interest. Presence-Absence gene Variations (PAVs) can contribute to individual differences within species, with dispensable genes carried by subsets of individuals possibly underpinning functional significance at individual or population-levels. We exploited 350 resequencing datasets of Dermacentor silvarum, Haemaphysalis longicornis, Ixodes persulcatus, Rhipicephalus microplus and Rhipicephalus sanguineus hard tick specimens to reveal the extension of PAV and the conservation of dispensable genes among individuals and, comparatively, between species. Overall, we traced 550-3,346 dispensable genes per species and were able to reconstruct 5.3-7 Mb of genomic regions not included in the respective reference genomes, as part of the tick pangenomes. Both dispensable genes and de novo predicted genes indicated that PAVs preferentially impacted mobile genetic elements in these tick species
The miRNA biogenesis in marine bivalves
No full textSmall non-coding RNAs include powerful regulators of gene expression, transposon
mobility and virus activity. Among the various categories, mature microRNAs (miRNAs)
guide the translational repression and decay of several targeted mRNAs. The
biogenesis of miRNAs depends on few gene products, essentially conserved from basal
to higher metazoans, whose protein domains allow specific interactions with dsRNA.
Here, we report the identification of key genes responsible of the miRNA biogenesis in
32 bivalves, with particular attention to the aquaculture species Mytilus galloprovincialis
and Crassostrea gigas. In detail, we have identified and phylogenetically compared eight
evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2,
AGO and PIWI. In mussels, we recognized several other proteins participating in the
miRNA biogenesis or in the subsequent RNA silencing. According to digital expression
analysis, these genes display low and not inducible expression levels in adult mussels
and oysters whereas they are considerably expressed during development. As miRNAs
play an important role also in the antiviral responses, knowledge on their production
and regulative effects can shed light on essential molecular processes and provide new
hints for disease prevention in bivalves
Toll-like receptors and MyD88 adaptors in Mytilus: complete cds and gene expression levels
No full textTLR- and MyD88-related sequences have been previously investigated in Mytibase and then in new transcript reads obtained by Illumina technology from the mussel, Mytilus galloprovincialis. Based on full cds and domain organizations of virtual translations, we identified 23 Toll-like receptors (TLRs) and 3 MyD88 adaptors. MgTLRs can be arranged in 4 clusters according to extra-cellular LRR domain content. MgTLR-b, -i and -k were the only ones containing a multiple cysteine cluster (mccTLR), a domain composition also found in Drosophila Toll-1 and 18-wheeler. The 3 MyD88 we identified in M. galloprovincialis were also retrieved from Mytilus edulis, as well as MgTLR-b and -i. All MgTLRs were constitutively expressed in digestive gland whereas only 4 of them were also present in hemocytes. On the opposite, the 3 MgMyD88s were constitutively expressed in all the tissues. In vivo challenge of M. galloprovincialis with bacteria caused the up regulation of only MgTLR-i, but of all the 3 MgMyD88s. Highest response was induced by Gram-negative Vibrio anguillarum at 9h p.i. Injection of filamentous fungus, Fusarium oxysporum, resulted in up regulation of MgTLR-i and MgMyD88-c at 9h p.i. Such similar pattern of responses suggested MgMyD88-c represents the intra cytoplasm partner of MgTLR-i. Their interaction constituted the first cellular event revealing the existence of a Toll-signalling pathway in Lophotrochozoa
Target capture and massive sequencing of genes transcribed in Mytilus galloprovincialis
No full textNext generation sequencing (NGS) allows fast and massive production of both genome and transcriptome sequence datasets. As the genome of the Mediterranean mussel Mytilus galloprovincialis is not available at present, we have explored the possibility of reducing the whole genome sequencing efforts by using capture probes coupled with PCR amplification and high-throughput 454-sequencing to enrich selected genomic regions. The enrichment of DNA target sequences was validated by Real-Time PCR, whereas the efficacy of the applied strategy was evaluated by mapping the 454-output reads on reference transcript data available for M. galloprovincialis and by measuring coverage, SNPs, number of de novo sequenced introns and complete gene sequences.
Focusing on a target size of nearly 1.5 Mbp we obtained a target coverage which allowed the identification of more than 250 complete introns, 10,741 SNPs and also complete gene sequences. This study confirms the transcriptome-based enrichment of gDNA regions as a good strategy to expand knowledge on specific subsets of genes also in non-model organisms
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