Phenotypic plasticity in the Caribbean sponge Callyspongia vaginalis (Porifera: Haplosclerida)

Sponge morphological plasticity has been a long-standing source of taxonomic difficulty. In the Caribbean, several morphotypes of the sponge Callyspongia vaginalis have been observed. To determine the taxonomic status of three of these morphotypes and their relationship with the congeneric species C. plicifera and C. fallax, we compared the spicule composition, spongin fiber skeleton and sequenced fragments of the mitochondrial genes 16S and COI and nuclear genes 28S and 18S ribosomal RNA. Phylogenetic analyses with ribosomal markers 18S and 28S rRNA confirmed the position of our sequences within the Callyspongiidae. None of the genetic markers provided evidence for consistent differentiation among the three morphotypes of C. vaginalis and C. fallax, and only C. plicifera stood as a distinct species. The 16S mtDNA gene was the most variable molecular marker for this group, presenting a nucleotide variability (π = 0.024) higher than that reported for COI. Unlike recent studies for other sponge genera, our results indicate that species in the genus Callyspongia maintain a high degree of phenotypic plasticity, and that morphological characteristics may not reflect reproductive boundaries in C. vaginalis.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.

Variability in ribosomal DNA genic and spacer regions in Verticillium dahliae isolates from different hosts

Using PCR-based assays with specific primers for amplification of the ribosomal DNA intergenic spacer region (IGS) and a portion of the mitochondrial DNA small subunit ribosomal RNA gene (mtDNA SSU rRNA), the genetic variability among Verticillium dahliae isolates from olive (Olea europaea) and other host species from Argentina and Brazil was estimated. The derived UPGMA-generated phenograms based upon the restriction fingerprinting data of rDNA IGS products revealed genetic differences, correlating with the host of origin. Isolates infecting olive genetically distinct from those from cocoa (Theobroma cacao) and sunflower (Helianthus annuus). Digestion of mitochondrial DNA SSU rRNA PCR products revealed less variability, distinguishing only one isolate from sunflower. Ribosomal DNA ITS restriction patterns were identical for all isolates of V. dahliae, irrespective of host of origin. These preliminary results may have relevance for Verticillium wilt control practices, possibly reflecting a different evolutionary origin, or reproductive isolation of the pathogen in olive, distinct from populations of other hosts.

Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae)

Leaf-cutting ants of the genera Atta and Acromyrmex (tribe Attini) are symbiotic with basidiomycete fungi of the genus Leucoagaricus (tribe Leucocoprineae), which they cultivate on vegetable matter inside their nests. We determined the variation of the 28S, 18S, and 5.8S ribosomal DNA (rDNA) gene loci and the rapidly evolving internal transcribed spacers 1 and 2 (ITS1 and ITS2) of 15 sympatric and allopatric fungi associated with colonies of 11 species of leafcutter ants living up to 2,600 km apart in Brazil. We found that the fungal rDNA and ITS sequences from different species of ants were identical (or nearly identical) to each other, whereas 10 GenBank Leucoagaricus species showed higher ITS variation. Our findings suggest that Atta and Acromyrmex leafcutters living in geographic sites that are very distant from each other cultivate a single fungal species made up of closely related lineages of Leucoagaricus gongylophorus. We discuss the strikingly high similarity in the ITS1 and ITS2 regions of the Atta and Acromyrmex symbiotic L. gongylophorus studied by us, in contrast to the lower similarity displayed by their non-symbiotic counterparts. We suggest that the similarity of our L. gongylophorus isolates is an indication of the recent association of the fungus with these ants, and propose that both the intense lateral transmission of fungal material within leafcutter nests and the selection of more adapted fungal strains are involved in the homogenization of the symbiotic fungal stock.

RNA recurrent motifs : identification and characterization

La détermination de la structure tertiaire du ribosome fut une étape importante dans la compréhension du mécanisme de la synthèse des protéines. Par contre, l’élucidation de la structure du ribosome comme tel ne permet pas une compréhension de sa fonction. Pour mieux comprendre la nature des relations entre la structure et la fonction du ribosome, sa structure doit être étudiée de manière systématique. Au cours des dernières années, nous avons entrepris une démarche systématique afin d’identifier et de caractériser de nouveaux motifs structuraux qui existent dans la structure du ribosome et d’autres molécules contenant de l’ARN. L’analyse de plusieurs exemples d’empaquetage de deux hélices d’ARN dans la structure du ribosome nous a permis d’identifier un nouveau motif structural, nommé « G-ribo ». Dans ce motif, l’interaction d’une guanosine dans une hélice avec le ribose d’un nucléotide d’une autre hélice donne naissance à un réseau d’interactions complexes entre les nucléotides voisins. Le motif G-ribo est retrouvé à 8 endroits dans la structure du ribosome. La structure du G-ribo possède certaines particularités qui lui permettent de favoriser la formation d’un certain type de pseudo-nœuds dans le ribosome. L’analyse systématique de la structure du ribosome et de la ARNase P a permis d’identifier un autre motif structural, nommé « DTJ » ou « Double-Twist Joint motif ». Ce motif est formé de trois courtes hélices qui s’empilent l’une sur l’autre. Dans la zone de contact entre chaque paire d’hélices, deux paires de bases consécutives sont surenroulées par rapport à deux paires de bases consécutives retrouvées dans l’ARN de forme A. Un nucléotide d’une paire de bases est toujours connecté directement à un nucléotide de la paire de bases surenroulée, tandis que les nucléotides opposés sont connectés par un ou plusieurs nucléotides non appariés. L’introduction d’un surenroulement entre deux paires de bases consécutives brise l’empilement entre les nucléotides et déstabilise l’hélice d’ARN. Dans le motif DTJ, les nucléotides non appariés qui lient les deux paires de bases surenroulées interagissent avec une des trois hélices qui forment le motif, offrant ainsi une stratégie élégante de stabilisation de l’arrangement. Pour déterminer les contraintes de séquences imposées sur la structure tertiaire d’un motif récurrent dans le ribosome, nous avons développé une nouvelle approche expérimentale. Nous avons introduit des librairies combinatoires de certains nucléotides retrouvés dans des motifs particuliers du ribosome. Suite à l’analyse des séquences alternatives sélectionnées in vivo pour différents représentants d’un motif, nous avons été en mesure d’identifier les contraintes responsables de l’intégrité d’un motif et celles responsables d’interactions avec les éléments qui forment le contexte structural du motif. Les résultats présentés dans cette thèse élargissent considérablement notre compréhension des principes de formation de la structure d’ARN et apportent une nouvelle façon d’identifier et de caractériser de nouveaux motifs structuraux d’ARN.

Stabilité de l’acide ribonucléique pour la datation des fluides corporels en biologie judiciaire

Projet de recherche réalisé en collaboration avec la section Biologie/ADN du Laboratoire de sciences judiciaires et de médecine légale (LSJML) de Montréal.

Structural rules for the formation of backbone-backbone interactions between closely packed RNA double helices

Les interactions entre les squelettes sucre-phosphate de nucléotides jouent un rôle important dans la stabilisation des structures tertiaires de larges molécules d’ARN. Elles sont régies par des règles particulières qui gouverne leur formation mais qui jusque là demeure quasiment inconnues. Un élément structural d’ARN pour lequel les interactions sucre-phosphate sont importantes est le motif d’empaquetage de deux doubles hélices d’ARN le long du sillon mineur. Ce motif se trouve à divers endroits dans la structure du ribosome. Il consiste en deux doubles hélices interagissant de manière à ce que le squelette sucre-phosphate de l’une se niche dans le sillon mineur de l’autre et vice versa. La surface de contact entre les deux hélices est majoritairement formée par les riboses et implique au total douze nucléotides. La présente thèse a pour but d’analyser la structure interne de ce motif et sa dépendance de stabilité résultant de l’association optimale ou non des hélices, selon leurs séquences nucléotidiques. Il est démontré dans cette thèse qu’un positionnement approprié des riboses leur permet de former des contacts inter-hélices, par l’entremise d’un choix particulier de l’identité des pairs de bases impliquées. Pour différentes pairs de bases participant à ce contact inter-hélices, l’identité optimale peut être du type Watson-Crick, GC/CG, or certaines pairs de bases non Watson-Crick. Le choix adéquat de paires de bases fournit une interaction inter-hélice stable. Dans quelques cas du motif, l’identité de certaines paires de bases ne correspond pas à la structure la plus stable, ce qui pourrait refléter le fait que ces motifs devraient avoir une liberté de formation et de déformation lors du fonctionnement du ribosome.

Plant U13 orthologues and orphan snoRNAs identified by RNomics of RNA from Arabidopsis nucleoli

Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs whose main function in eukaryotes is to guide the modification of nucleotides in ribosomal and spliceosomal small nuclear RNAs, respectively. Full-length sequences of Arabidopsis snoRNAs and scaRNAs have been obtained from cDNA libraries of capped and uncapped small RNAs using RNA from isolated nucleoli from Arabidopsis cell cultures. We have identified 31 novel snoRNA genes (9 box C/D and 22 box H/ACA) and 15 new variants of previously described snoRNAs. Three related capped snoRNAs with a distinct gene organization and structure were identified as orthologues of animal U13snoRNAs. In addition, eight of the novel genes had no complementarity to rRNAs or snRNAs and are therefore putative orphan snoRNAs potentially reflecting wider functions for these RNAs. The nucleolar localization of a number of the snoRNAs and the localization to nuclear bodies of two putative scaRNAs was confirmed by in situ hybridization. The majority of the novel snoRNA genes were found in new gene clusters or as part of previously described clusters. These results expand the repertoire of Arabidopsis snoRNAs to 188 snoRNA genes with 294 gene variants.

p90 ribosomal S6 kinases play a significant role in early gene regulation in the cardiomyocyte response to Gq protein-coupled receptor stimuli, endothelin-1 and α1-adrenergic receptor agonists

Extracellular signal-regulated kinases 1/2 (ERK1/2) and their substrates, p90 ribosomal S6 kinases (RSKs), phosphorylate different transcription factors, contributing differentially to transcriptomic profiles. In cardiomyocytes, ERK1/2 are required for >70% of the transcriptomic response to endothelin-1. Here, we investigated the role of RSKs in the transcriptomic responses to Gq protein-coupled receptor agonists, endothelin-1, phenylephrine (generic α1-adrenergic receptor agonist) and A61603 (α1A-adrenergic receptor selective). Phospho-ERK1/2 and phospho-RSKs appeared in cardiomyocyte nuclei within 2-3 min of stimulation (endothelin-1>a61603≈phenylephrine). All agonists increased nuclear RSK2, but only endothelin-1 increased nuclear RSK1 content. PD184352 (inhibits ERK1/2 activation) and BI-D1870 (inhibits RSKs) were used to dissect the contribution of RSKs to the endothelin-1-responsive transcriptome. Of 213 RNAs upregulated at 1 h, 51% required RSKs for upregulation whereas 29% required ERK1/2 but not RSKs. The transcriptomic response to phenylephrine overlapped with, but was not identical to, endothelin-1. As with endothelin-1, PD184352 inhibited upregulation of most phenylephrine-responsive transcripts, but the greater variation in effects of BI-D1870 suggests that differential RSK signalling influences global gene expression. A61603 induced similar changes in RNA expression in cardiomyocytes as phenylephrine, indicating that the signal was mediated largely through α1A-adrenergic receptors. A61603 also increased expression of immediate early genes in perfused adult rat hearts and, as in cardiomyocytes, upregulation of the majority of genes was inhibited by PD184352. PD184352 or BI-D1870 prevented the increased surface area induced by endothelin-1 in cardiomyocytes. Thus, RSKs play a significant role in regulating cardiomyocyte gene expression and hypertrophy in response to Gq protein-coupled receptor stimulation.

Distribution of mosquitoes in the South East of Argentina and first report on the analysis based on 18S rDNA and COI sequences

Although Mar del Plata is the most important city on the Atlantic coast of Argentina, mosquitoes inhabiting such area are almost uncharacterized. To increase our knowledge in their distribution, we sampled specimens of natural populations. After the morphological identification based on taxonomic keys, sequences of DNA from small ribosomal subunit (18S rDNA) and cytochrome c oxidase I (COI) genes were obtained from native species and the phylogenetic analysis of these sequences were done. Fourteen species from the genera Uranotaenia, Culex, Ochlerotatus and Psorophora were found and identified. Our 18S rDNA and COI-based analysis indicates the relationships among groups at the supra-species level in concordance with mosquito taxonomy. The introduction and spread of vectors and diseases carried by them are not known in Mar del Plata, but some of the species found in this study were reported as pathogen vectors.

Comparative Cytogenetics and Molecular Phylogeography in the Group Astyanax altiparanae - Astyanax aff. bimaculatus (Teleostei, Characidae)

The genus Astyanax comprises small characin fish of the neotropical region. The so-called `yellow-tailed characins` compose one of the most widely distributed Astyanax groups. A. altiparanae and A. aff. bimaculatus, are evolutionarily closely related and commonly found in several Brazilian hydrographic basins. In the present work, chromosomal data of specimens of A. altiparanae and A. aff. bimaculatus from 4 hydrographic basins in the states of Sao Paulo (Upper Tiete, Paranapanema, Ribeira de Iguape) and Rio de Janeiro (Guapimirim) are shown. All the populations showed 50 chromosomes, with different karyotypic formula. Although only a single Ag-NOR bearing chromosome pair was observed, all populations possess multiple cistrons of 18S rDNA. FISH with the 5S rDNA probe showed single signals at the interstitial position of one metacentric chromosome pair. C-bands are distributed in the terminal and interstitial regions of several chromosomes. However, the As-51 satDNA are frugally located in a few chromosomes of fishes from Upper Tiete, Paranapanema and Guapimirim Rivers, being absent in individuals of A. aff. bimaculatus from Ribeira de Iguape River basin. Beside these 4 populations, molecular phylogeography studies were also performed in individuals from Middle and Lower Tiete River basin and from 2 additional collection sites in the Paranapanema and Ribeira de Iguape River basins. The phylogeographic analysis using 2 mtDNA regions (totalizing 1.314 bp of ND2 and ATPase6/8 genes) of 8 populations of the group of `yellow-tailed characins` from 3 major hydrographic basins showed structuring of populations, suggesting a correlation between chromosomal (nuclear) and molecular (mitochondrial) data. Copyright (C) 2011 S. Karger AG, Basel

Comparative chromosomal analyses in species of the genus Pimelodella (Siluriformes, Heptapteridae): occurrence of structural and numerical polymorphisms

Cytogenetic analyses were carried out in five species of Pimelodella from the main sub-basins of Upper Parana River and Paraiba do Sul River. The diploid number ranged from 2n = 46 to 2n = 58 chromosomes, and all populations differed in the karyotype constitution. The presence of supernumerary chromosomes as well as the occurrence of a XX/XY sex chromosome system and heterochromatin polymorphisms were detected. The 18S rDNA FISH confirmed the presence of single NORs and revealed additional sites on supernumerary chromosomes. The number and location of 5S rDNA sites were variable. Aspects related to the karyotypic evolution within the genus are discussed.

The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5`-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

Characterization of spliced leader genes of Trypanosoma (Megatrypanum) theileri: phylogeographical analysis of Brazilian isolates from cattle supports spatial clustering of genotypes and parity with ribosomal markers

Trypanosoma (Megatrypanum) theileri from cattle and trypanosomes of other artiodactyls form a clade of closely related species in analyses using ribosomal sequences. Analysis of polymorphic sequences of a larger number of trypanosomes from broader geographical origins is required to evaluate the Clustering of isolates as suggested by previous studies. Here, we determined the sequences of the spliced leader (SL) genes of 21 isolates from cattle and 2 from water buffalo from distant regions of Brazil. Analysis of SL gene repeats revealed that the 5S rRNA gene is inserted within the intergenic region. Phylogeographical patterns inferred using SL sequences showed at least 5 major genotypes of T. theileri distributed in 2 strongly divergent lineages. Lineage TthI comprises genotypes IA and IB from buffalo and cattle, respectively, from the Southeast and Central regions, whereas genotype IC is restricted to cattle from the Southern region. Lineage Tth II includes cattle genotypes IIA, which is restricted to the North and Northeast, and IIB, found in the Centre, West, North and Northeast. PCR-RFLP of SL genes revealed valuable markers for genotyping T. theileri. The results of this study emphasize the genetic complexity and corroborate the geographical structuring of T. theileri genotypes found in cattle.

Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.