Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.

Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA

The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy. © 2007 Entomological Society of America.

Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution.

The internal transcribed spacers (ITS) of nuclear ribosomal DNA of 33 species of genus Paeonia (Paeoniaceae) were sequenced. In section Paeonia, different patterns of nucleotide additivity were detected in 14 diploid and tetraploid species at sites that are variable in the other 12 species of the section, suggesting that reticulate evolution has occurred. Phylogenetic relationships of species that do not show additivity, and thus ostensibly were not derived through hybridization, were reconstructed by parsimony analysis. The taxa presumably derived through reticulate evolution were then added to the phylogenetic tree according to additivity from putative parents. The study provides an example of successfully using ITS sequences to reconstruct reticulate evolution in plants and further demonstrates that the sequence data could be highly informative and accurate for detecting hybridization. Maintenance of parental sequences in the species of hybrid origin is likely due to slowing of concerted evolution caused by the long generation time of peonies. The partial and uneven homogenization of parental sequences displayed in nine species of putative hybrid origin may have resulted from gradients of gene conversion. The documented hybridizations may have occurred since the Pleistocene glaciations. The species of hybrid origin and their putative parents are now distantly allopatric. Reconstruction of reticulate evolution with sequence data, therefore, provides gene records for distributional histories of some of the parental species.

Radiation of the Australian flora: What can comparisons of molecular phylogenies across multiple taxa tell us about the evolution of diversity in present-day communities?

The Australian fossil record shows that from ca. 25 Myr ago, the aseasonal-wet biome (rainforest and wet heath) gave way to the unique Australian sclerophyll biomes dominated by eucalypts, acacias and casuarinas. This transition coincided with tectonic isolation of Australia, leading to cooler, drier, more seasonal climates. From 3 Myr ago, aridification caused rapid opening of the central Australian and zone. Molecular phylogenies with dated nodes have provided new perspectives on how these events could have affected the evolution of the Australian flora. During the Mid-Cenozoic (25-10 Myr ago) period of climatic change, there were rapid radiations in sclerophyll taxa, such as Banksia, eucalypts, pea-flowered legumes and Allocasuarina. At the same time, taxa restricted to the aseasonal-wet biome (Nothofagus, Podocarpaceae and Araucariaceae) did not radiate or were depleted by extinction. During the Pliocene aridification, two Eremean biome taxa (Lepidium and Chenopodiaceae) radiated rapidly after dispersing into Australia from overseas. It is clear that the biomes have different histories. Lineages in the aseasonal-wet biome are species poor, with sister taxa that are species rich, either outside Australia or in the sclerophyll biomes. In conjunction with the fossil record, this indicates depletion of the Australian aseasonal-wet biome from the Mid-Cenozoic. In the sclerophyll biomes, there have been multiple exchanges between the southwest and southeast, rather than single large endemic radiations after a vicariance event. There is need for rigorous molecular phylogenetic studies so that additional questions can be addressed, such as how interactions between biomes may have driven the speciation process during radiations. New studies should include the hither-to neglected monsoonal tropics.

Biogeography, host specificity, and molecular phylogeny of the basidiomycetous yeast phaffia rhodozyma and its sexual form, xanthophyllomyces dendrorhous

Applied and Environmental Microbiology, Vol. 73, No.4

CHARACTERIZATION OF NrsZ, A NOVEL SMALL REGULATORY NON-CODING RNA INVOLVED IN THE ADAPTATION OF PSEUDOMONAS AERUGINOSA PAO1

The opportunistic ubiquitous pathogen Pseudomonas aeruginosa strain PAOl is a versatile Gram-negative bacterium that has the extraordinary capacity to colonize a wide diversity of ecological niches and to cause severe and persistent infections in humans. To ensure an optimal coordination of the genes involved in nutrient utilization, this bacterium uses the NtrB/C and/or the CbrA/B two-component systems, to sense nutrients availability and to regulate in consequence the expression of genes involved in their uptake and catabolism. NtrB/C is specialized in nitrogen utilization, while the CbrA/B system is involved in both carbon and nitrogen utilization and both systems activate their target genes expression in concert with the alternative sigma factor RpoN. Moreover, the NtrB/C and CbrA/B two- component systems regulate the secondary metabolism of the bacterium, such as the production of virulence factors. In addition to the fine-tuning transcriptional regulation, P. aeruginosa can rapidly modulate its metabolism using small non-coding regulatory RNAs (sRNAs), which regulate gene expression at the post-transcriptional level by diverse and sophisticated mechanisms and contribute to the fast physiological adaptability of this bacterium. In our search for novel RpoN-dependent sRNAs modulating the nutritional adaptation of P. aeruginosa PAOl, we discovered NrsZ (Nitrogen regulated sRNA), a novel RpoN-dependent sRNA that is induced under nitrogen starvation by the NtrB/C two-component system. NrsZ has a unique architecture, formed of three similar stem-loop structures (SL I, II and II) separated by variant spacer sequences. Moreover, this sRNA is processed in short individual stem-loop molecules, by internal cleavage involving the endoribonuclease RNAse E. Concerning NrsZ functions in P. aeruginosa PAOl, this sRNA was shown to trigger the swarming motility and the rhamnolipid biosurfactants production. This regulation is due to the NrsZ-mediated activation of rhlA expression, a gene encoding for an enzyme essential for swarming motility and rhamnolipids production. Interestingly, the SL I structure of NrsZ ensures its regulatory function on rhlA expression, suggesting that the similar SLs are the functional units of this modular sRNA. However, the regulatory mechanism of action of NrsZ on rhlA expression activation remains unclear and is currently being investigated. Additionally, the NrsZ regulatory network was investigated by a transcriptome analysis, suggesting that numerous genes involved in both primary and secondary metabolism are regulated by this sRNA. To emphasize the importance of NrsZ, we investigated its conservation in other Pseudomonas species and demonstrated that NrsZ is conserved and expressed under nitrogen limitation in Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48 and Pseudomonas syringae pv. tomato DC3000, strains having different ecological features, suggesting an important role of NrsZ in the adaptation of Pseudomonads to nitrogen starvation. Interestingly the architecture of the different NrsZ homologs is similarly composed by SL structures and variant spacer sequences. However, the number of SL repetitions is not identical, and one to six SLs were predicted on the different NrsZ homologs. Moreover, NrsZ is processed in short molecules in all the strains, similarly to what was previously observed in P. aeruginosa PAOl, and the heterologous expression of the NrsZ homologs restored rhlA expression, swarming motility and rhamnolipids production in the P. aeruginosa NrsZ mutant. In many aspects, NrsZ is an atypical sRNA in the bacterial panorama. To our knowledge, NrsZ is the first described sRNA induced by the NtrB/C. Moreover, its unique modular architecture and its processing in similar short SL molecules suggest that NrsZ belongs to a novel family of bacterial sRNAs. -- L'agent pathogène opportuniste et ubiquitaire Pseudomonas aeruginosa souche PAOl est une bactérie Gram négative versatile ayant l'extraordinaire capacité de coloniser différentes niches écologiques et de causer des infections sévères et persistantes chez l'être humain. Afin d'assurer une coordination optimale des gènes impliqués dans l'utilisation de différents nutriments, cette bactérie se sert de systèmes à deux composants tel que NtrB/C et CbrA/B afin de détecter la disponibilité des ressources nutritives, puis de réguler en conséquence l'expression des gènes impliqués dans leur importation et leur catabolisme. Le système NtrB/C régule l'utilisation des sources d'azote alors que le système CbrA/B est impliqué à la fois dans l'utilisation des sources de carbone et d'azote. Ces deux systèmes activent l'expression de leurs gènes-cibles de concert avec le facteur sigma alternatif RpoN. En outre, NtrB/C et CbrA/B régulent aussi le métabolisme secondaire, contrôlant notamment la production d'importants facteurs de virulence. En plus de toutes ces régulations génétiques fines ayant lieu au niveau transcriptionnel, P. aeruginosa est aussi capable de moduler son métabolisme en se servant de petits ARNs régulateurs non-codants (ARNncs), qui régulent l'expression génétique à un niveau post- transcriptionnel par divers mécanismes sophistiqués et contribuent à rendre particulièrement rapide l'adaptation physiologique de cette bactérie. Au cours de nos recherches sur de nouveaux ARNncs dépendant du facteur sigma RpoN et impliqués dans l'adaptation nutritionnelle de P. aeruginosa PAOl, nous avons découvert NrsZ (Nitrogen regulated sRNA), un ARNnc induit par la cascade NtrB/C-RpoN en condition de carence en azote. NrsZ a une architecture unique, composée de trois structures en tige- boucle (TB I, II et III) hautement similaires et séparées par des « espaceurs » ayant des séquences variables. De plus, cet ARNnc est clivé en petits fragments correspondant au trois molécules en tige-boucle, par un processus de clivage interne impliquant l'endoribonucléase RNase E. Concernant les fonctions de NrsZ chez P. aeruginosa PAOl, cet ARNnc est capable d'induire la motilité de type « swarming » et la production de biosurfactants, nommés rhamnolipides. Cette régulation est due à l'activation par NrsZ de l'expression de rhlA, un gène essentiel pour la motilité de type swarming et pour la production de rhamnolipides. Étonnamment, la structure TB I est capable d'assurer à elle seule la fonction régulatrice de NrsZ sur l'expression de rhlA, suggérant que ces molécules TBs sont les unités fonctionnelles de cet ARNnc modulaire. Cependant, le mécanisme moléculaire par lequel NrsZ active l'expression de rhlA demeure à ce jour incertain et est actuellement à l'étude. En plus, le réseau de régulations médiées par NrsZ a été étudié par une analyse de transcriptome qui a indiqué que de nombreux gènes impliqués dans le métabolisme primaire ou secondaire seraient régulés par NrsZ. Pour accentuer l'importance de NrsZ, nous avons étudié sa conservation dans d'autres espèces de Pseudomonas. Ainsi, nous avons démontré que NrsZ est conservé et exprimé en situation de carence d'azote par les souches Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48, Pseudomonas syringae pv. tomato DC3000, quatre espèces ayant des caractéristiques écologiques très différentes, suggérant que NrsZ joue un rôle important dans l'adaptation du genre Pseudomonas envers la carence en azote. Chez toutes les souches étudiées, les différents homologues de NrsZ présentent une architecture similaire faite de TBs conservées et d'espaceurs. Cependant, le nombre de TBs n'est pas identique et peut varier de une à six copies selon la souche. Les différentes versions de NrsZ sont clivées en petites molécules dans ces quatre souches, comme il a été observé chez P. aeruginosa PAOl. De plus, l'expression hétérologue des différentes variantes de NrsZ est capable de restaurer l'expression de rhlA, la motilité swarming et la production de rhamnolipides dans une souche de P. aeruginosa dont nrsZ a été inactivé. Par bien des aspects, NrsZ est un ARNnc atypique dans le monde bactérien. À notre connaissance, NrsZ est le premier ARNnc décrit comme étant régulé par le système NtrB/C. De plus, son unique architecture modulaire et son clivage en petites molécules similaires suggèrent que NrsZ appartient à une nouvelle famille d'ARNncs bactériens.

Generic relationships and dating of lineages in Winteraceae based on nuclear (ITS) and plastid (rpS16 and psbA-trnH) sequence data

Phylogenetic analyses of representative species from the five genera of Winteraceae (Drimys, Pseudowintera, Takhtajania, Tasmannia, and Zygogynum s.l.) were performed using ITS nuclear sequences and a combined data-set of ITS + psbA-trnH + rpS16 sequences (sampling of 30 and 15 species, respectively). Indel informativity using simple gap coding or gaps as a fifth character was examined in both data-sets. Parsimony and Bayesian analyses support the monophyly of Drimys, Tasmannia, and Zygogynum s.l., but do not support the monophyly of Belliolum, Zygogynum s.s., and Bubbia. Within Drimys, the combined data-set recovers two subclades. Divergence time estimates suggest that the splitting between Drimys and its sister clade (Pseudowintera + Zygogynum s.l.) occurred around the end of the Cretaceous; in contrast, the divergence between the two subclades within Drimys is more recent (15.5-18.5 MY) and coincides in time with the Andean uplift. Estimates suggest that the earliest divergences within Winteraceae could have predated the first events of Gondwana fragmentation. (C) 2009 Elsevier Inc. All rights reserved.

Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes ?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct non-transcribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty-five accessions of the species-rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species-level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0-3 bp). Comparisons to previously published mitochondrial cox2-3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2-3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.

Análise de sequências da região intergência ITS-1 do rDNA em espécies de Drosophila do cluster buzzatti, (complexo Buzzatti, subgrupo Mullerri, grupo Repleta). -

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro.

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.

A synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes.

The 3' processing of histone pre-mRNAs is a nuclear event in which the U7 small nuclear ribonucleoprotein (snRNP) participates as an essential trans-acting factor. We have constructed a chimeric histone-U7 RNA that when injected into the cytoplasm of Xenopus laevis oocytes assembles into a snRNP-like particle and becomes cleaved at the correct site(s). RNP assembly is a prerequisite for cleavage, but, since neither the RNA nor the RNP appreciably enter the nucleus, cleavage occurs mostly, if not exclusively, in the cytoplasm. Consistent with this, cleavage also occurs in enucleated oocytes or in oocytes which have been depleted of U7 snRNPs. Thus all necessary components for cleavage must be present in the oocyte cytoplasm. The novel cleavage occurs in cis, involving only a single molecule of chimeric RNA with its associated proteins. This reaction is equally dependent upon base pairing interactions between histone spacer sequences and the 5'-end of the U7 moiety as the natural in trans reaction. These results imply that U7 is the only snRNP required for histone RNA processing. Moreover, the chimeric RNA is expected to be useful for further studies of the cleavage and assembly mechanisms of U7 snRNP.

3' end processing of mouse histone pre-mRNA: evidence for additional base-pairing between U7 snRNA and pre-mRNA.

We have analysed the extent of base-pairing interactions between spacer sequences of histone pre-mRNA and U7 snRNA present in the trans-acting U7 snRNP and their importance for histone RNA 3' end processing in vitro. For the efficiently processed mouse H4-12 gene, a computer analysis revealed that additional base pairs could be formed with U7 RNA outside of the previously recognised spacer element (stem II). One complementarity (stem III) is located more 3' and involves nucleotides from the very 5' end of U7 RNA. The other, more 5' located complementarity (stem I) involves nucleotides of the Sm binding site of U7 RNA, a part known to interact with snRNP structural proteins. These potential stem structures are separated from each other by short internal loops of unpaired nucleotides. Mutational analyses of the pre-mRNA indicate that stems II and III are equally important for interaction with the U7 snRNP and for processing, whereas mutations in stem I have moderate effects on processing efficiency, but do not impair complex formation with the U7 snRNP. Thus nucleotides near the processing site may be important for processing, but do not contribute to the assembly of an active complex by forming a stem I structure. The importance of stem III was confirmed by the ability of a complementary mutation in U7 RNA to suppress a stem III mutation in a complementation assay using Xenopus laevis oocytes. The main role of the factor(s) binding to the upstream hairpin loop is to stabilise the U7-pre-mRNA complex. This was shown by either stabilising (by mutation) or destabilising (by increased temperature) the U7-pre-mRNA base-pairing under conditions where hairpin factor binding was either allowed or prevented (by mutation or competition). The hairpin dependence of processing was found to be inversely related to the strength of the U7-pre-mRNA interaction.