Chromatin Structure of Ribosomal RNA Genes in Dipterans and Its Relationship to the Location of Nucleolar Organizers

Nucleoli, nuclear organelles in which ribosomal RNA is synthesized and processed, emerge from nucleolar organizers (NORs) located in distinct chromosomal regions. In polytene nuclei of dipterans, nucleoli of some species can be observed under light microscopy exhibiting distinctive morphology: Drosophila and chironomid species display well-formed nucleoli in contrast to the fragmented and dispersed nucleoli seen in sciarid flies. The available data show no apparent relationship between nucleolar morphology and location of NORs in Diptera. The regulation of rRNA transcription involves controlling both the transcription rate per gene as well as the proportion of rRNA genes adopting a proper chromatin structure for transcription, since active and inactive rRNA gene copies coexist in NORs. Transcription units organized in nucleosomes and those lacking canonical nucleosomes can be analyzed by the method termed psoralen gel retarding assay (PGRA), allowing inferences on the ratio of active to inactive rRNA gene copies. In this work, possible connections between chromosomal location of NORs and proportion of active rRNA genes were studied in Drosophila melanogaster, and in chironomid and sciarid species. The data suggested a link between location of NORs and proportion of active rRNA genes since the copy number showing nucleosomal organization predominates when NORs are located in the pericentric heterochromatin. The results presented in this work are in agreement with previous data on the chromatin structure of rRNA genes from distantly related eukaryotes, as assessed by the PGRA.

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas

Background: Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. Objective: The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. Methods: Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. Results: Computer-generated genomic analysis tools identified 13 722 and 14 993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. Conclusion: We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.

Bacterial diversity in rhizosphere soil from Antarctic vascular plants of Admiralty Bay, maritime Antarctica

The Antarctic is a pristine environment that contributes to the maintenance of the global climate equilibrium. The harsh conditions of this habitat are fundamental to selecting those organisms able to survive in such an extreme habitat and able to support the relatively simple ecosystems. The DNA of the microbial community associated with the rhizospheres of Deschampsia antarctica Desv (Poaceae) and Colobanthus quitensis (Kunth) BartI (Caryophyllaceae), the only two native vascular plants that are found in Antarctic ecosystems, was evaluated using a 16S rRNA multiplex 454 pyrosequencing approach. This analysis revealed similar patterns of bacterial diversity between the two plant species from different locations, arguing against the hypothesis that there would be differences between the rhizosphere communities of different plants. Furthermore, the phylum distribution presented a peculiar pattern, with a bacterial community structure different from those reported of many other soils. Firmicutes was the most abundant phylum in almost all the analyzed samples, and there were high levels of anaerobic representatives. Also, some phyla that are dominant in most temperate and tropical soils, such as Acidobacteria, were rarely found in the analyzed samples. Analyzing all the sample libraries together, the predominant genera found were Bifidobacterium (phylum Actinobacteria), Arcobacter (phylum Proteobacteria) and Faecalibacterium (phylum Firmicutes). To the best of our knowledge, this is the first major bacterial sequencing effort of this kind of soil, and it revealed more than expected diversity within these rhizospheres of both maritime Antarctica vascular plants in Admiralty Bay, King George Island, which is part of the South Shetlands archipelago. The ISME Journal (2010) 4, 989-1001; doi:10.1038/ismej.2010.35; published online 1 April 2010

Revealing archaeal diversity patterns and methane fluxes in Admiralty Bay, King George Island, and their association to Brazilian Antarctic Station activities

The study of Antarctic archaeal communities adds information on the biogeography of this group and helps understanding the dynamics of biogenic methane production in such extreme habitats. Molecular methods were combined to methane flux determinations in Martel Inlet, Admiralty Bay, to assess archaeal diversity, to obtain information about contribution of the area to atmospheric methane budget and to detect possible interferences of the Antarctic Brazilian Station Comandante Ferraz (EACF) wastewater discharge on local archaeal communities and methane emissions. Methane fluxes in Martel Inlet ranged from 3.2 to 117.9 mu mol CH(4) m(-2) d(-1), with an average of 51.3 +/- 8.5 mu mol CH(4) m(-2) d(-1) and a median of 57.6 mu mol CH(4) m(-2)d(-1). However, three negative fluxes averaging -11.3 mu mol CH(4) m(-2) d(-1) were detected in MacKellar Inlet, indicating that Admiralty Bay can be either a source or sink of atmospheric methane. Denaturing gradient gel electrophoresis (DGGE) showed that archaeal communities at EACF varied with depth and formed a group separated from the reference sites. Granulometric analysis indicated that differences observed may be mostly related to sediment type. However, an influence of wastewater input could not be discarded, since higher methane fluxes were found at CF site. suggesting stimulation of local methanogenesis. DGGE profile of the wastewater sample grouped separated from all other samples, suggesting that methanogenesis stimulation may be due to changes in environmental conditions rather than to the input of allochtonous species from the wastewater. 16S ribosomal DNA clone libraries analysis showed that all wastewater sequences were related to known methanogenic groups belonging to the hydrogenotrophic genera Methanobacterium and Methanobrevibacter and the aceticlastic genus Methanosaeta. EACF and Botany Point sediment clone libraries retrieved only groups of uncultivated Archaea, with predominance of Crenarchaeota representatives (MCG, MG1, MBG-B, MBG-C and MHVG groups). Euryarchaeota sequences found were mostly related to the LDS and RC-V groups, but MBG-D and DHVE-5 were also present. No representatives of cultivated methanogenic groups were found, but coverage estimates suggest that a higher number of clones would have to be analyzed in order to cover the greater archaeal diversity of Martel Inlet sediment. Nevertheless, the analysis of the libraries revealed groups not commonly found by other authors in Antarctic habitats and also indicated the presence of groups of uncultivated archaea previously associated to methane rich environments or to the methane cycle. (C) 2010 Elsevier Ltd. All rights reserved.

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.

Gastrotricha: A Marine Sister for a Freshwater Puzzle

Background: Within an evolutionary framework of Gastrotricha Marinellina flagellata and Redudasys fornerise bear special interest, as they are the only Macrodasyida that inhabit freshwater ecosystems. Notwithstanding, these rare animals are poorly known; found only once (Austria and Brazil), they are currently systematised as incertae sedis. Here we report on the rediscovery of Redudasys fornerise, provide an account on morphological novelties and present a hypothesis on its phylogenetic relationship based on molecular data. Methodology/Principal Findings: Specimens were surveyed using DIC microscopy and SEM, and used to obtain the 18 S rRNA gene sequence; molecular data was analyzed cladistically in conjunction with data from 42 additional species belonging to the near complete Macrodasyida taxonomic spectrum. Morphological analysis, while providing new information on taxonomically relevant traits (adhesive tubes, protonephridia and sensorial bristles), failed to detect elements of the male system, thus stressing the parthenogenetic nature of the Brazilian species. Phylogenetic analysis, carried out with ML, MP and Bayesian approaches, yielded topologies with strong nodal support and highly congruent with each other. Among the supported groups is the previously undocumented clade showing the alliance between Redudasys fornerise and Dactylopodola agadasys; other strongly sustained clades include the densely sampled families Thaumastodermatidae and Turbanellidae and most genera. Conclusions/Significance: A reconsideration of the morphological traits of Dactylopodola agadasys in light of the new information on Redudasys fornerise makes the alliance between these two taxa very likely. As a result, we create Anandrodasys gen. nov. to contain members of the previously described D. agadasys and erect Redudasyidae fam. nov. to reflect this novel relationship between Anandrodasys and Redudasys. From an ecological perspective, the derived position of Redudasys, which is deeply nested within the Macrodasyida clade, unequivocally demonstrates that invasion of freshwater by gastrotrichs has taken place at least twice, in contrast with the single event hypothesis recently put forward.

Genome Sequence of the Bacterioplanktonic, Mixotrophic Vibrio campbellii Strain PEL22A, Isolated in the Abrolhos Bank

Vibrio campbellii PEL22A was isolated from open ocean water in the Abrolhos Bank. The genome of PEL22A consists of 6,788,038 bp (the GC content is 45%). The number of coding sequences (CDS) is 6,359, as determined according to the Rapid Annotation using Subsystem Technology (RAST) server. The number of ribosomal genes is 80, of which 68 are tRNAs and 12 are rRNAs. V. campbellii PEL22A contains genes related to virulence and fitness, including a complete proteorhodopsin cluster, complete type II and III secretion systems, incomplete type I, IV, and VI secretion systems, a hemolysin, and CTX Phi.

Genetic Variability of Beauveria bassiana and a DNA Marker for Environmental Monitoring of a Highly Virulent Isolate Against Cosmopolites sordidus

The banana weevil Cosmopolites sordidus (Germar) is one of a number of pests that attack banana crops. The use of the entomopathogenic fungus Beauveria bassiana as a biological control agent for this pest may contribute towards reducing the application of chemical insecticides on banana crops. In this study, the genetic variability of a collection of Brazilian isolates of B. bassiana was evaluated. Samples were obtained from various geographic regions of Brazil, and from different hosts of the Curculionidae family. Based on the DNA fingerprints generated by RAPD and AFLP, we found that 92 and 88 % of the loci were polymorphic, respectively. The B. bassiana isolates were attributed to two genotypic clusters based on the RAPD data, and to three genotypic clusters, when analyzed with AFLP. The nucleotide sequences of nuclear ribosomal DNA intergenic spacers confirmed that all isolates are in fact B. bassiana. Analysis of molecular variance showed that variability among the isolates was not correlated with geographic origin or hosts. A RAPD-specific marker for isolate CG 1024, which is highly virulent to C. sordidus, was cloned and sequenced. Based on the sequences obtained, specific PCR primers BbasCG1024F (5'-TGC GGC TGA GGA GGA CT-3') and BbasCG1024R (5'-TGC GGC TGA GTG TAG AAC-3') were designed for detecting and monitoring this isolate in the field.

Characterization of Immobilized Biomass by Amplified rDNA Restriction Analysis (ARDRA) in an Anaerobic Sequencing-Batch Biofilm Reactor (ASBBR) for the Treatment of Industrial Wastewater

The performance of an anaerobic sequencing-batch biofilm reactor (ASBBR-laboratory scale- 14L) containing biomass immobilized on coal was evaluated for the removal of elevated concentrations of sulfate (between 200 and 3,000 mg SO4-2.L-1) from industrial wastewater effluents. The ASBBR was shown to be efficient for removal of organic material (between 90% and 45%) and sulfate (between 95% and 85%). The microbiota adhering to the support medium was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). The ARDRA profiles for the Bacteria and Archaea domains proved to be sensitive for the determination of microbial diversity and were consistent with the physical-chemical monitoring analysis of the reactor. At 3,000 mg SO4-2.L-1, there was a reduction in the microbial diversity of both domains and also in the removal efficiencies of organic material and sulfate.

The contrasting role of heterochromatin in the differentiation of sex chromosomes: an overview from Neotropical fishes

During the evolutionary process of the sex chromosomes, a general principle that arises is that cessation or a partial restriction of recombination between the sex chromosome pair is necessary. Data from phylogenetically distinct organisms reveal that this phenomenon is frequently associated with the accumulation of heterochromatin in the sex chromosomes. Fish species emerge as excellent models to study this phenomenon because they have much younger sex chromosomes compared to higher vertebrates and many other organisms making it possible to follow their steps of differentiation. In several Neotropical fish species, the heterochromatinization, accompanied by amplification of tandem repeats, represents an important step in the morphological differentiation of simple sex chromosome systems, especially in the ZZ/ZW sex systems. In contrast, multiple sex chromosome systems have no additional increase of heterochromatin in the chromosomes. Thus, the initial stage of differentiation of the multiple sex chromosome systems seems to be associated with proper chromosomal rearrangements, whereas the simple sex chromosome systems have an accumulation of heterochromatin. In this review, attention has been drawn to this contrasting role of heterochromatin in the differentiation of simple and multiple sex chromosomes of Neotropical fishes, highlighting their surprising evolutionary dynamism.

Antibacterial compound from the endophytic fungus Phomopsis longicolla isolated from the tropical red seaweed Bostrychia radicans

Endophytic fungi isolated from the red seaweed Bostrychia radicans were studied to identify their molecularly diverse and biologically active natural chemical products. According to 28S ribosomal DNA-based identification, the strain named C81 was 98% identical to Phomopsis longicolla. This strain was cultivated in solid rice medium and produced three major metabolites identified as 18-deoxycytochalasin H (1), mycophenolic acid (2), and dicerandrol C (3). The chemical structures of these compounds were elucidated by 1D and 2D nuclear magnetic resonance as well as by mass spectrometry. Dicerandrol C had significant antimicrobial activity against Staphylococcus aureus (ATCC 6538) and Staphylococcus saprophyticus (ATCC 15305), with minimum inhibitory concentrations of 1 and 2 mu g ml(-1) (1.33 and 2.66 mu M), respectively. These results show the presence of promising metabolites and indicate that these natural products should be considered in the development of new antibiotics.

NEW RECORDS OF ANOPHELES HOMUNCULUS IN CENTRAL AND SERRA DO MAR BIODIVERSITY CORRIDORS OF THE ATLANTIC FOREST, BRAZIL

Two new records of Anopheles homunculus in the eastern part of the Atlantic Forest are reported. This species was found for the first time in Barra do Ouro district, Maquine municipality, Rio Grande do Sul state, located in the southern limit of the Atlantic Forest. The 2nd new record was in the Serra Bonita Reserve, Camacan municipality, southeast Bahia state. These records extend the geographical distribution of An. homunculus, suggesting that the species may be widely distributed in coastal areas of the Atlantic Forest. It is hypothesized that the disjunct distribution of the species may be caused by inadequate sampling, and also difficulties in species identification based only on female external characteristics. Species identification was based on morphological characters of the male, larva, and pupa, and corroborated by DNA sequence analyses, employing data from both 2nd internal transcribed spacer of nuclear ribosomal DNA and of mitochondrial cytochrome c oxidase subunit I.

Levels of Selenomonas species in generalized aggressive periodontitis

Goncalves LFH, Fermiano D, Feres M, Figueiredo LC, Teles FRP, Mayer MPA, Faveri M. Levels of Selenomonas species in generalized aggressive periodontitis. J Periodont Res 2012; 47: 711718. (c) 2012 John Wiley & Sons A/S Background and Objective: To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from periodontally healthy subjects and from subjects with generalized aggressive periodontitis. Material and Methods: Fifteen periodontally healthy subjects and 15 subjects with generalized aggressive periodontitis were recruited and their clinical periodontal parameters were evaluated. Nine subgingival plaque samples were collected from each subject and all were individually analyzed for the levels of 10 bacterial taxa, including cultured and uncultivated/unrecognized microorganisms, using the RNA-oligonucleotide quantification technique. Between-group differences in the levels of the test taxa were determined using the MannWhitney U-test. Results: Subjects with generalized aggressive periodontitis showed significantly higher mean counts of Porphyromonas gingivalis, S. sputigena and the Mitsuokella sp. Human Oral Taxon (HOT) 131 (previously described as Selenomonas sp. oral clone CS002), while higher mean counts of Actinomyces gerencseriae and Streptococcus sanguinis were found in periodontally healthy subjects (p < 0.01). Selenomonas sp. HOT 146 was only detected in the generalized aggressive periodontitis group. In the generalized aggressive periodontitis group, the levels of P.gingivalis and S.sputigena were higher in deep sites (probing depth = 5 mm) than in shallow sites (probing depth = 3 mm) (p < 0.01). Furthermore, in subjects with generalized aggressive periodontitis, sites with probing depth of = 3 mm harbored higher levels of these two species than sites with the same probing depth in periodontally healthy subjects. There were positive correlations between probing depth and the levels of P.gingivalis (r = 0.77; p < 0.01), S.sputigena (r = 0.60; p < 0.01) and Selenomonas dianae (previously described as Selenomonas sp. oral clone EW076) (r = 0.42, p < 0.05). Conclusion: S. sputigena and Mitsuokella sp. HOT 131 may be associated with the pathogenesis of generalized aggressive periodontitis, and their role in the onset and progression of this infection should be investigated further.

Haploinsufficiency of a Spliceosomal GTPase Encoded by EFTUD2 Causes Mandibulofacial Dysostosis with Microcephaly

Mandibulofacial dysostosis with microcephaly (MFDM) is a rare sporadic syndrome comprising craniofacial malformations, microcephaly, developmental delay, and a recognizable dysmorphic appearance. Major sequelae, including choanal atresia, sensorineural hearing loss, and cleft palate, each occur in a significant proportion of affected individuals. We present detailed clinical findings in 12 unrelated individuals with MFDM; these 12 individuals compose the largest reported cohort to date. To define the etiology of MFDM, we employed whole-exome sequencing of four unrelated affected individuals and identified heterozygous mutations or deletions of EFTUD2 in all four. Validation studies of eight additional individuals with MFDM demonstrated causative EFTUD2 mutations in all affected individuals tested. A range of EPTUD2-mutation types, including null alleles and frameshifts, is seen in MFDM, consistent with haploinsufficiency; segregation is de novo in all cases assessed to date. U5-116kD, the protein encoded by EFTUD2, is a highly conserved spliceosomal GTPase with a central regulatory role in catalytic splicing and post-splicing-complex disassembly. MFDM is the fast multiple-malformation syndrome attributed to a defect of the major spliceosome. Our findings significantly extend the range of reported spliceosomal phenotypes in humans and pave the way for further investigation in related conditions such as Treacher Collins syndrome.

Karyotype characterization reveals an up and down of 45S and 5S rDNA sites in Crotalaria (Leguminosae-Papilionoideae) species of the section Hedriocarpae subsection Macrostachyae

The genus Crotalaria is one of the largest within the family Leguminosae-Papilionoideae, with more than 600 species. However, few karyotypes have been described. In the present paper, five species belonging to the section Hedriocarpae were studied (subsection Machrostachyae), in order to better understand chromosomal evolution in Crotalaria. The results reveals that all species presented 2n = 2x = 16 with symmetrical karyotypes, and slight differences in the chromosome morphology. A secondary constriction was identified at short arm of the pair 1. The 45S rDNA was mapped in the secondary constriction and adjacent heterochromatin (NOR-heterochromatin) and a minor site was identified in C. ochroleuca. The 5S rDNA was mapped linked to 45S rDNA at chromosome 1 short arm in all species. Additional sites for 5S rDNA were identified in C. pallida, C. striata and C. mucronata. Heterochromatin blocks around the centromeres are not CMA(+) neither DAPI(+). The karyotypes of the subsection Macrostachyae are characterized by an inversion at chromosome pair one in relation to previous specialized floral species analyzed. Additional sites of 45S and 5S rDNA were assumed to be a result of transposition events by different ways. The results suggest heterochromatin differentiation and the position of ribosomal genes indicates chromosomal rearrangements during evolution. Karyotype characteristics corroborate the morphological infrageneric classification.