Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)

Heterosigma akashiwo (Hada) is a fragile, fish-killing alga. Efforts to understand and prevent blooms due to this harmful species to mitigate the impact on aquaculture require the development of methods for rapid and precise identification and quantification, so that adequate warning of a harmful algal bloom may be given. Here, we report the development and application of rRNA and rDNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection and enumeration of H. akashiwo. The designed probes were species specific, showing no cross-reactivity with four common HAB causative species: Prorocentrum micans Ehrenberg, P. minimum (Pavillard) Schiller, Alexandrium tarmarense (Lebour) Balech, and Skeletonema costatum (Greville) Cleve, or with four other microalgae, including Gymnodinium sp. Stein, Platy-monas cordiformis (Karter) Korsch, Skeletonema sp.1 Greville and Skeletonema sp.2. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. (C) 2008 Elsevier B.V. All rights reserved.

Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies.

Molecular characterisation of Alternaria linicola and its detection in linseed

Nucleotide sequences of the ribosomal DNA (rDNA) internal transcribed spacers (ITS) 1 and 2 and a 1068 bp section of the beta-tubulin gene divided seven designated species of Alternaria into five taxa. Stemphylium botryosum formed a sixth closely related taxon. Isolates of A. linicola possessed an identical ITS sequence to one group of A. solani isolates, and two clusters of A. linicola isolates, revealed from beta-tubulin gene data to show minor variation, were as genetically similar to isolates of A. solani as they were to each other. We suggest, therefore, that A. linicola falls within the species A. solani. Similar results suggest that A. lini falls within the species A. alternata. RAPD analysis of the total genomic DNA from the Alternaria spp. concurred with the nucleotide sequence analyses. An oligonucleotide primer (ALP) was selected from the rDNA ITS1 region of A. linicola/A. solani. PCR with primers ALP and ITS4 (from a conserved region of the rDNA) amplified a c. 536 bp fragment from isolates of A. linicola and A. solani but not from other Alternaria spp. nor from other fungi which may be associated with linseed. These primers amplified an identical fragment, confirmed by Southern hybridization, from DNA released from infected linseed seed and leaf tissues. These primers have the potential to be used also for the detection of A. solani in host tissues.

Molecular comparison of Scytalidium thermophilum isolates using RAPD and ITS nucleotide sequence analyses

Scytalidium thermophilum plays an important role in determining selectivity of compost produced for growing Agaricus bisporus. The objective of this study was to characterise S. thermophilum isolates by random amplified polymorphic DNA (RAPD) analysis and sequence analysis of internally transcribed spacer (ITS) regions of the rDNA, to assess the genetic variation exhibited by this species complex and to compare this with existing morphological and thermogravimetric data. RAPD analysis of 34 isolates from various parts of the world revealed two distinct groups, which could be separated on the basis of the differences in the banding patterns produced with five random primers. Nucleotide sequence analysis of the ITS region, which was ca 536 bp in length, revealed only very minor variation among S. thermophilum isolates examined. Several nucleotide base changes within this region demonstrated variation. Genetic distance values among type 1 and 2 S. thermophilum isolates, as determined by ITS sequence analysis, varied by a value of 0.005 %. Molecular analyses carried out in the present study would suggest that isolates within this species complex exhibit genetic differences which correlate well with morphological variation and thermogravimetric data previously determined.

Phylogenetic insights of Avena genus based on genomic analysis of 45S rDNA molecular organization

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014

Elucidating the crosstalk between condensin subunits and its relevance in chromosome condensation

ADN subit une série de transformations structurelles complexes au cours de la division cellulaire, ce qui entraîne dans son compactage chromosomes mitotiques par un processus appelé la condensation des chromosomes. Le complexe de condensine pentamérique est fortement impliqué comme un effecteur majeur de ce phénomène. Il s'agit d'un complexe protéine de sous-unités multiples avec deux sous-unités catalytiques [SMC- Structural Maintenance of Chromosomes] et de trois sous-unités de régulation, hautement conservés de la levure à l'homme. Le complexe de condensine dans Saccharomyces cerevisiae est constitué de deux sous-unités de SMC [Smc2 et Smc4] et trois protéines non réglementaires [Brn1, Ycs4, Ycg1]. Malgré son importance, le mécanisme d'action de condensine reste largement inconnu. Par conséquent, l'objectif de cette recherche est de comprendre le mécanisme d'action de condensine et comment elle est affectée par l'interaction entre ses sous-unités réglementaires et non-réglementaires. Cette thèse identifie quatre morphologies dépendants du cycle cellulaire distincts du locus d'ADNr. Cette transformation du phénotype ADNr de G1 à la mitose dépend condensine. Afin de déterminer le rôle de l'interaction entre les sous-unités catalytiques et réglementaires de condensine dans la régulation du complexe condensine, nous avons identifié six résidus positifs sur l'extrémité C-terminale de BRN1 qui affectent la formation du complexe condensine, l'activité de la condensation et l'interaction avec tubuline, ce qui suggère que ces résidus ont un rôle dans la régulation de condensine. Ensemble, nos résultats suggèrent un modèle de règlement du condensine par l'interaction entre les sous-unités de condensine.

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Molecular identification of marine yeast and its spectroscopic analysis establishes unsaturated fatty acid accumulation

Marine microbes are competent organisms, some of which can accumulate large amounts of lipids. A yeast strain, Rhodotorula mucilaginosa AMCQ8A was isolated from the marine water of the Queenscliff region, Victoria, Australia. The yeast isolate was identified by sequencing 18s rDNA genes. Scanning electron microscopy images revealed scars on the surface of the yeast cells. The Fourier transform infrared spectroscopy microspectroscopy studies demonstrated the presence of unsaturated fatty acids by differential microscopic analysis. The sharp band at 1745 cm^-1 was represented by ν(C=O) stretches of ester functional groups from lipids and fats, and therefore indicated the presence of total lipids produced by the cells. Over 65% of the fatty acids from the yeast strain were analyzed as C¹⁶ and C^18:1 with omega-3 content from about 6% to 7%. Thus, this marine-derived yeast could be a potential source of lipids, including omega-3 fatty acids. 2012, The Society for Biotechnology, Japan. All rights reserved.

Cytogenetic characterisation of the ornamental freshwater fish, Piabucus melanostomus (Iguanodectinae) from Brazilian wetlands and its relation with species of Characidae basal group

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Multiple nucleolus organizer regions in Leptodactylus mystacinus (Amphibia, Anura) and comments on its systematic position in the L fuscus group based on cytogenetic and molecular analyses

Specimens of Leptodactylus mystacinus from Brazil were karyotyped with conventional and differential staining. The 2n = 22 karyotype is similar to that found for the majority of the Leptodactylus, the karyotypic conservatism also confirmed by the similarity of the replication banding patterns with those previously described. L. mystacinus has a small amount of C-banded heterochromatin, located mainly at the centromeres, although telomeric or interstitial bands have also been noticed. With DA/CMA(3) some chromosome regions showed slightly bright fluorescence, and with DA/DAPI, no particular AT-rich repetitive region was observed. Silver staining showed an extensive inter- and intraindividual variation in the number and position of Ag-positive regions, in 1p, 4p, 8p, 8q, and 11p. Nevertheless, FISH using rDNA probes confirmed only the signals on the short arms of chromosomes 4 and 8 as true NORs. The remaining silver stained regions are probably due to the heterochromatin with some affinity to the Ag-staining. Phylogenetic analysis based on partial cytochrome b sequence revealed that L. mystacinus forms a basal branch, so that the presence of multiple NORs in pairs 4 and 8 in this species indicates an autapomorphy.

Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Ribosomal DNA ITS-1 intergenic spacer polymorphism in triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.

rDNA-based characterization of a new binucleate Rhizoctonia spp. causing root rot on kale in Brazil

In this paper we present the first report of the occurrence of a binucleate Rhizoctonia spp. causing hypocotyl and root rot in kale in Brazil. Rhizoctonia spp. were isolated from kale (Brassica oleracea var. acephala) with symptoms of hypocotyl and root rot. The isolates, characterized as binucleate Rhizoctonia spp., did not show an anastomosis reaction with any of the binucleate Rhizoctonia spp. testers used. The pathogenicity of the isolates was tested under greenhouse conditions; all isolates were pathogenic and showed different symptom severities on kale. The ITS-5.8S rDNA sequences of kale isolates and 50 testers (25 binucleate Rhizoctonia spp. and 25 Rhizoctonia solani) were compared in order to characterize the genetic identity of Rhizoctonia spp. infecting kale. The kale isolates showed genetic identities ranging from 99.3 to 99.8% and were phylogenetically closely related to CAG 7 (AF354084), with identities of 98.5 and 98.7%. It is suggested that the binucleate Rhizoctonia spp. causing hypocotyl and root rot on kale Brazil comprises a new AG not yet described.

Análise filogenética de linhagens de Xylella fastidiosa de diferentes hospedeiros baseada na sequencia da região intergênica 16S-23S rDNA

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC