Identification of Biomphalaria havanensis and Biomphalaria obstructa populations from Cuba using polymerase chain reaction and restriction fragment length polymorphism of the ribosomal RNA intergenic spacer

In Cuba, several Biomphalaria species have been reported such as B. orbignyi, B. schrammi, B. helophila, B. havanensis and B. peregrina; only the latter three are considered as potential hosts of Schistosoma mansoni. The specific identification of Biomphalaria species is based on anatomical and morphological characters of genital organs and shells. The correct identification of these snails is complicated by the high variation in these characters, similarity among species and in some cases by the small size of the snails. In this paper, we reported the classical morphological identification, the use of PCR and RFLP analysis of the internal transcribed spacer region of the ribosomal RNA genes for molecular identification of seven snail populations from different localities in Cuba. Using morphological and molecular analysis, we showed that among the studied Cuban Biomphalaria populations only B. havanensis and B. obstructa species were found.

A new mtDNA COI gene lineage closely related to Anopheles janconnae of the Albitarsis complex in the Caribbean region of Colombia

An understanding of the taxonomic status and vector distribution of anophelines is crucial in controlling malaria. Previous phylogenetic analyses have supported the description of six species of the Neotropical malaria vector Anopheles (Nyssorhynchus) albitarsis s.l. (Diptera: Culicidae): An. albitarsis, Anopheles deaneorum, Anopheles marajoara, Anopheles oryzalimnetes, Anopheles janconnae and An. albitarsis F. To evaluate the taxonomic status of An. albitarsis s.l. mosquitoes collected in various localities in the Colombian Caribbean region, specimens were analyzed using the complete mitochondrial DNA cytochrome oxidase I (COI) gene, the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) region and partial nuclear DNA white gene sequences. Phylogenetic analyses of the COI gene sequences detected a new lineage closely related to An. janconnae in the Caribbean region of Colombia and determined its position relative to the other members of the complex. However, the ITS2 and white gene sequences lacked sufficient resolution to support a new lineage closely related to An. janconnae or the An. janconnae clade. The possible involvement of this new lineage in malaria transmission in Colombia remains unknown, but its phylogenetic closeness to An. janconnae, which has been implicated in local malaria transmission in Brazil, is intriguing.

Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species

As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequencing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as targets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, genotypic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirmation of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species.

Characterization of a new clinical yeast species, Candida tunisiensis sp. nov., isolated from a strain collection from Tunisian hospitals.

From a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and metabolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylogenic analyses suggested that this isolate clustered at the base of the Dipodascaceae (Saccharomycetales, Saccharomycetes, and Ascomycota). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohyphal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromosomes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC = 8 μg/ml), voriconazole (MIC = 0.5 μg/ml), itraconazole (MIC = 16 μg/ml), and amphotericin B (MIC = 4 μg/ml) but still susceptible to posaconazole (MIC = 0.008 μg/ml) and caspofungin (MIC = 0.5 μg/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classified based on phylogenetic approaches.

Can eukaryotic cells monitor the presence of unreplicated DNA?

Completion of DNA replication before mitosis is essential for genome stability and cell viability. Cellular controls called checkpoints act as surveillance mechanisms capable of detecting errors and blocking cell cycle progression to allow time for those errors to be corrected. An important question in the cell cycle field is whether eukaryotic cells possess mechanisms that monitor ongoing DNA replication and make sure that all chromosomes are fully replicated before entering mitosis, that is whether a replication-completion checkpoint exists. From recent studies with smc5–smc6 mutants it appears that yeast cells can enter anaphase without noticing that replication in the ribosomal DNA array was unfinished. smc5–smc6 mutants are proficient in all known cellular checkpoints, namely the S phase checkpoint, DNA-damage checkpoint, and spindle checkpoint, thus suggesting that none of these checkpoints can monitor the presence of unreplicated segments or the unhindered progression of forks in rDNA. Therefore, these results strongly suggest that normal yeast cells do not contain a DNA replication-completion checkpoint.

Análise de restrição de DNA ribossomal amplificado (ARDRA) pode diferenciar Fusarium solani f. sp. phaseoli de F. solani f. sp. glycines

Métodos moleculares têm sido utilizados para caracterizar a diversidade entre isolados de Fusarium spp. patogênicos e não patogênicos a uma cultura e, para determinar relações genéticas entre formae speciales. Testes de patogenicidade realizados em soja (Glycine max) e feijoeiro (Phaseolus vulgaris) com 17 isolados de Fusarium solani não demonstraram especificidade de hospedeiros. Utilizou-se a técnica ARDRA (Amplified Ribosomal DNA Restriction Analysis) para analisar a região ITS1 - 5,8S rDNA - ITS2, amplificada com os primers ITS5 e ITS4. Os produtos amplificados foram digeridos com as enzimas de restrição Hae III e Msp I. Os padrões de bandas gerados pela digestão com a enzima Hae III permitiram diferenciar três grupos entre os isolados de F. solani, sendo um grupo específico para isolados de F. solani f. sp. phaseoli com 100% de similaridade entre os 11 isolados. Entre os isolados de F. solani f. sp glycines foram observados dois padrões distintos de restrição. A técnica de ARDRA utilizando a enzima Hae III apresenta, portanto, potencial para utilização como um marcador para diferenciação entre as formae specialesphaseoli e glycines, dentro do complexo F. solani.

Molecular systematics of Thorea (Rhodophyta, Thoreales) species in Brazil

This study aimed to evaluate species level taxonomy and phylogenetic relationship among Thorea species in Brazil and other regions of the world using two molecular markers - RUBISCO large subunit plastid gene (rbcL) and nuclear small-subunit ribosomal DNA (SSU rDNA). Three samples of Thorea from Brazil (states of Mato Grosso do Sul and São Paulo) and one sample from Dominican Republic (DR) were sequenced. Analyses based on partial sequences of rbcL (1,282 bp) and complete sequences of SSU (1,752 bp) were essentially congruent and revealed that Thoreales formed a distinct monophyletic clade, which had two major branches with high support, representing the genera Thorea and Nemalionopsis. Thorea clade had four main branches with high support for all analyses, each one representing the species: 1) T. gaudichaudii C. Agardh from Asia (Japan and Philippines) - this clade occurred only in the rbcL analyses; 2) T. violacea Bory from Asia (Japan) and North America (U.S.A. and DR); 3) T. hispida (Thore) Desvaux from Europe (England) and Asia (Japan); 4) a distinct group with the three Brazilian samples (sequence identity: rbcL 97.2%, 1,246 bp; SSU 96.0-98.1%, 1,699-1,720 bp). The Brazilian samples clearly formed a monophyletic clade based on both molecular markers and was interpreted as a separate species, for which we resurrected the name T. bachmannii Pujals. Morphological and molecular evidences indicate that the Thoreales is well-resolved at ordinal and generic levels. In contrast, Thorea species recognized by molecular data require additional characters (e.g. reproductive and chromosome numbers) to allow consistent and reliable taxonomic circumscription aiming at a world revision based on molecular and morphological evidences.

Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo

It is generally accepted that mitochondria are able to proliferate even in postmitotic cells due to their natural turnover and also to satisfy increased cell energy requirements. However, no detailed studies are available, particularly with respect to specific cell types. Since [3H]-thymidine is incorporated not only into nuclear (n) DNA but also into the DNA of cytoplasmic mitochondria, an autoradiographic approach was developed at the light microscopy level in order to study basic questions of mitochondrial (mt) proliferation in organs of rodents in situ via the cytoplasmic incorporation of [3H]-thymidine injected into the animals 1 h before sacrifice. Experiments carried out on mice after X-irradiation showed that cytoplasmic labeling was not due to a process such as unscheduled nuclear DNA synthesis (nUDS). Furthermore, half-lives of mitochondria between 8-23 days were deduced specifically in relation to cell types. The phase of mtDNA synthesis was about 75 min. Finally, mt proliferation was measured in brain cells of mice as a function of age. While all neurons showed a decreasing extent of mtDNA synthesis during old age, nUDS decreased only in distinct cell types of the cortex and hippocampus. We conclude that the leading theories explaining the phenomenon of aging are closely related, i.e., aging is due to a decreasing capacity of nDNA repair, which leads to unrepaired nDNA damage, or to an accumulation of mitochondria with damaged mtDNA, which leads to a deficit of cellular energy production

The nucleolus: a paradigm for cell proliferation and aging

The nucleolus is the cellular site of ribosome biosynthesis. At this site, active ribosomal DNA (rDNA) genes are rapidly transcribed by RNA polymerase I (pol I) molecules. Recent advances in our understanding of the pol I transcription system have indicated that regulation of ribosomal RNA (rRNA) synthesis is a critical factor in cell growth. Importantly, the same signaling networks that control cell growth and proliferation and are deregulated in cancer appear to control pol I transcription. Therefore, the study of the biochemical basis for growth regulation of pol I transcription can provide basic information about the nuclear signaling network. Hopefully, this information may facilitate the search for drugs that can inhibit the growth of tumor cells by blocking pol I activation. In addition to its function in ribosome biogenesis, recent studies have revealed the prominent role of the nucleolus in cell senescence. These findings have stimulated a new wave of research on the functional relationship between the nucleolus and aging. The aim of this review is to provide an overview of some current topics in the area of nucleolus biology, and it has been written for a general readership.

Phylogeny, biogeography, and ecology of Ficus section Malvanthera (Moraceae)

We conducted the first molecular phylogenetic study of Ficus section Malvanthera (Moraceae; subgenus Urostigma) based on 32 Malvanthera accessions and seven outgroups representing other sections of Ficus subgenus Urostigma. We used DNA sequences from the nuclear ribosomal internal and external transcribed spacers (ITS and ETS), and the glyceraldehyde-3-phosphate dehydrogenase (G3pdh) region. Phylogenetic analysis using maximum parsimony, maximum likelihood and Bayesian methods recovered a monophyletic section Malvanthera to the exclusion of the rubber fig, Ficus elastica. The results of the phylogenetic analyses do not conform to any previously proposed taxonomic subdivision of the section and characters used for previous classification are homoplasious. Geographic distribution, however, is highly conserved and Melanesian Malvanthera are monophyletic. A new subdivision of section Malvanthera reflecting phylogenetic relationships is presented. Section Malvanthera likely diversified during a period of isolation in Australia and subsequently colonized New Guinea. Two Australian series are consistent with a pattern of dispersal out of rainforest habitat into drier habitats accompanied by a reduction in plant height during the transition from hemi-epiphytic trees to lithophytic trees and shrubs. In contradiction with a previous study of Pleistodontes phylogeny suggesting multiple changes in pollination behaviour, reconstruction of changes in pollination behaviour on Malvanthera, suggests only one or a few gains of active pollination within the section. (C) 2008 Elsevier Inc. All rights reserved.

Diversification of a large genus in a continental biodiversity hotspot: temporal and spatial origin of Muraltia (Polygalaceae) in the Cape of South Africa

Phylogenetic relationships in the largely South African genus Muraltia (Polygalaceae) are assessed based on DNA sequence data (nuclear ribosomal ITS, plastid atpB-rbcL spacer, trnL intron, and trnL-F spacer) for 73 of the 117 currently recognized species in the genus. The previously recognised subgenus Muraltia is monophyletic, but the South African endemic genus Nylandtia is embedded in Muraltia subgenus Psiloclada. Subgenus Muraltia is found to be sister to subgenus Psiloclada. Estimates show the beginning of diversification of the two subgenera in the early Miocene (Psiloclada, 19.3+/-3.4 Ma; Muraltia, 21.0+/-3.5 Ma) pre-dating the establishment of the Benguela current (intermittent in the middle to late Oligocene and markedly intensifying in the late Miocene), and summer-dry climate in the Cape region. However, the later increase in species numbers is contemporaneous with these climatic phenomena. Results of dispersal-vicariance analyses indicate that major clades in Muraltia diversified from the southwestern and northwestern Cape, where most of the species are found today.

Evolution of micromorphological and chemical characters in the lichen-forming fungal family Pertusariaceae

Micromorphological characters of the fruiting bodies, such as ascus-type and hymenial amyloidity, and secondary chemistry have been widely employed as key characters in Ascomycota classification. However, the evolution of these characters has yet not been studied using molecular phylogenies. We have used a combined Bayesian and maximum likelihood based approach to trace character evolution on a tree inferred from a combined analysis of nuclear and mitochondrial ribosomal DNA sequences. The maximum likelihood aspect overcomes simplifications inherent in maximum parsimony methods, whereas the Markov chain Monte Carlo aspect renders results independent of any particular phylogenetic tree. The results indicate that the evolution of the two chemical characters is quite different, being stable once developed for the medullary lecanoric acid, whereas the cortical chlorinated xanthones appear to have been lost several times. The current ascus-types and the amyloidity of the hymenial gel in Pertusariaceae appear to have been developed within the family. The basal ascus-type of pertusarialean fungi remains unknown. (c) 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 615-626.

Analysis of DNA polymorphism in ancient barley herbarium material: validation of the KASP SNP genotyping platform

The availability of crop specimens archived in herbaria and old seed collections represent valuable resources for the analysis of plant genetic diversity and crop domestication. The ability to extract ancient DNA (aDNA) from such samples has recently allowed molecular genetic investigations to be undertaken in ancient materials. While analyses of aDNA initially focused on the use of markers which occur in multiple copies such as the internal transcribed spacer region (ITS) within ribosomal DNA and those requiring amplification of short DNA regions of variable length such as simple sequence repeats (SSRs), emphasis is now moving towards the genotyping of single nucleotide polymorphisms (SNPs), traditionally undertaken in aDNA by Sanger sequencing. Here, using a panel of barley aDNA samples previously surveyed by Sanger sequencing for putative causative SNPs within the flowering-time gene PPD-H1, we assess the utility of the Kompetitive Allele Specific PCR (KASP) genotyping platform for aDNA analysis. We find KASP to out-perform Sanger sequencing in the genotyping of aDNA samples (78% versus 61% success, respectively), as well as being robust to contamination. The small template size (≥46 bp) and one-step, closed-tube amplification/genotyping process make this platform ideally suited to the genotypic analysis of aDNA, a process which is often hampered by template DNA degradation and sample cross-contamination. Such attributes, as well as its flexibility of use and relatively low cost, make KASP particularly relevant to the genetic analysis of aDNA samples. Furthermore, KASP provides a common platform for the genotyping and analysis of corresponding SNPs in ancient, landrace and modern plant materials. The extended haplotype analysis of PPD-H1 undertaken here (allelic variation at which is thought to be important for the spread of domestication and local adaptation) provides further resolution to the previously identified geographic cline of flowering-time allele distribution, illustrating how KASP can be used to aid genetic analyses of aDNA from plant species. We further demonstrate the utility of KASP by genotyping ten additional genetic markers diagnostic for morphological traits in barley, shedding light on the phenotypic traits, alleles and allele combinations present in these unviable ancient specimens, as well as their geographic distributions.

Insight into Anopheles (Nyssorhynchus) (Diptera: Culicidae) Species from Brazil

Anopheles (Nyssorhynchus) benarrochi s.l., Anopheles (Nyssorhynchus) oswaldoi s.l., and Anopheles (Nyssorhynchus) konderi s.l. collected in Acrelandia, state of Acre, Brazil, were identified based on morphological characters of the male genitalia, fourth-instar larvae, and pupae. Morphological variation was observed in the male genitalia of these species in comparison with specimens from other localities in Brazil. DNA sequence from the nuclear ribosomal second internal transcribed spacer of individuals identified as An. benarrochi s.l. by using male genitalia characteristics showed that the various morphological forms are conspecific but are distinct from An. benarrochi B from Colombia. Anopheles konderi s.l. and An. oswaldoi s.l. both misidentified as An. oswaldoi s.s. (Peryassu) throughout Brazil, may actually comprise at least two undescribed species. Diagnostic morphological characteristics of the male genitalia are provided to distinguish Anopheles benarrochi s.l., Anopheles oswaldoi s.l., and Anopheles konderi s.l. from morphologically similar species. Incrimination of An. oswaldoi s.s. in malaria transmission in Brazil needs further investigation because other undescribed species from Acre may have been confounded with this taxon.

PHYLOGENETIC AFFINITIES OF "CHANTRANSIA" STAGES IN MEMBERS OF THE BATRACHOSPERMALES AND THOREALES (RHODOPHYTA)

This study evaluated the phylogenetic relationship among samples of ""Chantransia"" stage of the Batrachospermales and Thoreales from several regions of the world based on sequences of two genes-the plastid-encoded RUBISCO LSU gene (rbcL) and the nuclear SSU ribosomal DNA gene (SSU rDNA). All sequences of ""Chantransia macrospora"" were shown to belong to Batrachospermum macrosporum based on both molecular markers, confirming evidence from previous studies. In contrast, nine species are now associated with ""Chantransia pygmaea,"" including seven species of the Batrachospermales and two of the Thoreales. Therefore, the presence of ""C. macrospora"" in a stream can be considered reliable evidence that it belongs to B. macrosporum, whereas the occurrence of ""C. pygmaea"" does not allow the recognition of any particular species, since it is associated with at least nine species. Affinities of ""Chantransia"" stages to particular taxa were congruent for 70.5% of the samples comparing the rbcL and SSU analyses, which were associated with the same or closely related species for both markers. Sequence divergences have been reported in the ""Chantransia"" stage in comparison to the respective gametophyte, and this matter deserves further attention.