Initial description of the phylogeography, population structure and genetic diversity of Atlantic spotted dolphins from Brazil and the Caribbean, inferred from analyses of mitochondrial and nuclear DNA

We provide initial information regarding the population structure and genetic diversity of Stenella frontalis from the Caribbean and southeastern Brazil from analyses of mitochondrial control region sequences and sequences from the first intron of the α-lactalbumin gene. Comparisons with previously described S. frontalis sequences showed a high number of haplotypes shared between populations throughout their distribution range. High diversity was found for southeastern Brazil and Caribbean samples, and population structure analyses indicate significant differentiation among population units at the FST level, but not at the ΦST level. Significant differentiation at the FST level was found between the Caribbean population unit and all other populations units. These results suggest historical or present connectivity between the Azores and Madeira and the southeastern Brazil groups and population differentiation between the Caribbean and southeastern Brazil, supporting the notion of two separate stocks in the waters around the Atlantic coast of South America. © 2013 Elsevier Ltd.

Composition and interrelationships of a large Neotropical freshwater fish group, the subfamily Cheirodontinae (Characiformes: Characidae): A case study based on mitochondrial and nuclear DNA sequences

•Relationships of Cheirodontinae based on a broad taxonomic sample.•Results reject the monophyly of Cheirodontinae as previously conceived.•Exclusion of Amazonspinther and Spintherobolus from the subfamily Cheirodontinae.•The removal of Leptagoniates pi of the genus Leptagoniates and inclusion in Cheirodontinae.•Division of Cheirodontinae in three newly defined monophyletic tribes. Characidae is the most species-rich family of freshwater fishes in the order Characiformes, with more than 1000 valid species that correspond to approximately 55% of the order. Few hypotheses about the composition and internal relationships within this family are available and most fail to reach an agreement. Among Characidae, Cheirodontinae is an emblematic group that includes 18 genera (1 fossil) and approximately 60 described species distributed throughout the Neotropical region. The taxonomic and systematic history of Cheirodontinae is complex, and only two hypotheses about the internal relationships in this subfamily have been reported to date. In the present study, we test the composition and relationships of fishes assigned to Cheirodontinae based on a broad taxonomic sample that also includes some characid incertae sedis taxa that were previously considered to be part of Cheirodontinae. We present phylogenetic analyses of a large molecular dataset of mitochondrial and nuclear DNA sequences. Our results reject the monophyly of Cheirodontinae as previously conceived, as well as the tribes Cheirodontini and Compsurini, and the genera Cheirodon, Compsura, Leptagoniates, Macropsobrycon, Odontostilbe, and Serrapinnus. On the basis of these results we propose: (1) the exclusion of Amazonspinther and Spintherobolus from the subfamily Cheirodontinae since they are the sister-group of all remaining Characidae; (2) the removal of Macropsobrycon xinguensis of the genus Macropsobrycon; (3) the removal of Leptagoniates pi of the genus Leptagoniates; (4) the inclusion of Leptagoniates pi in the subfamily Cheirodontinae; (5) the removal of Cheirodon stenodon of the genus Cheirodon and its inclusion in the subfamily Cheirodontinae under a new genus name; (6) the need to revise the polyphyletic genera Compsura, Odontostilbe, and Serrapinnus; and (7) the division of Cheirodontinae in three newly defined monophyletic tribes: Cheirodontini, Compsurini, and Pseudocheirodontini. Our results suggest that our knowledge about the largest Neotropical fish family, Characidae, still is incipient. © 2013 Elsevier Inc..

Mitochondrial DNA-like sequence in the nuclear genome of Saguinus (Callitrichinae, Primates): transfer estimation

Seqüências tipo mitocondriais têm comumente sido encontradas no genoma nuclear de diversos organismos. Quando acidentalmente incluídas em estudos de seqüências mitocondriais, diversas conclusões errôneas podem ser obtidas. No entanto, estes pseudogenes nucleares tipo mitocondriais podem ser usados para a estimativa da taxa relativa de evolução de genes mitocondriais e também como grupo externo em análises filogenéticas. No presente trabalho, seqüências mitocondriais com características do tipo de pseudogene, tais como deleções e/ou inserções e códons de parada, foram encontradas em tamarins (Saguinus spp., Callitrichinae, Primates). A análise filogenética permitiu a estimativa do tempo da migração da seqüência mitocondrial para o genoma nuclear e algumas inferências filogenéticas. A escolha de um grupo externo não adequado (Aotus infulatus) não permitiu uma reconstrução filogenética confiável da subfamília Callitrichinae. A divergência bastante antiga de Cebidae (Callitrichinae, Aotinae e Cebinae) pode ter favorecido o aparecimento de homoplasias, obscurecendo a análise.

Análise das relações filogenéticas entre espécies da subfamília Bryconinae (Ostariophysi: Characiformes: Characidae) utiliando sequeências de DNA mitocrondrial e nuclear

Pós-graduação em Ciências Biológicas (Zoologia) - IBB

Análise das relações filogenéticas entre as subfamílias de characidae (Ostariophysi: Characoformes) utilizando sequências de DNA mitocondrial e nuclear

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise das relações filogenéticas entre os gêneros de Cheirodontinae (Ostariophysi: Characiformes: Charasidae) utilizando sequências de DNA mitocondrial e nuclear

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Oxidative stress and nuclear dna damage in hyperglycemic pregnancies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inference of Raja miraletus population structure using mitochondrial and nuclear DNA: comparing the resolution power of molecular markers in exploring species and population boundaries

This study focused on the role of oceanographic discontinuities and the presence of transitional areas in shaping the population structure and the phylogeography of the Raja miraletus species complex, coupled with the test of the effective occurrence of past speciation events. The comparisons between the Atlantic African and the North-Eastern Atlantic-Mediterranean geographic populations were unravelled using both Cytochrome Oxidase I and eight microsatellite loci. This approach guaranteed a robust dataset for the identification of a speciation event between the Atlantic African clade, corresponding to the ex Raja ocellifera nominal species, and the NE Atlantic-Mediterranean R. miraletus clade. As a matter of fact, the origin of the Atlantic Africa and the NE Atlantic-Mediterranean deep split dated about 11.74MYA and was likely due to the synergic influence currents and two upwelling areas crossing the Western African Waters. Within the Mediterranean Sea, particular attention was also paid to the transitional area represented by Adventura and Maltese Bank, that might have contributed in sustaining the connectivity of the Western and the Eastern Mediterranean geographical populations. Furthermore, the geology of the easternmost part of Sicily and the geo-morphological depression of the Calabrian Arc could have driven the differentiation of the Eastern Mediterranean Sea. Although bathymetric and oceanographic discontinuity could represent barriers to dispersal and migration between Eastern and Western Mediterranean samples, a clear and complete genetic separation among them was not detected. Results produced by this work identified a speciation event defining Raja ocellifera and R. miraletus as two different species, and describing the R. miraletus species complex as the most ancient cryptic speciation event in the family Rajidae, representing another example of how strictly connected the environment, the behavioural habits and the evolutionary and ecologic drivers are.

Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: A comparison of tricyclo-DNA with LNA

The nuclear antisense properties of a series of tricyclo(tc)-DNA oligonucleotide 9-15mers, targeted against the 3' and 5' splice sites of exon 4 of cyclophilin A (CyPA) pre-mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA-oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence and dose dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc-oligonucleotides was found to be superior to that of the LNA-oligonucleotides in all cases by a factor of at least 4-5. A tc-oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2‘M concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction of CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc-15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc- and LNA-oligonucleotides. The obtained results confirm the power of tricyclo-DNA for nuclear antisense applications. Morover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV-1.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

LOCALIZATION OF DNA REPAIR FUNCTIONS TO THE NUCLEAR MATRIX

The nucleus of a eukaryotic cell contains both structural and functional elements that contribute to the controlled operation of the cell. In this context, functional components refers to those nuclear constituents that perform metabolic activities such as DNA replication and RNA transcription. Structural nuclear components, designated nuclear matrix, organize the DNA into loops or domains and appear to provide a framework for nuclear DNA organization. However, the boundary between structural and functional components is not clear cut as evinced by reports of associations between metabolic functions and the nuclear matrix. The studies reported here attempt to determine the relationship of another nuclear function, DNA repair, to the nuclear matrix.^ One objective of these studies was to study the initiation of DNA repair by directly measuring the UV-incision activities in human cells and determine the influence of various extractable nuclear components on these activities. The assay for incision activities required the development of a nuclear isolation protocol that produced nuclei with intact DNA; the conformation of the nuclear DNA and its physical characteristics in response to denaturing conditions were determined.^ The nuclei produced with this protocol were then used as substrates for endogenous UV-specific nuclease activities. The isolated nuclei were shown to contain activities that cause breaks in nuclear DNA in response to UV-irradiation. These UV-responsive activities were tightly associated with nuclear components, being unextractable with salt concentration of up to 0.6 M.^ The tight association of the incision activities with salt-extracted nuclei suggested that other repair function might also be associated with salt-stable components of the nucleus. The site of unscheduled DNA synthesis (UDS) was determined in salt-extracted nuclei (nucleoids) using autoradiography and fluorescent microscopy. UDS was found to occur in association with the nuclear matrix following low-doses (2.55 J/M('2)) of ultraviolet light, but the association became looser after higher doses of ultraviolet light (10-30 J/m('2)). ^

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

An interaction between DNA ligase I and proliferating cell nuclear antigen: Implications for Okazaki fragment synthesis and joining

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.