Aplicação de redes neurais artificiais na classificação de padrões filogeográficos com base na variabilidade genômica do DNA mitocondrial

Historicamente, o processo de formação das populações da Amazônia, assim como de todo território brasileiro, envolveu três grupos étnicos principais: o ameríndio, o europeu e o africano. Como conseqüência, estas populações possuem em geral constituição miscigenada do ponto de vista social e biológico. Desde o final do século passado, estudos do DNA mitocondrial (mtDNA) tem sido desenvolvidos com o propósito de estimar a mistura interétnica presente nestas populações. Para isto, é de fundamental importância a classificação de uma determinada linhagem de mtDNA em um dos mais de 250 haplogrupos/subclados propostos na literatura. Com o objetivo de desenvolver um sistema automatizado, preciso e acurado de classificação de seqüências (linhagens) de mtDNA, o presente trabalhou lançou mão da técnica de Redes Neurais Artificiais (RNA’s) tendo como base os estudos de filogeografia. Para esta classificação, foram desenvolvidas quatro redes neurais artificiais diretas, com múltiplas camadas e algoritmo de aprendizagem de retropropagação. As entradas de cada rede equivalem às posições nucleotídicas polimórficas da região hipervariável do DNA mitocondrial, as quais retornam como saída a classificação específica de cada linhagem. Posterior ao treinamento, todas as redes apresentaram índices de acerto de 100%, demonstrando que a técnica de Rede Neural Artificial pode ser utilizada, com êxito, na classificação de padrões filogeográficos com base no DNA mitocondrial.

A test of the utility of DNA barcoding in the radiation of the freshwater stingray genus Potamotrygon (Potamotrygonidae, Myliobatiformes)

DNA barcoding is a recently proposed global standard in taxonomy based on DNA sequences. The two main goals of DNA barcoding methodology are assignment of specimens to a species and discovery of new species. There are two main underlying assumptions: i) reciprocal monophyly of species, and ii) intraspecific divergence is always less than interspecific divergence. Here we present a phylogenetic analysis of the family Potamotrygonidae based on mitochondrial cytochrome c oxidase I gene, sampling 10 out of the 18 to 20 valid species including two non-described species. Potamotrygonidae systematics is still not fully resolved with several still-to-be-described species while some other species are difficult to delimit due to overlap in morphological characters and because of sharing a complex color patterns. Our results suggest that the family passed through a process of rapid speciation and that the species Potamotrygon motoro, P. scobina, and P. orbignyi share haplotypes extensively. Our results suggest that systems of identification of specimens based on DNA sequences, together with morphological and/or ecological characters, can aid taxonomic studies, but delimitation of new species based on threshold values of genetic distances are overly simplistic and misleading.

Multiple mitochondrial genes of some sylvatic Brazilian Triatoma: Non-monophyly of the T. brasiliensis subcomplex and the need for a generic revision in the Triatomini

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

Análise de SNPs do DNA mitocondrial em indivíduos residentes no estado do Espírito Santo para aplicação na Identificação Humana

Pós-graduação em Biotecnologia - IQ

Evolution of the Metazoan Mitochondrial Replicase

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

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

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

DNA Barcoding em Utricularia (Lentibulariaceae)

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Estimating the number of whales entering trade using DNA profiling and capture-recapture analysis of market products

Surveys of commercial markets combined with molecular taxonomy (i.e. molecular monitoring) provide a means to detect products from illegal, unregulated and/or unreported (IUU) exploitation, including the sale of fisheries bycatch and wild meat (bushmeat). Capture-recapture analyses of market products using DNA profiling have the potential to estimate the total number of individuals entering the market. However, these analyses are not directly analogous to those of living individuals because a ‘market individual’ does not die suddenly but, instead, remains available for a time in decreasing quantities, rather like the exponential decay of a radioactive isotope. Here we use mitochondrial DNA (mtDNA) sequences and microsatellite genotypes to individually identify products from North Pacific minke whales (Balaenoptera acutorostrata ssp.) purchased in 12 surveys of markets in the Republic of (South) Korea from 1999 to 2003. By applying a novel capture-recapture model with a decay rate parameter to the 205 unique DNA profiles found among 289 products, we estimated that the total number of whales entering trade across the five-year survey period was 827 (SE, 164; CV, 0.20) and that the average ‘half-life’ of products from an individual whale on the market was 1.82 months (SE, 0.24; CV, 0.13). Our estimate of whales in trade (reflecting the true numbers killed) was significantly greater than the officially reported bycatch of 458 whales for this period. This unregulated exploitation has serious implications for the survival of this genetically distinct coastal population. Although our capture-recapture model was developed for specific application to the Korean whale-meat markets, the exponential decay function could be modified to improve the estimates of trade in other wildmeat or fisheries markets or abundance of living populations by noninvasive genotyping.

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae)

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus. (c) 2011 Elsevier GmbH. All rights reserved.

A molecular phylogeny of bivalve mollusks: ancient radiations and divergences as revealed by mitochondrial genes

The main scope of my PhD is the reconstruction of the large-scale bivalve phylogeny on the basis of four mitochondrial genes, with samples taken from all major groups of the class. To my knowledge, it is the first attempt of such a breadth in Bivalvia. I decided to focus on both ribosomal and protein coding DNA sequences (two ribosomal encoding genes -12s and 16s -, and two protein coding ones - cytochrome c oxidase I and cytochrome b), since either bibliography and my preliminary results confirmed the importance of combined gene signals in improving evolutionary pathways of the group. Moreover, I wanted to propose a methodological pipeline that proved to be useful to obtain robust results in bivalves phylogeny. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the importance of molding and implementing non-trivial evolutionary models. In the line of a more rigorous approach to data analysis, I also proposed a new method to assess taxon sampling, by developing Clarke and Warwick statistics: taxon sampling is a major concern in phylogenetic studies, and incomplete, biased, or improper taxon assemblies can lead to misleading results in reconstructing evolutionary trees. Theoretical methods are already available to optimize taxon choice in phylogenetic analyses, but most involve some knowledge about genetic relationships of the group of interest, or even a well-established phylogeny itself; these data are not always available in general phylogenetic applications. The method I proposed measures the "phylogenetic representativeness" of a given sample or set of samples and it is based entirely on the pre-existing available taxonomy of the ingroup, which is commonly known to investigators. Moreover, it also accounts for instability and discordance in taxonomies. A Python-based script suite, called PhyRe, has been developed to implement all analyses.

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.

The active digestion of uniparental chloroplast DNA in a single zygote of Chlamydomonas reinhardtii is revealed by using the optical tweezer

The non-Mendelian inheritance of organelle genes is a phenomenon common to almost all eukaryotes, and in the isogamous alga Chlamydomonas reinhardtii, chloroplast (cp) genes are transmitted from the mating type positive (mt+) parent. In this study, the preferential disappearance of the fluorescent cp nucleoids of the mating type negative (mt−) parent was observed in living young zygotes. To study the change in cpDNA molecules during the preferential disappearance, the cpDNA of mt+ or mt− origin was labeled separately with bacterial aadA gene sequences. Then, a single zygote with or without cp nucleoids was isolated under direct observation by using optical tweezers and investigated by nested PCR analysis of the aadA sequences. This demonstrated that cpDNA molecules are digested completely during the preferential disappearance of mt− cp nucleoids within 10 min, whereas mt+ cpDNA and mitochondrial DNA are protected from the digestion. These results indicate that the non-Mendelian transmission pattern of organelle genes is determined immediately after zygote formation.

Specific molecular recognition of mixed nucleic acid sequences: An aromatic dication that binds in the DNA minor groove as a dimer

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

Mitochondrial diversity and the origins of African and European cattle.

The nature of domestic cattle origins in Africa are unclear as archaeological data are relatively sparse. The earliest domesticates were humpless, or Bos taurus, in morphology and may have shared a common origin with the ancestors of European cattle in the Near East. Alternatively, local strains of the wild ox, the aurochs, may have been adopted by peoples in either continent either before or after cultural influence from the Levant. This study examines mitochondrial DNA displacement loop sequence variation in 90 extant bovines drawn from Africa, Europe, and India. Phylogeny estimation and analysis of molecular variance verify that sequences cluster significantly into continental groups. The Indian Bos indicus samples are most markedly distinct from the others, which is indicative of a B. taurus nature for both European and African ancestors. When a calibration of sequence divergence is performed using comparisons with bison sequences and an estimate of 1 Myr since the Bison/Bos Leptobos common ancestor, estimates of 117-275,000 B.P. and 22-26,000 B.P. are obtained for the separation between Indians and others and between African and European ancestors, respectively. As cattle domestication is thought to have occurred approximately 10,000 B.P., these estimates suggest the domestication of genetically discrete aurochsen strains as the origins of each continental population. Additionally, patterns of variation that are indicative of population expansions (probably associated with the domestication process) are discernible in Africa and Europe. Notably, the genetic signatures of these expansions are clearly younger than the corresponding signature of African/European divergence.

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.