Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

Human pythiosis, Brazil

Pythiosis, caused by Pythium insidiosum, occurs in humans and animals and is acquired from aquatic environments that harbor the emerging pathogen. Diagnosis is difficult because clinical and histopathologic features are not pathognomonic. We report the first human case of pythiosis from Brazil, diagnosed by using culture and rDNA sequencing.

Reproductive ecology of the freshwater red alga Batrachospermum delicatulum (Batrachospermales, Rhodophyta) in three tropical streams

Batrachospermum delicatulum specimens from three stream segments were analyzed from a tropical region in south-eastern Brazil (20°18′- 20°49′S, 49°13′-49°46′W). Physical and chemical parameters and the spatial placement of thalli were investigated along with the reproductive characteristics of the gametophytic phase. Sequence data of the cox 2-3 spacer region was also utilized to evaluate genetic variation in individuals within and among stream segments. Gametophyte occurred under relatively diverse environmental conditions, whereas thalli abundance was weakly or not correlated to environmental variables within the stream segments. All specimens examined were dioecious. The ratio of male/female plants was relatively low (0.5 to 1.3) and male plants tended to occur as clumps (two or three plants together). High reproductive success was observed, as indicated by the occurrence of 100% fertilized (carposporophytic) female plants. This is similar to previous reports for this and other dioecious species, which is remarkable considering the relatively low proportion of male/female plants. Results support the two hypotheses to explain the high reproductive success in dioecious species. The occurrence of male plants in clumps was evidence for a strict spatial relationship (i.e. male plants located in upstream position of female plants in order to release spermatia, which would be carried by eddies through female plants). In contrast, the occurrence of male and female plants adjacent to each other allowed outcrossing among neighboring plants with intermingled male and female branches, which seemed more applicable to some situations (low turbulence habitats). The cox 2-3 spacer region from the 18 individuals sequenced was 376 bp and the DNA sequence was identical with no base pair substitutions. Likewise, a previous study of another Batrachospermum species showed that the same haplotypes were present in all stream segments from the same drainage basin, even though the stream segments were a considerable distance apart. Short distance dispersal either by small birds or waterway connectivity might explain these findings.

Systematic review of the frog family Hylidae, with special reference to Hylinae: Phylogenetic analysis and taxonomic revision

Hylidae is a large family of American, Australopapuan, and temperate Eurasian treefrogs of approximately 870 known species, divided among four subfamilies. Although some groups of Hylidae have been addressed phylogenetically, a comprehensive phylogenetic analysis has never been presented. The first goal of this paper is to review the current state of hylid systematics. We focus on the very large subfamily Hylinae (590 species), evaluate the monophyly of named taxa, and examine the evidential basis of the existing taxonomy. The second objective is to perform a phylogenetic analysis using mostly DNA sequence data in order to (1) test the monophyly of the Hylidae; (2) determine its constituent taxa, with special attention to the genera and species groups which form the subfamily Hylinae, and c) propose a new, monophyletic taxonomy consistent with the hypothesized relationships. We present a phylogenetic analysis of hylid frogs based on 276 terminals, including 228 hylids and 48 outgroup taxa. Included are exemplars of all but 1 of the 41 genera of Hylidae (of all four nominal subfamilies) and 39 of the 41 currently recognized species groups of the species-rich genus Hyla. The included taxa allowed us to test the monophyly of 24 of the 35 nonmonotypic genera and 25 species groups of Hyla. The phylogenetic analysis includes approximately 5100 base pairs from four mitochondrial (12S, tRNA valine, 16S, and cytochrome b) and five nuclear genes (rhodopsin, tyrosinase, RAG-1, seventh in absentia, and 28S), and a small data set from foot musculature. Concurring with previous studies, the present analysis indicates that Hemiphractinae are not related to the other three hylid subfamilies. It is therefore removed from the family and tentatively considered a subfamily of the paraphyletic Leptodactylidae. Hylidae is now restricted to Hylinae, Pelodryadinae, and Phyllomedusinae. Our results support a sister-group relationship between Pelodryadinae and Phyllomedusinae, which together form the sister taxon of Hylinae. Agalychnis, Phyllomedusa, Litoria, Hyla, Osteocephalus, Phrynohyas, Ptychohyla, Scinax, Smilisca, and Trachycephalus are not monophyletic. Within Hyla, the H. albomarginata, H. albopunctata, H. arborea, H. boons, H. cinerea, H. eximia, H. geographica, H. granosa, H. microcephala, H. miotympanum, H. tuberculosa, and H. versicolor groups are also demonstrably nonmonophyletic. Hylinae is composed of four major clades. The first of these includes the Andean stream-breeding Hyla, Aplastodiscus, all Gladiator Frogs, and a Tepuian clade. The second clade is composed of the 30-chromosome Hyla, Lysapsus, Pseudis, Scarthyla, Scinax (including the H. uruguaya group), Sphaenorhynchus, and Xenohyla. The third major clade is composed of Nyctimantis, Phrynohyas, Phyllodytes, and all South American/West Indian casque-headed frogs: Aparasphenodon, Argenteohyla, Corythomantis, Osteocephalus, Osteopilus, Tepuihyla, and Trachycephalus. The fourth major clade is composed of most of the Middle American/Holarctic species groups of Hyla and the genera Acris, Anotheca, Duellmanohyla, Plectrohyla, Pseudacris, Ptychohyla, Pternohyla, Smilisca, and Triprion. A new monophyletic taxonomy mirroring these results is presented where Hylinae is divided into four tribes. Of the species currently in Hyla, 297 of the 353 species are placed in 15 genera; of these, 4 are currently recognized, 4 are resurrected names, and 7 are new. Hyla is restricted to H. femoralis and the H. arborea, H. cinerea, H. eximia, and H. versicolor groups, whose contents are redefined. Phrynohyas is placed in the synonymy of Trachycephalus, and Pternohyla is placed in the synonymy of Smilisca. The genus Dendropsophus is resurrected to include all former species of Hyla known or suspected to have 30 chromosomes. Exerodonta is resurrected to include the former Hyla sumichrasti group and some members of the former H. miotympanum group. Hyloscirtus is resurrected for the former Hyla armata, H. bogotensis, and H. larinopygion groups. Hypsiboas is resurrected to include several species groups - many of them redefined here - of Gladiator Frogs. The former Hyla albofrenata and H. albosignata complexes of the H. albomarginata group are included in Aplastodiscus. New generic names are erected for (1) Agalychnis calcarifer and A. craspedopus; (2) Osteocephalus langsdorffii; the (3) Hyla aromatica, (4) H. bromeliacia, (5) H. godmani, (6) H. mixomaculata, (7) H. taeniopus, (8) and H. tuberculosa groups; (9) the clade composed of the H. pictipes and H. pseudopuma groups; and (10) a clade composed of the H. circumdata, H. claresignata, H. martinsi, and H. pseudopseudis groups. Copyright © American Museum of Natural History 2005.

Tuberculose e o estudo molecular da sua epidemiologia

Systems that can distinguish epidemiologically-related Mycobacterium tuberculosis strains from unrelated ones are extremely valuable. Molecular biology techniques have allowed a great deal of information to be acquired about the infectious disease tuberculosis (TB) that was very hard or impossible to obtain by conventional epidemiology. A typing method based on bacterial DNA genome differences, known as RFLP (restriction fragment length polymorphism), is widely used to discriminate strains in the epidemiologic study of TB. However, RFLP is laborious and there is a tendency to replace it by other methods. Thus, other DNA sequences have been employed as epidemiological markers, as in Spoligotyping, a fast technique based on PCR followed by differential hybridization of amplified products. The polymorphism observed among different isolates is probably the product of strain-dependent recombination. MIRU (mycobacterial interspersed repetitive unit) typing is a reproducible and fast assay, involving the generation of genotypes based on the study of 12 loci containing VNTRs (variable-number tandem repeats) in strains of the M. tuberculosis complex. It compares strains from different geographic areas and allows the movement of individual lineages to be tracked, as in RFLP. This approach enables a greater number of isolates to be analyzed, leading to the identification of a larger number of foci of transmission within the population and thus to improved ways of slowing the progress of the disease.

Evaluation of HPFH and δβ-thalassemia mutations in a Brazilian group with high Hb F levels.

Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (δβ-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in δβ-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish δβ-thalassemia and none were heterozygous for Sicilian δβ- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the δβ-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.

Illegal hunting cases detected with molecular forensics in Brazil

Background: Illegal hunting is one of the major threats to vertebrate populations in tropical regions. This unsustainable practice has serious consequences not only for the target populations, but also for the dynamics and structure of tropical ecosystems. Generally, in cases of suspected illegal hunting, the only evidence available is pieces of meat, skin or bone. In these cases, species identification can only be reliably determined using molecular technologies. Here, we reported an investigative study of three cases of suspected wildlife poaching in which molecular biology techniques were employed to identify the hunted species from remains of meat.Findings: By applying cytochrome b (cyt-b) and cytochrome oxidase subunit I (COI) molecular markers, the suspected illegal poaching was confirmed by the identification of three wild species, capybara (Hydrochoerus hydrochaeris), Chaco Chachalaca (Ortalis canicollis) and Pampas deer (Ozotoceros bezoarticus). In Brazil, hunting is a criminal offense, and based on this evidence, the defendants were found guilty and punished with fines; they may still be sentenced to prison for a period of 6 to 12 months.Conclusions: The genetic analysis used in this investigative study was suitable to diagnose the species killed and solve these criminal investigations. Molecular forensic techniques can therefore provide an important tool that enables local law enforcement agencies to apprehend illegal poachers. © 2012 Sanches et al.; licensee BioMed Central Ltd.

Filamentous fungi from the Atlantic marine sponge Dragmacidon reticulatum

Dragmacidon reticulatum is a marine sponge of wide occurrence in the Eastern and Western Atlantic. Little is known about D. reticulatum fungal diversity. Filamentous fungi recovered from D. reticulatum were assessed in the present study using a polyphasic taxonomic approach, including classical morphology, molecular biology and MALDI-TOF ICMS. Ninety-eight fungal strains were isolated from two D. reticulatum samples by using six different culture media, which were identified up to the genus level. Sixty-four distinct fungal ribotypes were obtained, distributed among twenty-four different genera belonging to the Ascomycota and Zygomycota. Representatives of Penicillium and Trichoderma were the most diverse and abundant fungi isolated. Amongst Penicillium spp. three isolates belonged to the same ribotype can be considered as a putative new species. Data derived from the present study highlight the importance of using a polyphasic approach to get an accurate identification in order to structure a reliable culture collection. © 2012 Springer-Verlag Berlin Heidelberg.

Allelic variants of XRCC1 and XRCC3 repair genes and susceptibility of oral cancer in Brazilian patients

Background: The capacity for DNA repair is essential in maintaining cellular functions and homeostasis; however, this capacity can be altered based on DNA sequence variations in DNA repair genes, which may contribute to the onset of cancer. Many single-nucleotide polymorphisms (SNPs) in repair genes have been found to be associated with oral cancer. The aim of this study was to investigate the relationship between the presence of allelic variants Arg194Trp (rs:1799782) and Arg399Gln (rs: 25487) of XRCC1 gene and Thr241Met (rs: 861539) of XRCC3 gene and susceptibility to oral cancer. We also attempted to correlate the frequencies obtained for each of the SNPs to histopathological parameters. Methods: A case-control study was conducted with genomic DNA from 150 patients with oral squamous cell carcinomas and 150 controls. SNPs were genotyped by RFLP-PCR. Results: The presence of the polymorphic variants of the XRCC1 gene within codon 194 (OR 0.82, 95% CI: 0.44-1.51) and codon 399 (OR 0.94, 95% CI: 0.59-1.50) and within the XRCC3 gene (OR 0.72; 95% CI: 0.45-1.16) were not associated with an increased risk of oral cancer. A combinational analysis of SNPs in both genes indicated no association. The presence of the allelic variants of these two genes had no statistically significant effect on tumor differentiation, lymph node invasion or tumor size. Conclusions: These results suggest that allelic variants of XRCC1 and XRCC3 are not suitable markers for susceptibility to carcinomas of the oral cavity and are also not related to the later stages of such tumors. © 2012 John Wiley & Sons A/S.

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd.

Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework

Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) [1] to be typed using SNaPShotTM (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) [1] was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons performed. The assay was validated with casework samples. Results show that the method is straightforward and can be used for exclusionary purposes, saving time and laboratory resources. The assay confirms the theoretic prediction suggested by Salas and Amigo (2010) [1]. All forensic advantages, such as high sensitivity and power of discrimination, as also the disadvantages, such as the occurrence of allele dropouts, are discussed throughout the article. © 2013 Elsevier B.V.

Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure. © 2013 Lima et al.

Description of larva of Amblyomma romitii (Acari: Ixodidae) by optical and scanning electron microscopy, including porotaxy and phylogenetic analysis

The description of the larva of Amblyomma romitii Tonelli-Rondelli is based on optical and scanning electron microscopy. Larvae were obtained under laboratory conditions from an engorged female collected on capybara from Rurópolis municipality, State of Pará, Northern Brazil. Several characters are presented including the chaetotaxy of idiosoma, palpi and Haller's organ. The larval porotaxy (topographical and numerical patterns of integumentary structures) was presented and compared to that of the other Amblyomma spp. larvae. The mitochondrial 16S rDNA partial sequence of A. romitii generated in the present study was aligned with sequences previously determined for other Amblyomma species available in Genbank and with some species presently sequenced. The larval morphology of A. romitii and other Neotropical Amblyomma spp. larvae is discussed as well as the DNA sequence and its phylogenetic position among other species of the genus. © 2012 Springer Science+Business Media Dordrecht.

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C0t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes. © 2013 Bueno et al.