Genetic diversity in wild (Sus scrofa scrofa) and domestic (Sus scrofa domestica) pigs and their hybrids based on polymorphism of a fragment of the D-loop region in the mitochondrial DNA

We examined the variation in mitochondrial DNA by sequencing the D-loop region in wild and domestic (large-white breed) pigs, in hybrids between domestic and wild pigs, and in Monteiro pigs. A D-loop fragment of approximately 330 bp was amplified by PCR. Sequencing of DNA amplicons identified haplotypes previously described as European and Asian types. Monteiro pigs and wild pigs had European haplotypes and domestic pigs had both European and Asian haplotypes. ©FUNPEC-RP.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background. The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results. In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN ox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion. This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. © 2008 Ely et al; licensee BioMed Central Ltd.

Human interferon B1 ser17: Coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein

A protocol to produce large amounts of bioactive homogeneous human interferon β1 expressed in Escherichia coli was developed. Human interferon β1 ser17 gene was constructed, cloned and subcloned, and the recombinant protein expressed in E. coli cells. Solubilization of recombinant human interferon β1 ser17 (rhIFN-β1 ser17) was accomplished by employing a brief shift to high alkaline pH in the presence of non-ionic detergent. The recombinant protein was purifi ed by three chromatographic steps. N-terminal amino acid sequencing and mass spectrometry analysis provided experimental evidence for the identity of the recombinant protein. Reverse phase liquid chromatography demonstrated that the content of deamidates and sulphoxides was similar to a commercial standard. Size exclusion chromatography demonstrated the absence of high molecular mass aggregates and dimers. The protocol represents an effi cient and high-yield method to obtain bioactive homogeneous monomeric rhIFN-β1 ser17 protein. It may thus represent an important step towards scaling up for rhIFN-β1 ser17 large-scale production. © 2010 Villela AD, et al.

Implementation of DNA markers to produce genomically - Enhanced EPDs in nellore cattle

Background: The sequencing and publication of the cattle genome and the identification of single nucleotide polymorphism (SNP) molecular markers have provided new tools for animal genetic evaluation and genomic-enhanced selection. These new tools aim to increase the accuracy and scope of selection while decreasing generation interval. The objective of this study was to evaluate the enhancement of accuracy caused by the use of genomic information (Clarifide® - Pfizer) on genetic evaluation of Brazilian Nellore cattle. Review: The application of genome-wide association studies (GWAS) is recognized as one of the most practical approaches to modern genetic improvement. Genomic selection is perhaps most suited to the improvement of traits with low heritability in zebu cattle. The primary interest in livestock genomics has been to estimate the effects of all the markers on the chip, conduct cross-validation to determine accuracy, and apply the resulting information in GWAS either alone [9] or in combination with bull test and pedigree-based genetic evaluation data. The cost of SNP50K genotyping however limits the commercial application of GWAS based on all the SNPs on the chip. However, reasonable predictability and accuracy can be achieved in GWAS by using an assay that contains an optimally selected predictive subset of markers, as opposed to all the SNPs on the chip. The best way to integrate genomic information into genetic improvement programs is to have it included in traditional genetic evaluations. This approach combines traditional expected progeny differences based on phenotype and pedigree with the genomic breeding values based on the markers. Including the different sources of information into a multiple trait genetic evaluation model, for within breed dairy cattle selection, is working with excellent results. However, given the wide genetic diversity of zebu breeds, the high-density panel used for genomic selection in dairy cattle (Ilumina Bovine SNP50 array) appears insufficient for across-breed genomic predictions and selection in beef cattle. Today there is only one breed-specific targeted SNP panel and genomic predictions developed using animals across the entire population of the Nellore breed (www.pfizersaudeanimal.com), which enables genomically - enhanced selection. Genomic profiles are a way to enhance our current selection tools to achieve more accurate predictions for younger animals. Material and Methods: We analyzed the age at first calving (AFC), accumulated productivity (ACP), stayability (STAY) and heifer pregnancy at 30 months (HP30) in Nellore cattle fitting two different animal models; 1) a traditional single trait model, and 2) a two-trait model where the genomic breeding value or molecular value prediction (MVP) was included as a correlated trait. All mixed model analyses were performed using the statistical software ASREML 3.0. Results: Genetic correlation estimates between AFC, ACP, STAY, HP30 and respective MVPs ranged from 0.29 to 0.46. Results also showed an increase of 56%, 36%, 62% and 19% in estimated accuracy of AFC, ACP, STAY and HP30 when MVP information was included in the animal model. Conclusion: Depending upon the trait, integration of MVP information into genetic evaluation resulted in increased accuracy of 19% to 62% as compared to accuracy from traditional genetic evaluation. GE-EPD will be an effective tool to enable faster genetic improvement through more dependable selection of young animals.

Estrutura populacional de Lutzomya longipalpis através da amplificação e sequenciamento do segmento ribossomal 12S de DNA mitocondrial

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

Utlização do DNA mitocondrial no contexto forense brasileiro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Variabilidade genética de três colônias de Triatoma rubrovaria (Blanchard, 1843), (Hemiptera, Reduviidae), oriundas do estado do Rio Grande do Sul, avaliadas por meio do seqüenciamento de genes do DNA mitocondrial e ribossomal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise da variabilidade e estruturação genética do tubarão azul, Prionace glauca (Chondrichthyes, Carcharhinidae) no Oceano Atlântico Sul Ocidental utilizando marcador molecular do DNA mitocondrial

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise da expressão gênica global da bactéria Xylella fastidiosa em laranja doce por microarranjos de DNA

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)

Chromosomal Mapping of Repetitive DNA and Cytochrome C Oxidase I Sequence Analysis Reveal Differentiation among Sympatric Samples of Astyanax fasciatus (Characiformes, Characidae)

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

Origin and distribution of AT-rich repetitive DNA families in Triatoma infestans (Heteroptera)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Characterization of immobilized biomass by amplified rDNA restriction analysis (ARDRA) in an anaerobic sequencing-batch biofilm reactor (ASBBR) for the treatment of industrial wastewater

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