Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Prospecção de genes com interesse biotecnológico

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

Random regression models using different functions to model test-day milk yield of Brazilian Holstein cows

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

Genetic parameters for first lactation test-day milk flow in Holstein cows

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Random regression models to estimate genetic parameters for test-day milk yield in Brazilian Murrah buffaloes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Random regression models to estimate test-day milk yield genetic parameters Holstein cows in Southeastern Brazil

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

Genetic parameters for milk yield analyzed by test-day models in Murrah buffaloes in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genetic parameter estimates for live weight and daily live weight gain obtained for Nellore bulls in a test station using different models

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

Genetic parameters for test-day yield of milk, fat and protein in buffaloes estimated by random regression models

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

Estimative of genetic parameters in progeny test of Pinus caribaea Morelet var. hondurensis Barret & Golfari by quantitative traits and microsatellite markers

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

Genetic Parameters For the Somatic Cells Count In the Milk of Buffaloes Using Ordinary Test Day Models

The buffaloes dairy milk production (BDMP) has increased in the last 20 years, mainly for the manufacturing of mozzarella cheese, which is recognized by its high nutritional quality. However, this quality can be affected by several factors i. e. high somatic cells count (SCC) provokes changes in the milk's constituents. As in bovine dairy milk, the SCC is used as diagnostic tool for milk quality; because it enables the diagnosis of sub-clinic mastitis and also allows the selection of individuals genetically resistant to that disease. Based on it, we collected information about SCC and BDMP along the lactation in Murrah breed buffaloes, during the period between 1997 and 2005. Curves were designed to estimate genetic parameters. These parameters were estimated by ordinary test-day models. There were observed variations in the estimated heritability for both characteristics the lowest score for somatic cells count (SSCC) was seen at first month (0.01) and the highest at sixth months (0.29 the genetic correlation between these traits varied from -1 at the 1 and 9(th) months to 0.31 and 0.30 in the2 and 4(th) month of lactation. Phenotypic correlations were all negative (-0.07 in the second month and up to -0.35 in the eighth month of lactation). These results showed that environmental factors are more important than genetics in explain SCC, for this reason, selection for genetic resistance to mastitis in buffalos based in SCC should not be done. In the other hand, negative phenotypic correlations demonstrated that as the SCC increased, the milk production decreased.

Genetic parameters estimates for milk, fat and protein yield analyzed by test day models for Murrah buffaloes in Brazil

The objectives of this study were to estimate genetic parameters for test-day milk, fat and protein yields, in Murrah buffaloes. In this study 4,757 complete lactations of Murrah buffaloes were analyzed. The (co) variance components were estimated by restricted maximum likelihood using MTDFREML software. The bi-trait animal test-day models included genetic additive direct and permanent environment effects, as random effects, and the fixed effects of contemporary group (herds-year-month of control) and age of the cow at calving as linear and quadratic covariable. The heritability estimate at first control was 0.19, increased until the third control (0.24), decreasing thereafter, reaching the lowest value at the ninth control (0.09). The highest heritability estimates for fat and protein yield were 0.23 (first control) and 0.33 (third control), respectively. For milk yield, genetic and phenotypic correlation estimates ranged from 0.37 to 0.99 and from 0.52 to 0.94, respectively. Genetic correlations were higher than phenotypic ones. For fat and protein yields, genetic correlation estimates ranged from 0.42 to 0.97.

Genetic Variation in an International Provenance-Progeny Test of Pinus caribaea Mor. var. bahamensis Bar. et Gol., in São Paulo, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genetic parameters for test-day milk yield, 305-day milk yield, and lactation length in Guzerat cows

Milk production in tropical environments requires the use of crossbreeding systems including breeds well adapted to harsh conditions, but with lower productivities when compared to specialized breeds. Besides the genetic improvement for milk production, lactation lengths also need to be studied for most of these breeds. Accordingly, genetic parameters were estimated for 305-day cumulative milk yield (MY305), test-day milk yield (TDMY), and lactation length (LL) using information from the first lactations of 2816 Guzerat cows selected for milk production in 28 herds in Brazil. Contemporary groups were defined as herd, year and season of the test for TDMY, and as herd, year and season of calving for MY305 and LL. Variance components were estimated with the restricted maximum likelihood method under a multi-trait animal model. Heritabilities estimated for TDMY ranged from 0.16 to 0.24, and were 0.24 and 0.12 for MY305 and LL, respectively. Genetic correlations were high and positive, ranging from 0.51 to 0.99 among TDMY records, from 0.81 to 0.98 between each TDMY and MY305, and from 0.71 to 0.94 between each TDMY and LL. Genetic parameters obtained in this study indicated the possibility of using test-day records for the prediction of breeding values for milk yield in this population of the Guzerat breed. The use of TDMY as selection criteria would result in indirect gains in MY305 and LL. However, the highest response to selection for MY305 would be obtained by direct selection for this trait. © 2012 Elsevier B.V.