Sequencing and promoter analysis of the nifENXorf3orf5fdxAnifQ operon from Azospirillum brasilense Sp7

A 40-kb DNA region containing the major cluster of nif genes has been isolated from the Azospirillum brasilense Sp7 genome. In this region three nif operons have been identified: nifHDKorf1Y, nifENXorf3orf5fdxAnifQ and orf2nifUSVorf4. The operons containing nifENX and nifUSV genes are separated from the structural nifHDKorf1Y operon by about 5 kb and 10 kb, respectively. The present study shows the sequence analysis of the 6045-bp DNA region containing the nifENX genes. The deduced amino acid sequences from the open reading frames were compared to the nif gene products of other diazotrophic bacteria and indicate the presence of seven ORFs, all reading in the same direction as that of the nifHDKorf1Y operon. Consensus sigma54 and NifA-binding sites are present only in the promoter region upstream of the nifE gene. This promoter is activated by NifA protein and is approximately two-times less active than the nifH promoter, as indicated by the ß-galactosidase assays. This result suggests the differential expression of the nif genes and their respective products in Azospirillum.

Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8% identity for nucleotides and 96.8% identity for amino acids) and more similar to the BCoV-ENT strain (98.7% for nucleotides and 98.7% for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

Parâmetros de estrusão para produção de "snacks" de farinha de cará (Dioscorea alata)

Farinha de cará (Dioscorea alata) foi processada em extrusor mono-rosca, para a manufatura de "snacks". Foi empregada a Metodologia de Superfície de Resposta para avaliar os efeitos da umidade (16, 20 e 24%), temperatura (120, 150 e 180ºC) e diâmetro da matriz (3, 4 e 5mm) nas características dos extrusados. A temperatura foi a variável mais importante, influenciando todas as propriedades estudadas. O volume específico e o comprimento específico aumentaram quando a temperatura aumentou e a umidade e o diâmetro da matriz diminuíram, enquanto, ao mesmo tempo, fraturabilidade e dureza diminuíram. A expansão diminuiu, quando a temperatura aumentou de 120 para 180ºC. Foram selecionadas duas condições experimentais que possibilitavam obter "snacks" com propriedades desejáveis (alto volume específico e baixa dureza e fraturabilidade) e os produtos foram avaliados por equipes sensoriais não treinadas. Estas apontaram que o melhor produto foi aquele extrusado com um teor de umidade de 17%, a 170ºC, com uma matriz de 4mm de abertura. Este produto obteve um índice de aceitabilidade de 80%, quando avaliado por 140 consumidores.