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Multiplex PCR Assay for Detection of Bacterial Pathogens Associated with Warm-Water Streptococcosis in Fish. A. I. Mata, 2004.A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae) . Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism . The sensitivity of the multiplex PCR using purified DNA was 25 pg for S . iniae, 12.5 pg for S . difficilis, 50 pg for S . parauberis, and 30 pg for L . garvieae . The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish . Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish . Inferring Genome Trees by Using a Filter To Eliminate Phylogenetically Discordant Sequences and a Distance Matrix Based on Mean Normalized BLASTP Scores. G. D. Paul Clarke, 2002.Darwin's paradigm holds that the diversity of present-day organisms has arisen via a process of genetic descent with modification, as on a bifurcating tree . Evidence is accumulating that genes are sometimes transferred not along lineages but rather across lineages . To the extent that this is so, Darwin's paradigm can apply only imperfectly to genomes, potentially complicating or perhaps undermining attempts to reconstruct historical relationships among genomes (i.e., a genome tree) . Whether most genes in a genome have arisen via treelike (vertical) descent or by lateral transfer across lineages can be tested if enough complete genome sequences are used . We define a phylogenetically discordant sequence (PDS) as an open reading frame (ORF) that exhibits patterns of similarity relationships statistically distinguishable from those of most other ORFs in the same genome . PDSs represent between 6.0 and 16.8% (mean, 10.8%) of the analyzable ORFs in the genomes of 28 bacteria, eight archaea, and one eukaryote (Saccharomyces cerevisiae) . In this study we developed and assessed a distance-based approach, based on mean pairwise sequence similarity, for generating genome trees . Exclusion of PDSs improved bootstrap support for basal nodes but altered few topological features, indicating that there is little systematic bias among PDSs . Many but not all features of the genome tree from which PDSs were excluded are consistent with the 16S rRNA tree . Transcriptional Analysis of the sfa Determinant Revealing Multiple mRNA Processing Events in the Biogenesis of S Fimbriae in Pathogenic Escherichia coli. Carlos Balsalobre, 2003.Among the virulence factors present in pathogenic extraintestinal Escherichia coli strains, expression of fimbrial adhesins is necessary for attachment to the host tissues and subsequent colonization . Occurrence of the sfa determinant coding for the S fimbriae is widespread among the uropathogens and meningitis isolates . The sfa operon consists of nine genes . In the biogenesis of S fimbriae, the proteins encoded by the sfa genes are presumably required in a specific stoichiometry . In the present work we studied how differential expression of the sfa operon genes occurs . Our findings indicate that a number of endoribonucleolytic cleavages occur in the mRNA from the sfa operon, and we detected the presence of different distinct transcriptional products, including sfaBA, sfaA, sfaADE, and sfaGSH . The sfaGSH transcript represents the three distal genes of the sfa operon, which code for the minor subunits of the S fimbriae . Analysis of the proteins in S fimbriae suggested that expression of the sfaGSH transcript provides equimolar amounts of the minor subunits . Furthermore, we showed that in the generation of the major sfaA transcript, the processing included RNase E endoribonuceolytic cleavage of the precursor sfaBA transcript . We suggest that posttranscriptional mRNA processing events result in differential gene expression important to achieve the stoichiometry necessary for fimbrial adhesin biogenesis . Enhanced Production of Insulin-Like Growth Factor I Fusion Protein in Escherichia coli by Coexpression of the Down-Regulated Genes Identified by Transcriptome Profiling. Jong Hyun Choi, 2003.The transcriptome profiles of recombinant Escherichia coli producing human insulin-like growth factor I fusion protein (IGF-If) during the high-cell-density fed-batch culture were analyzed using DNA microarrays . The expression levels of 529 genes were significantly altered after induction . About 200 genes were significantly down-regulated during the production of IGF-If after induction . Among these down-regulated genes, we rationally selected and coexpressed in E . coli producing IGF-If the prsA gene (encoding a phosphoribosyl pyrophosphate synthetase) and the glpF gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively . As a result, the production of IGF-If could be increased from 1.8 ± 0.13 (± standard deviation) to 4.3 ± 0.24 g/liter . The volumetric productivity was also increased from 0.36 ± 0.027 to 0.82 ± 0.048 g/liter/h . These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance .
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