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• W103471815 abstract "In this chapter we will analyse the results available on the characterization of the Brucella transcriptome. After a summary of earlier work on transcription, two technical approaches will be mainly described, on one side the use microarrays, specially that derived from the Brucella ORFeome that allows hybridization with mRNA derived cDNA to determine the relative abundance of transcripts from each B. melitensis ORF. On the other, RNAseq, consisting in the massive sequencing of cDNA libraries derived from mRNA obtained from B. abortus grown in culture medium. Sequencing with the Illumina Genome-Analyser II platform produced 3 millions of 35-nt-long reads that annealed with single copy coding regions of the genome. This allowed a good coverage for every CDS and produced a new data set on the transcription of Brucella. We obtained a good correlation for the set of highly expressed genes from the microarrays and confirmed the observations obtained on the asymmetry between chromosome transcription. Preliminary conclusions on intracellular transcription have been drawn from real-time polymerase chain reaction (RT-PCR) on selected candidate genes and from microarray data sets obtained from virulence related conditions. The RNAseq derived data allowed more versatile data mining, giving some new details on transcription from pseudogenes or intergenic regions. Introduction During the last 50 years, to understand the mechanisms working in bacterial cells, we have applied a reductionist approach consisting in the analysis of isolated parts of the organism at different complexity levels ranging from the genetics to the three-dimensional determination of complex molecular structures. This approach has been really fruitful and undoubtedly it will be our principal source of information in the near future. Newer approaches to the study of biological systems want to analyse living organisms as a complex unit by looking at all the components of the organism as well as the interactions among them that result in an observable behaviour at the same time. This methodology is the hallmark of the field of systems biology. These procedures are being applied already into microbiology. Perhaps, the most appealing case has been the analysis of Mycoplasma, the free-living bacteria with the smallest genome (Ochman and Raghavan, 2009). After a lot of genomic data on Mycoplasma were available, a multinational team has analysed in depth the transcriptome of the bacteria using both, RNA sequencing and tiling microarrays (Guell et al., 2009). They have also studied the proteome of the bacteria, including protein interactions (Kuhner et al., 2009) and finally they have reconstructed the metabolism by analysing any metabolic pathway integrating transcriptional, proteomic and metabolic flux data sets (Yus et al., 2009). This piece of work signals the path for the study of more complex organisms using the Garcia-Lobo et al. 90 | same methodologies. Perhaps, the most important conclusion to take into account is that some classic paradigms of molecular microbiology are changing. This is mainly the case for bacterial transcription. The analysis of the transcriptome of Mycoplasma has revealed extensive transcription from both DNA strands of the same DNA, alternative transcripts from operons including only some of the operon genes, and existence of many short RNA’s usually non-detected by automatic annotation pipelines. As a desirable objective we should consider performing a similar study on Brucella. Taking into account genome size only, we expect a much more complex problem to solve. While this effort is organized, the progressive application of high throughput technologies to Brucella may build a set of data that could be later used to sketch the blueprint of our beloved bacteria. Early transcriptional analysis Application of molecular analysis to genetic information flow in Brucella started near 30 years ago. To our knowledge the first paper published on this field (that somehow inspired work of our group) was a description of mutagenesis produced by transposon Tn5 in B. abortus published in 1987 (Smith and Heffron, 1987). The first nucleotide sequences of Brucella rRNA were deposited in GenBank in 1989. Focus on transcription started soon after, targeting first surface antigen genes with a clear interest in the improvement of brucellosis vaccines and diagnostic tests. For instance, the analysis of the 36-kDa outer membrane protein gene of B. abortus, included the detection of promoter activity by fusion to LacZ, identification of Shine-Dalgarno sequences, and rho-independent terminators (Ficht et al., 1989). Besides the transcriptional analysis of a few dozens of individual genes performed in the following years, a first experiment addressing global analysis of transcription was performed by R.C. Essemberg, who made a library of Sau3AI generated DNA fragments fused to a promoter-less luciferase reporter and studied transcription arising from the cloned fragments and the effect that different sugars produced on the transcription levels. In this way fragments containing promoters with activity dependent on erythritol, glucose, galactose and succinate were detected. This work was not published, but was communicated at the Chicago Brucellosis meeting and the sequence of the cloned fragments was deposited in GenBank (accession numbers AF075168, AF072121, AF072119, AF074323, AF073884, AF072580, AF072569, AF072120, AF072570, AF072571, AF072572, AF072573, AF072574, AF072575, AF072576, AF072577, AF072578, AF072579). Methodological overview of massive transcriptomic" @default.
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• W103471815 date "2012-01-01" @default.
• W103471815 modified "2023-09-22" @default.
• W103471815 title "The exploration of Brucella transcriptome: From the ORFeome to RNAseq" @default.
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