The exploration of Brucella transcriptome: From the ORFeome to RNAseq

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