A rapid method for RNA preparation from Gram-positive bacteria

Oh, Eun Taex; So, Jae-Seong

doi:10.1016/s0167-7012(02)00218-x

Cited by 37 publications

(21 citation statements)

References 5 publications

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“…RNA extraction was performed as described in ref. 8. DNA was removed by treatment with 10 units of FPLCpure DNase I (Amersham Pharmacia) at 37°C for 1 h in 40 mM Tris⅐HCl͞6 mM MgCl 2 , pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains

Pichon

Felden

2005

Proc. Natl. Acad. Sci. U.S.A.

239

271

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Small RNA (sRNA) genes are expressed in all organisms, primarily as regulators of translation and message stability. We have developed comparative genomic approaches to identify sRNAs that are expressed by Staphylococcus aureus , the most common cause of hospital-acquired infections. This study represents an in-depth analysis of the RNome of a Gram-positive bacterium. A set of sRNAs candidates were identified in silico within intergenic regions, and their expression levels were monitored by using microarrays and confirmed by Northern blot hybridizations. Two sRNAs were also detected directly from purification and RNA sequence determination. In total, at least 12 sRNAs are expressed from the S. aureus genome, five from the core genome and seven from pathogenicity islands that confer virulence and antibiotic resistance. Three sRNAs are present in multiple (two to five) copies. For the sRNAs that are conserved throughout the bacterial phylogeny, their secondary structures were inferred by phylogenetic comparative methods. In vitro binding assays indicate that one sRNA encoded within a pathogenicity island is a trans-encoded antisense RNA regulating the expression of target genes at the posttranscriptional level. Some of these RNAs show large variations of expression among pathogenic strains, suggesting that they are involved in the regulation of staphylococcal virulence.

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Section: Methodsmentioning

confidence: 99%

Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains

Pichon

Felden

2005

Proc. Natl. Acad. Sci. U.S.A.

239

271

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“…The S. coelicolor M145 and W10 strains were grown on cellophane sheets placed on plates of ONA medium containing 200 mM 2,29-dipyridyl. After 48 h growth at 30 uC, the mycelium was collected and RNA was extracted using the method described by Oh & So (2003). The purified RNA samples were treated with DNase (Ambion) followed by extraction twice with phenol and once with chloroform.…”

Section: Methodsmentioning

confidence: 99%

MbtH-like protein-mediated cross-talk between non-ribosomal peptide antibiotic and siderophore biosynthetic pathways in Streptomyces coelicolor M145

et al. 2007

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“…Overnight staphylococcal cultures in BHI medium were diluted 1000-fold in the same medium and grown at 37°C. Total RNAs were extracted at the indicated optical density as previously described (Oh and So 2003;Bohn et al 2007). Northern blot experiments were performed on samples separated by denaturing polyacrylamide gel electrophoresis.…”

Section: Experimental Validationmentioning

confidence: 99%

Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles

Marchais¹,

Naville²,

Bohn³

et al. 2009

Genome Res.

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Identification and characterization of functional elements in the noncoding regions of genomes is an elusive and timeconsuming activity whose output does not keep up with the pace of genome sequencing. Hundreds of bacterial genomes lay unexploited in terms of noncoding sequence analysis, although they may conceal a wide diversity of novel RNA genes, riboswitches, or other regulatory elements. We describe a strategy that exploits the entirety of available bacterial genomes to classify all noncoding elements of a selected reference species in a single pass. This method clusters noncoding elements based on their profile of presence among species. Most noncoding RNAs (ncRNAs) display specific signatures that enable their grouping in distinct clusters, away from sequence conservation noise and other elements such as promoters. We submitted 24 ncRNA candidates from Staphylococcus aureus to experimental validation and confirmed the presence of seven novel small RNAs or riboswitches. Besides offering a powerful method for de novo ncRNA identification, the analysis of phylogenetic profiles opens a new path toward the identification of functional relationships between co-evolving coding and noncoding elements.[Supplemental material is available online at www.genome.org.]In all living organisms, the genome regions located between protein-coding sequences are home to a wide diversity of functional elements that include noncoding RNA (ncRNA) genes, DNA regulatory elements, untranslated regions (UTRs) of genes, transposable and self-replicating elements, and a variety of other transcribed or nontranscribed functional sequences. As these elements are often key players in gene regulation and thus in the global cell interaction network, their systematic identification and characterization has become a major challenge in biology.Computational protocols developed to collect and characterize noncoding elements in genomic sequences rely, to a large extent, on comparative genomics. The most common strategies involve, first, collecting sequences under selective pressure and, second, analyzing the aligned sequences using various classifiers that exploit criteria such as nucleotide composition, folding potential, fold conservation, or covariation between distant positions (Rivas and Eddy 2001;Washietl et al. 2005;Pedersen et al. 2006;Torarinsson et al. 2006). In general, such classifiers are designed to detect structured RNAs among noncoding elements with no further distinction between regulatory elements, repeats, or artifacts produced by sequence comparison algorithms.Comparative genomics entails a significant amount of expert intervention, especially in obtaining the right genome set to optimize the specificity and sensitivity of RNA detection. Although a number of studies have successfully identified ncRNAs in several animal (Missal et al. 2005;Washietl et al. 2007) and microbial genomes (Altuvia 2007), the pace at which such studies are performed and published lags far behind the rate of genome sequence output. Most of the complete...

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A rapid method for RNA preparation from Gram-positive bacteria

Cited by 37 publications

References 5 publications

Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains

Small RNA genes expressed from Staphylococcus aureus genomic and pathogenicity islands with specific expression among pathogenic strains

MbtH-like protein-mediated cross-talk between non-ribosomal peptide antibiotic and siderophore biosynthetic pathways in Streptomyces coelicolor M145

Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles

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