Friday, March 30, 2012

Downstream CpG islands control transcription initiation and elongation of ncRNA

A recent study by Koerner et al. cites the use of Epicentre's most popular products: the Ribo-Zero™ Kit (Human/Mouse/Rat) to deplete rRNA, and the ScriptSeq™ Kit to prepare RNA-Seq libraries. The authors explored the presence of CpG islands at the 5' end of the Airn noncoding RNA (ncRNA). This region contains some unusual features, such as a GC-rich region just downstream from the promoter, and tandem direct repeats (TDR) in its second half.

The authors used homologous recombination to generate embryonic stem cells that carry deletions at the endogenous locus of the entire CpG island or only the TDRs. The TDRs were shown to play a small part in the Airn transcription elongation process, but a much larger part in methylation of the Airn promoter that comes from the maternal allele. Further, the CpG island is essential for transcription initiation of Airn, and in maintaining the unmethylated state of the Airn promoter. The research provides insight into how the imprinted Airn ncRNA CpG island (normally methylated on the maternal and unmethylated on the paternal chromosome) regulates its associated promoter, suggesting that a class of CpG islands can exhibit regulatory effects on upstream transcriptional elements.

ResearchBlogging.orgKoerner, M. et al. (2012). A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter PLoS Genetics, 8 (3) DOI: 10.1371/journal.pgen.1002540

Tuesday, March 20, 2012

Transposon mutagenesis enables determination of pathways involved in biofilm formation

Transposon mutagenesis is still considered to be an excellent tool for probing the genomics of bacterial species to create gene-silencing mutations. This approach can help elucidate the genetic markers responsible for various bioactivities.

A report by Garavaglia et al. explores the pathway of biosynthetic genes responsible for biofilm formation in the bacterium E. coli MG1655. Using the EZ-Tn5™ <R6Kγori/KAN-2> Transposome, insertional mutants were characterized that allowed the researchers to determine that blocking transcription of the csgDEFG operon in the uridine monophosphate (UMP) biosynthesis pathway inhibits biofilm formation. The transposon mutation was found to be in the carB gene, encoding a subunit of carbamoyl phosphate synthetase, which catalyzes the first step in the de novo pyrimidine nucleotide biosynthetic pathway.

A requirement of biofilm formation is the production of cellulose by E. coli, and this biosynthesis is modulated by the UMP pathway. Mutations in the carB gene also shut down cellulose production, as shown by lack of biofilm formation. Adding back exogenous uracil (which can be converted to UMP through the pyrimidine nucleotide salvage pathway) restored cellulose production and biofilm formation. Thus, the authors conclude that there exist tight links between pyrimidine metabolism and cellulose production/biofilm formation.

ResearchBlogging.orgGaravaglia, M. et al. (2012). The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031252

Friday, March 9, 2012

Epicentre posters at ABRF 2012

Epicentre will be presenting the following posters at the ABRF 2012 meeting in Orlando, Florida March 17-20:
  • #129: Eliminating Ultracentrifugation in the Deep Sequencing of Ribosome-Protected mRNA Fragments Using Polysomes and Monosomes
  • #131: The ScriptSeq™ v2 Library Preparation Method: Directional RNA-Seq Libraries in 4 Hours
  • #204: Superior rRNA Removal for RNA-Seq Library Preparation
  • #205: Enhanced Methods To Capture the Entire Small-RNA Transcriptome for RNA-Seq
We hope to see you there!

Tuesday, March 6, 2012

Sensitive detection of pathogens for biosecurity analysis

Göransson et al. report a new method of finding pathogens as part of a biosecurity study that is able to detect pathogens down to the single-molecule/organism level. The method combines a padlock probe approach, using Ampligase® Thermostable DNA Ligase, with rolling-circle amplification (RCA).

The authors used an environmental air sampling unit to trap particulate material on a membrane, followed by a rapid extraction of the DNA using magnetic beads. After clean-up, the DNA-containing solution was placed into an "on-bead" padlock probe/proximity ligation assay (PLA) catalyzed by Ampligase enzyme. Reacted probes were then subjected to two further rounds of RCA, first on beads and then in solution. Probes were then tagged with fluorescent dye and detected using an optical system with sensitivity down to 30 bacteria or 5 spores. The authors have improved the performance of the system by reducing the time required for the RCA step and are working improve the sensitivity of the process.

The combination of improved RCA and sensitive detection represent a significant improvement over previous methods for pathogen detection. The method can also be adapted to detect proteins.

ResearchBlogging.orgGöransson, J. et al. (2012). Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031068