Micrococcal nuclease does not substantially bias nucleosome mapping.

We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms.

Functional analysis of a mitochondrial phosphopantetheinyl transferase (PPTase) gene pptB in Aspergillus fumigatus.

The mitochondrial phosphopantetheinyl transferase gene pptB of the opportunistic pathogen Aspergillus fumigatus has been identified and characterised. Unlike pptA, which is required for lysine biosynthesis, secondary metabolism, and iron assimilation, pptB is essential for viability. PptB is located in the mitochondria.

Detection of single nucleotide polymorphisms using a DNA Holliday junction nanoswitch--a high-throughput fluorescence lifetime assay.

We report a simple DNA sensor device, using a combination of binding and conformational switching, capable of rapid detection of specific single nucleotide polymorphisms in an unlabelled nucleic acid target sequence. The detection is demonstrated using fluorescence lifetime measurements in a high-throughput micro plate reader instrument based on the time-correlated single-photon counting technique. The sensor design and instrumental architecture are capable of detecting perturbations in the molecular structure of the probe-target complex (which is similar to that of a Holliday junction), due to a single base pair mismatch in a synthetic target.

High-resolution mapping of sequence-directed nucleosome positioning on genomic DNA.

We have mapped in vitro nucleosome positioning on the sheep beta-lactoglobulin gene using high-throughput sequencing to characterise the DNA sequences recovered from reconstituted nucleosomes. This methodology surpasses previous approaches for coverage, accuracy and resolution and, most importantly, offers a simple yet rapid and relatively inexpensive method to characterise genomic DNA sequences in terms of nucleosome positioning capacity. We demonstrate an unambiguous correspondence between in vitro and in vivo nucleosome positioning around the promoter of the gene; identify discrete, sequence-specific nucleosomal structures above the level of the canonical core particle-a feature that has implications for regulatory protein access and higher-order chromatin packing; and reveal new insights into the involvement of periodically organised dinucleotide sequence motifs of the type GG and CC and not AA and TT, as determinants of nucleosome positioning-an observation that supports the idea that the core histone octamer can exploit different patterns of sequence organisation, or structural potential, in the DNA to bring about nucleosome positioning.

The npgA/ cfwA gene encodes a putative 4'-phosphopantetheinyl transferase which is essential for penicillin biosynthesis in Aspergillus nidulans.

Non-ribosomal peptide synthetases, polyketides and fatty acid synthetases have a modular organisation of multi-enzymatic activities. In all of them, the acyl or peptidyl carrier proteins have 4'-phosphopantetheine (P-pant) as an essential prosthetic group. This is added by 4'-phosphopantetheinyl transferases (PPTases) that derive the P-pant group from coenzyme A.