High-field NMR spectroscopy has been used to study the complex formed by the tetrasaccharide sialyl Lewis X and its receptor, E-selectin. Transferred NOEs demonstrate a specific interaction between the protein and ligand and enable measurement of the dissociation constant for the complex to be between approximately 1.1 and 2.0 mM. Differences between Overhauser spectra for free and bound sialyl Lewis X highlight a conformational change upon binding. This can be pinpointed to a change in the torsion angle of the glycosidic link between the sialyl and galactosyl residues and used to select a likely "bound" conformation from four low-energy species. Docking the bound form of sialyl Lewis X onto a model of the lectin domain of E-selectin suggests that the conformational change upon binding results primarily from steric interactions.

A 7.3 kbp DNA fragment, encompassing the erythromycin (Em) resistance gene (ermE) and a portion of the gene cluster encoding the biosynthetic genes for erythromycin biosynthesis in Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has been cloned in Streptomyces lividans using the plasmid vector pIJ702, and its nucleotide sequence has been determined using a modified dideoxy chain-termination procedure. In particular, we have examined the region immediately 5' of the resistance determinant, where the tandem promoters for ermE overlap the promoters for a divergently transcribed coding sequence (ORF). Disruption of this ORF using an integrational pIJ702-based plasmid vector gave mutants which were specifically blocked in erythromycin biosynthesis, and which accumulated 3-O-alpha-L-mycarosylerythronolide B: this behaviour is identical to that of previously described eryC1 mutants. The eryC1-gene product, a protein of subunit Mr 39,200, is therefore involved either as a structural or as a regulatory gene in the formation of the deoxyamino-sugar desosamine or in its attachment to the macrolide ring.

Many of the effects of calcium ions in eukaryotic cells are mediated by calcium-binding regulatory proteins such as calmodulin, in which each calcium-binding site has a distinctive helix-loop-helix conformation termed the EF hand. Protein S from the spore coat of the Gram-negative bacterium Myxococcus xanthus has been shown to resemble calmodulin in its internally-duplicated structure and ability to bind calcium. However, it has a beta-sheet secondary structure rather than the helix-loop-helix arrangement of the eukaryotic proteins. We have determined the complete amino-acid sequence of a calcium-binding protein from the Gram-positive bacterium "Streptomyces erythraeus" by cloning and sequencing the corresponding gene. It contains four EF-hand motifs bearing remarkable sequence similarity to the calcium-binding sites in calmodulin. This implies that the EF-hand super-family may have evolved from ancient proteins present in prokaryotes.

No abstract

We have evaluated a multiplex STR system for routine forensic use, which co-amplifies six short tandem repeat (STR) loci; HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA), in conjunction with the X-Y homologous gene Amelogenin. Analysis of PCR products employs denaturing polyacrylamide gels coupled with fluorescent labelled primers and detection is undertaken on ABD 373A automated sequencers. The technique was shown to be robust and reproducible when samples were analysed under conditions consistent with those encountered in a forensic environment. The system was demonstrated to be human specific and is suitable for use with both aged and degraded material. Somatic stability was proven with a wide range of tissue types and we were able to detect mixtures at ratios between 1:10 and 10:1. During this study no incidence of sample mis-typing due to allelic or locus drop-out was observed. Furthermore, although additional artefact bands were occasionally encountered these did not interfere with the interpretation of results. The performance of the system with poor quality samples demonstrated its suitability as a powerful tool in forensic investigation.

No abstract

A heat-stable factor, required for de novo synthesis of fatty acids in the erythromycin-producing organism Streptomyces erythraeus, has been purified to homogeneity and identified as an acyl carrier protein (ACP). We conclude that, contrary to previous belief, fatty acid synthase in S. erythraeus more closely resembles the dissociable complex of E. coli than the tightly associated, multifunctional enzyme complex found in the related actinomycete Mycobacterium smegmatis.Fatty acid synthase; Acyl carrier protein; (Streptomycetes)

The emergence of variants of SARS-CoV-2 has created challenges for the testing infrastructure. Although large-scale genome sequencing of SARS-CoV-2 has facilitated hospital and public health responses, access to sequencing facilities globally is variable and turnaround times can be significant, so there is a requirement for rapid and cost-effective alternatives. Applying a polymerase chain reaction (PCR)-based single nucleotide polymorphism (SNP) approach enables rapid (<4 h) identification of SARS-CoV-2 lineages from nucleic acid extracts, through the presence or absence of a panel of defined of genomic polymorphisms. For example, the B.1.1.7 lineage (“UK”, “Alpha”, or “Kent” variant) is characterised by 23 mutations compared to the reference strain, and the most biologically significant of these are found in the S gene. We have developed a SARS-CoV-2 typing assay focused on five positions in the S gene (HV69/70, N501, K417, E484 and P681). This configuration can identify a range of variants, including all the “Variants of Concern” currently designated by national and international public health bodies. The panel has been evaluated using a range of clinical isolates and standardised control materials at four UK hospitals and shows excellent concordance with the known lineage information derived from full sequence analysis. The assay has a turnaround time of about three hours for a set of up to 24 samples and has been utilised to identify emerging variants in a clinical setting.