HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling

Type I restriction-modification (RM) systems are large, multifunctional enzymes composed of three different subunits. HsdS and HsdM form a complex in which HsdS recognizes the target DNA sequence, and HsdM carries out methylation of adenosine residues. The HsdR subunit, when associated with the HsdS-HsdM complex, translocates DNA in an ATP-dependent process and cleaves unmethylated DNA at a distance of several thousand base-pairs from the recognition site. The molecular mechanism by which these enzymes translocate the DNA is not fully understood, in part because of the absence of crystal structures. To date, crystal structures have been determined for the individual HsdS and HsdM subunits and models have been built for the HsdM-HsdS complex with the DNA. However, no structure is available for the HsdR subunit. In this work, the gene coding for the HsdR subunit of EcoR124I was re-sequenced, which showed that there was an error in the published sequence. This changed the position of the stop codon and altered the last 17 amino acid residues of the protein sequence. An improved purification procedure was developed to enable HsdR to be purified efficiently for biophysical and structural analysis. Analytical ultracentrifugation shows that HsdR is monomeric in solution, and the frictional ratio of 1.21 indicates that the subunit is globular and fairly compact. Small angle neutron-scattering of the HsdR subunit indicates a radius of gyration of 3.4 nm and a maximum dimension of 10 nm. We constructed a model of the HsdR using protein fold-recognition and homology modelling to model individual domains, and small-angle neutron scattering data as restraints to combine them into a single molecule. The model reveals an ellipsoidal shape of the enzymatic core comprising the N-terminal and central domains, and suggests conformational heterogeneity of the C-terminal region implicated in binding of HsdR to the HsdS-HsdM complex.     

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Background  

Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).     

The occurrence of Lepeophtheirus salmonis and Caligus clemensi (Copepoda: Caligidae) on three-spine stickleback Gasterosteus aculeatus in coastal British Columbia

Infections with sea lice species belonging to Lepeophtheirus and Caligus are reported from examinations of 1,309 three-spine sticklebacks collected in coastal British Columbia. Over 97% of the 19,960 Lepeophtheirus specimens and nearly 96% of the 2,340 Caligus specimens were in the copepodid and chalimus developmental stages. The parasites were identified as Lepeophtheirus salmonis and Caligus clemensi based on morphology of adult stages. Between 1,763 and 1,766 base pairs (bp) of 18S rDNA from adult specimens collected from sticklebacks and salmon differed from the GenBank L. salmonis reference sequence by a single bp and were distinct from those of 2 other Lepeophtheirus species. A 530-bp region of 18S rDNA from chalimus stages of Lepeophtheirus obtained from sticklebacks and salmon was identical to that of the L. salmonis reference sequence. The three-spine stickleback is a new host record for L. salmonis. The prevalence of L. salmonis was 83.6% and that of C. clemensi was 42.8%. The intensities of these infections were 18.3 and 4.2, respectively. There was no significant relationship between sea lice abundance and stickleback condition factor. Significant spatial and temporal variations both in abundance of sea lice and surface seawater salinities were measured. The abundance of both sea lice species was lowest in zones in which surface seawater salinity was also lowest. Sticklebacks appear to serve as temporary hosts, suggesting a role of this host in the epizootiology of L. salmonis. The stickleback may be a useful sentinel species with which to monitor spatial and temporal changes in the abundance of L. salmonis and C. clemensi.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.     

The realigment of the orchid flora in the mountain range of Guamuahaya,Cienfuegos, Cuba

The present study of the Orchidaceae family was carried out in Guamuahaya’s mountain range, from 2000 to March 2013. Fifteen districts were explored after 33 expeditions in the Province of Cienfuegos. Ninety two plant species were identified in the studied area, taking into account the ecological parameters of the mountainous areas of Cienfuegos and Cumanayagua municipalities.     

PRIMARY ADHESION OF ENTEROMORPHA (CHLOROPHYTA,ULVALES) PROPAGULES: QUANTITATIVE SETTLEMENT STUDIES AND VIDEO MICROSCOPY1

Quantitative methods and associated kinetic analyses have been used for the first time to study detailed aspects of the settlement and adhesion of various types of Enteromorpha popagule. Time course experiments showed that quadri and biflagellate zoospores and zygotes adhered rapidly, but a proportion within any one population appeared to be incompetent at adhering to the substratum. Kinetic (Scatchard) analysis of adhesion experiments performed at a range of zoospore concentrations revealed density-dependent effects not previously reported, with positive cooperativity at low spore densities and negative cooperativity at high spore densities. High-resolution video microscopy was used for the first time to reveal details of the various stages in the settlement and adhesion of zoospores and zygotes. Novel observations were made of an initial, temporary phase of attachment via the apical papilla, followed by a permanent phase of commitment, characterized by discharge of adhesive-containing cytoplasmic vesicles, as the cell contracted against the surface, and adsorption of flagella. The phase of commitment was followed by exploitation of the surface through amoeboid-like movements at the interface. Gregarious settlement behavior was frequently observed leading to the formation of rafts of cells. The possible mechanisms and significance of density-dependent spore adhesion are discussed.