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).     

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called "atypical") restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene.     

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.     

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.     

Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes

Aeromonas hydrophila strains recovered from clinical samples and ambient sources were phenotypically and genetically identified. In addition, the distribution of putative virulence factors was assayed. To determine the genetic diversity of these strains, random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers were used. The discriminatory ability of the techniques, using Simpson's index, was 0.96 for both methods. The most consistent dendrogram was obtained when RAPD and ERIC data were combined. The genetic diversity revealed a high intra-specific genetic diversity (h=0.364+/-0.024 and I=0.538+/-0.030). The strains showed a tendency to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). The present study demonstrates and quantifies the high intra-specific diversity within this species and reveals a clear differentiation of strains according to their ecological origin. The distribution of virulence-related genes confirm that A. hydrophila is a genetically heterogeneous species that harbour ecotypes which have different pathogenic potential to human and other animals.     

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.     

Complete Type III Secretion System of a Mesophilic Aeromonas hydrophila Strain

We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.     

Intraspecific DNA sequence variation of the mitochondrial control region of white sturgeon (Acipenser transmontanus)

Intraspecific sequence variation in the D-loop region of mtDNA in white sturgeon (Acipenser transmontanus), a relict North American fish species, was examined in 27 individuals from populations of the Columbia and Fraser rivers. Thirty-three varied nucleotide positions were present in a 462-nucleotide D-loop sequence, amplified using the polymerase chain reaction. Bootstrapped neighbor-joining and maximum- parsimony trees of sequences from 19 haplotypes suggest that the two populations have recently diverged. This is consistent with the hypothesis that the Columbia River, a Pleistocene refugium habitat, was the source of founders for the Fraser River after the last glacial recession. On the basis of a divergence time of 10-12 thousand years ago, the estimated substitution rate of the white sturgeon D-loop region is 1.1-1.3 x 10(-7) nucleotides/site/year, which is comparable to rates for hypervariable sequences in the human D-loop region. Furthermore, the ratio of mean percent nucleotide differences in the D- loop (2.27%) to that in whole mtDNA (0.54%, as estimated from restriction-enzyme data) is 4.3, which is similar to the fourfold-to- fivefold-higher substitution rate estimated for the human D-loop. The high nucleotide substitution rate of the hypervariable region indicates that the vertebrate D-loop has potential as a genetic marker in molecular population studies.      

Ancient large-scale genome duplications: phylogenetic and linkage analyses shed light on chordate genome evolution

Paralogous genes from several families were found in four human chromosome regions (4p16, 5q33-35, 8p12-21, and 10q24-26), suggesting that their common ancestral region underwent several rounds of large- scale duplication. Searches in the EMBL databases, followed by phylogenetic analyses, showed that cognates (orthologs) of human duplicated genes can be found in other vertebrates, including bony fishes. In contrast, within each family, only one gene showing the same high degree of similarity with all the duplicated mammalian genes was found in nonvertebrates (echinoderms, insects, nematodes). This indicates that large-scale duplications occurred after the echinoderms/chordates split and before the bony vertebrate radiation. It has been suggested that two rounds of gene duplication occurred in the vertebrate lineage after the separation of Amphioxus and craniate (vertebrates + Myxini) ancestors. Before these duplications, the genes that have led to the families of paralogous genes in vertebrates must have been physically linked in the craniate ancestor. Linkage of some of these genes can be found in the Drosophila melanogaster and Caenorhabditis elegans genomes, suggesting that they were linked in the triploblast Metazoa ancestor.