Molecular Biology and Biochemistry, Department of

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A Novel Approach to Modelling Water Transport and Drug Diffusion Through the Stratum Corneum

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: The potential of using skin as an alternative path for systemicallyadministering active drugs has attracted considerable interest, since the creation ofnovel drugs capable of diffusing through the skin would provide a great steptowards easily applicable -and more humane- therapeutic solutions. However, fordrugs to be able to diffuse, they necessarily have to cross a permeability barrier: thestratum corneum (SC), the uppermost set of skin layers. The precise mechanism bywhich drugs penetrate the skin is generally thought to be diffusion of moleculesthrough this set of layers following a “tortuous pathway” around corneocytes, i.e.impermeable dead cells.Results: In this work, we simulate water transport and drug diffusion using a threedimensionalporous media model. Our numerical simulations show that diffusiontakes place through the SC regardless of the direction and magnitude of the fluidpressure gradient, while the magnitude of the concentrations calculated areconsistent with experimental studies.Conclusions: Our results support the possibility for designing arbitrary drugs capableof diffusing through the skin, the time-delivery of which is solely restricted by theirdiffusion and solubility properties.

Document type: 
Article

Characterization of the Octamer, A Cis-Regulatory Element that Modulates Excretory Cell Gene-Expression in Caenorhabditis Elegans

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: We have previously demonstrated that the POU transcription factor CEH-6 is required for drivingaqp-8 expression in the C. elegans excretory (canal) cell, an osmotic regulatory organ that is functionally analogousto the kidney. This transcriptional regulation occurs through a CEH-6 binding to a cis-regulatory element called theoctamer (ATTTGCAT), which is located in the aqp-8 promoter.Results: Here, we further characterize octamer driven transcription in C. elegans. First, we analyzed the positionalrequirements of the octamer. To do so, we assayed the effects on excretory cell expression by placing the octamerwithin the well-characterized promoter of vit-2. Second, using phylogenetic footprinting between threeCaenorhabditis species, we identified a set of 165 genes that contain conserved upstream octamers in theirpromoters. Third, we used promoter::GFP fusions to examine the expression patterns of 107 of the 165 genes. Thisanalysis demonstrated that conservation of octamers in promoters increases the likelihood that the gene isexpressed in the excretory cell. Furthermore, we found that the sequences flanking the octamers may havefunctional importance. Finally, we altered the octamer using site-directed mutagenesis. Thus, we demonstrated thatsome nucleotide substitutions within the octamer do not affect the expression pattern of nearby genes, butchange their overall expression was changed. Therefore, we have expanded the core octamer to include flankingregions and variants of the motif.Conclusions: Taken together, we have demonstrated that octamer-containing regions are associated withexcretory cell expression of several genes that have putative roles in osmoregulation. Moreover, our analysis of theoctamer sequence and its sequence variants could aid in the identification of additional genes that are expressedin the excretory cell and that may also be regulated by CEH-6.

Document type: 
Article

Curating the Innate Immunity Interactome

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: The innate immune response is the first line of defence against invading pathogens and is regulatedby complex signalling and transcriptional networks. Systems biology approaches promise to shed new light on theregulation of innate immunity through the analysis and modelling of these networks. A key initial step in thisprocess is the contextual cataloguing of the components of this system and the molecular interactions thatcomprise these networks. InnateDB (http://www.innatedb.com) is a molecular interaction and pathway databasedeveloped to facilitate systems-level analyses of innate immunity.Results: Here, we describe the InnateDB curation project, which is manually annotating the human and mouseinnate immunity interactome in rich contextual detail, and present our novel curation software system, which hasbeen developed to ensure interactions are curated in a highly accurate and data-standards compliant manner. Todate, over 13,000 interactions (protein, DNA and RNA) have been curated from the biomedical literature. Here, wepresent data, illustrating how InnateDB curation of the innate immunity interactome has greatly enhanced networkand pathway annotation available for systems-level analysis and discuss the challenges that face such curationefforts. Significantly, we provide several lines of evidence that analysis of the innate immunity interactome has thepotential to identify novel signalling, transcriptional and post-transcriptional regulators of innate immunity.Additionally, these analyses also provide insight into the cross-talk between innate immunity pathways and otherbiological processes, such as adaptive immunity, cancer and diabetes, and intriguingly, suggests links to otherpathways, which as yet, have not been implicated in the innate immune response.Conclusions: In summary, curation of the InnateDB interactome provides a wealth of information to enablesystems-level analysis of innate immunity.

Document type: 
Article

Genomic Organization of Duplicated Major Histocompatibility Complex Class I Regions in Atlantic Salmon (Salmo Salar)

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2007
Abstract: 

Background: We have previously identified associations between major histocompatibility complex(MHC) class I and resistance towards bacterial and viral pathogens in Atlantic salmon. To evaluate if onlyMHC or also closely linked genes contributed to the observed resistance we ventured into sequencing ofthe duplicated MHC class I regions of Atlantic salmon.Results: Nine BACs covering more than 500 kb of the two duplicated MHC class I regions of Atlanticsalmon were sequenced and the gene organizations characterized. Both regions contained the proteasomecomponents PSMB8, PSMB9, PSMB9-like and PSMB10 in addition to the transporter for antigen processingTAP2, as well as genes for KIFC1, ZBTB22, DAXX, TAPBP, BRD2, COL11A2, RXRB and SLC39A7. TheIA region contained the recently reported MHC class I Sasa-ULA locus residing approximately 50 kbupstream of the major Sasa-UBA locus. The duplicated class IB region contained an MHC class I locusresembling the rainbow trout UCA locus, but although transcribed it was a pseudogene. No other MHCclass I-like genes were detected in the two duplicated regions. Two allelic BACs spanning the UBA locushad 99.2% identity over 125 kb, while the IA region showed 82.5% identity over 136 kb to the IB region.The Atlantic salmon IB region had an insert of 220 kb in comparison to the IA region containing threechitin synthase genes.Conclusion: We have characterized the gene organization of more than 500 kb of the two duplicatedMHC class I regions in Atlantic salmon. Although Atlantic salmon and rainbow trout are closely related,the gene organization of their IB region has undergone extensive gene rearrangements. The Atlanticsalmon has only one class I UCA pseudogene in the IB region while trout contains the four MHC UCA, UDA,UEA and UFA class I loci. The large differences in gene content and most likely function of the salmon andtrout class IB region clearly argues that sequencing of salmon will not necessarily provide informationrelevant for trout and vice versa.

Document type: 
Article

Comprehensive Analysis of MHC Class I Genes from the U-, S-, and Z-Lineages in Atlantic Salmon

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: We have previously sequenced more than 500 kb of the duplicated MHC class I regions in Atlanticsalmon. In the IA region we identified the loci for the MHC class I gene Sasa-UBA in addition to a soluble MHCclass I molecule, Sasa-ULA. A pseudolocus for Sasa-UCA was identified in the nonclassical IB region. Both regionscontained genes for antigen presentation, as wells as orthologues to other genes residing in the human MHCregion.Results: The genomic localisation of two MHC class I lineages (Z and S) has been resolved. 7 BACs weresequenced using a combination of standard Sanger and 454 sequencing. The new sequence data extended the IAregion with 150 kb identifying the location of one Z-lineage locus, ZAA. The IB region was extended with 350 kbincluding three new Z-lineage loci, ZBA, ZCA and ZDA in addition to a UGA locus. An allelic version of the IB regioncontained a functional UDA locus in addition to the UCA pseudolocus. Additionally a BAC harbouring two MHCclass I genes (UHA) was placed on linkage group 14, while a BAC containing the S-lineage locus SAA (previouslyknown as UAA) was placed on LG10. Gene expression studies showed limited expression range for all class I geneswith exception of UBA being dominantly expressed in gut, spleen and gills, and ZAA with high expression in blood.Conclusion: Here we describe the genomic organization of MHC class I loci from the U-, Z-, and S-lineages inAtlantic salmon. Nine of the described class I genes are located in the extension of the duplicated IA and IBregions, while three class I genes are found on two separate linkage groups. The gene organization of the tworegions indicates that the IB region is evolving at a different pace than the IA region. Expression profiling,polymorphic content, peptide binding properties and phylogenetic relationship show that Atlantic salmon has onlyone MHC class Ia gene (UBA), in addition to a multitude of nonclassical MHC class I genes from the U-, S- andZ-lineages.

Document type: 
Article

Comparative Genomic Analysis of Atlantic Salmon, Salmo Salar, from Europe and North America

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: Several lines of evidence including allozyme analysis, restriction digest patterns and sequencing ofmtDNA as well as mini- and micro-satellite allele frequencies indicate that Atlantic salmon (Salmo salar) from NorthAmerica and Europe are genetically distinct. These observations are supported by karyotype analysis, whichrevealed that North American Atlantic salmon have 27 pairs of chromosomes whereas European salmon have 29pairs. We set out to construct a linkage map for a North American Atlantic salmon family and to compare this mapwith the well developed map for European Atlantic salmon.Results: We used microsatellite markers, which had previously been mapped in the two Atlantic salmon SALMAPmapping families from the River Tay, Scotland, to carry out linkage analysis in an Atlantic salmon family (NB1)whose parents were derived from the Saint John River stock in New Brunswick, Canada. As large differences inrecombination rates between female and male Atlantic salmon have been noted, separate genetic maps wereconstructed for each sex. The female linkage map comprises 218 markers in 37 linkage groups while the male maphas 226 markers in 28 linkage groups. We combined 280 markers from the female and male maps into 27composite linkage groups, which correspond to the haploid number of chromosomes in Atlantic salmon from theWestern Atlantic.Conclusions: A comparison of the composite NB1 and SALMAP linkage maps revealed the reason for thedifference in the chromosome numbers between European and North American Atlantic salmon: Linkage groupsAS-4 and AS-32 in the Scottish salmon, which correspond to chromosomes Ssa-6 and Ssa-22, are combined into asingle NB1 linkage group as are linkage groups AS-21 and AS-33 (corresponding to chromosomes Ssa-26 and Ssa-28). The comparison of the linkage maps also suggested some additional chromosomal rearrangements, but it willrequire finer mapping, potentially using SNPs, to test these predictions. Our results provide the first comparison ofthe genomic architecture of Atlantic salmon from North America and Europe with respect to chromosomeorganization.

Document type: 
Article

A Dense SNP-Based Linkage Map for Atlantic Salmon (Salmo salar) Reveals extended Chromosome Homeologies and Striking Differences in Sex-Specific Recombination Patterns

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2011
Abstract: 

Background: The Atlantic salmon genome is in the process of returning to a diploid state after undergoing awhole genome duplication (WGD) event between 25 and100 million years ago. Existing data on the proportion ofparalogous sequence variants (PSVs), multisite variants (MSVs) and other types of complex sequence variationsuggest that the rediplodization phase is far from over. The aims of this study were to construct a high densitylinkage map for Atlantic salmon, to characterize the extent of rediploidization and to improve our understandingof genetic differences between sexes in this species.Results: A linkage map for Atlantic salmon comprising 29 chromosomes and 5650 single nucleotidepolymorphisms (SNPs) was constructed using genotyping data from 3297 fish belonging to 143 families. Of these,2696 SNPs were generated from ESTs or other gene associated sequences. Homeologous chromosomal regionswere identified through the mapping of duplicated SNPs and through the investigation of syntenic relationshipsbetween Atlantic salmon and the reference genome sequence of the threespine stickleback (Gasterosteusaculeatus). The sex-specific linkage maps spanned a total of 2402.3 cM in females and 1746.2 cM in males,highlighting a difference in sex specific recombination rate (1.38:1) which is much lower than previously reportedin Atlantic salmon. The sexes, however, displayed striking differences in the distribution of recombination siteswithin linkage groups, with males showing recombination strongly localized to telomeres.Conclusion: The map presented here represents a valuable resource for addressing important questions of interestto evolution (the process of re-diploidization), aquaculture and salmonid life history biology and not least as aresource to aid the assembly of the forthcoming Atlantic salmon reference genome sequence.

Document type: 
Article

Interaction Profile-Based Protein Classification of Death Domain

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2004
Abstract: 

Background: The increasing number of protein sequences and 3D structure obtained fromgenomic initiatives is leading many of us to focus on proteomics, and to dedicate our experimentaland computational efforts on the creation and analysis of information derived from 3D structure.In particular, the high-throughput generation of protein-protein interaction data from a feworganisms makes such an approach very important towards understanding the molecularrecognition that make-up the entire protein-protein interaction network. Since the generation ofsequences, and experimental protein-protein interactions increases faster than the 3D structuredetermination of protein complexes, there is tremendous interest in developing in silico methodsthat generate such structure for prediction and classification purposes. In this study we focused onclassifying protein family members based on their protein-protein interaction distinctiveness.Structure-based classification of protein-protein interfaces has been described initially by Ponstinglet al. [1] and more recently by Valdar et al. [2] and Mintseris et al. [3], from complex structures thathave been solved experimentally. However, little has been done on protein classification based onthe prediction of protein-protein complexes obtained from homology modeling and dockingsimulation.Results: We have developed an in silico classification system entitled HODOCO (Homologymodeling, Docking and Classification Oracle), in which protein Residue Potential InteractionProfiles (RPIPS) are used to summarize protein-protein interaction characteristics. This systemapplied to a dataset of 64 proteins of the death domain superfamily was used to classify eachmember into its proper subfamily. Two classification methods were attempted, heuristic andsupport vector machine learning. Both methods were tested with a 5-fold cross-validation. Theheuristic approach yielded a 61% average accuracy, while the machine learning approach yielded an89% average accuracy.Conclusion: We have confirmed the reliability and potential value of classifying proteins via theirpredicted interactions. Our results are in the same range of accuracy as other studies that classifyprotein-protein interactions from 3D complex structure obtained experimentally. While ourclassification scheme does not take directly into account sequence information our results are inagreement with functional and sequence based classification of death domain family members.

Document type: 
Article

Salmo Salar and Esox Lucius Full-Length cDNA Sequences Reveal Changes in Evolutionary Pressures on a Post-Tetraploidization Genome

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010
Abstract: 

Background: Salmonids are one of the most intensely studied fish, in part due to their economic and environmentalimportance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. Thisduplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomicresources have recently become available for Atlantic salmon (Salmo salar), but documentation of allelic versusduplicate reference genes remains a major uncertainty in the complete characterization of its genome and itsevolution.Results: From existing expressed sequence tag (EST) resources and three new full-length cDNA libraries, 9,057reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-lengthclones were annotated from 29,221 northern pike (Esox lucius) ESTs. Pairwise dN/dS comparisons within each of 408 setsof duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection onsalmon duplicates.Conclusions: 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles andgene family members. Comparisons of duplicated genes show that while purifying selection is the predominant forceacting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure ongene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs,allowing one of the pair to diverge at a faster rate.

Document type: 
Article

The Bovine Lactation Genome: Insights into the Evolution of Mammalian Milk

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009
Abstract: 

Background: The newly assembled Bos taurus genome sequence enables the linkage of bovine milkand lactation data with other mammalian genomes.Results: Using publicly available milk proteome data and mammary expressed sequence tags, 197milk protein genes and over 6,000 mammary genes were identified in the bovine genome.Intersection of these genes with 238 milk production quantitative trait loci curated from theliterature decreased the search space for milk trait effectors by more than an order of magnitude.Genome location analysis revealed a tendency for milk protein genes to be clustered with othermammary genes. Using the genomes of a monotreme (platypus), a marsupial (opossum), and fiveplacental mammals (bovine, human, dog, mice, rat), gene loss and duplication, phylogeny, sequenceconservation, and evolution were examined. Compared with other genes in the bovine genome,milk and mammary genes are: more likely to be present in all mammals; more likely to be duplicatedin therians; more highly conserved across Mammalia; and evolving more slowly along the bovine lineage. The most divergent proteins in milk were associated with nutritional and immunologicalcomponents of milk, whereas highly conserved proteins were associated with secretory processes.Conclusions: Although both copy number and sequence variation contribute to the diversity ofmilk protein composition across species, our results suggest that this diversity is primarily due toother mechanisms. Our findings support the essentiality of milk to the survival of mammalianneonates and the establishment of milk secretory mechanisms more than 160 million years ago.

Document type: 
Article