FIGENIX: Intelligent automation of genomic annotation: expertise integration in a new software platform

BACKGROUND: Two of the main objectives of the genomic and post-genomic era are to structurally and functionally annotate genomes which consists of detecting genes' position and structure, and inferring their function (as well as of other features of genomes). Structural and functional annotation both require the complex chaining of numerous different software, algorithms and methods under the supervision of a biologist. The automation of these pipelines is necessary to manage huge amounts of data released by sequencing projects. Several pipelines already automate some of these complex chaining but still necessitate an important contribution of biologists for supervising and controlling the results at various steps. RESULTS: Here we propose an innovative automated platform, FIGENIX, which includes an expert system capable to substitute to human expertise at several key steps. FIGENIX currently automates complex pipelines of structural and functional annotation under the supervision of the expert system (which allows for example to make key decisions, check intermediate results or refine the dataset). The quality of the results produced by FIGENIX is comparable to those obtained by expert biologists with a drastic gain in terms of time costs and avoidance of errors due to the human manipulation of data. CONCLUSION: The core engine and expert system of the FIGENIX platform currently handle complex annotation processes of broad interest for the genomic community. They could be easily adapted to new, or more specialized pipelines, such as for example the annotation of miRNAs, the classification of complex multigenic families, annotation of regulatory elements and other genomic features of interest

STATAWAARS: a promoter motif associated with spatial expression in the major effector-producing tissues of the plant-parasitic nematode Bursaphelenchus xylophilus.

BACKGROUND: Plant-parasitic nematodes cause severe damage to a wide range of crop and forest species worldwide. The migratory endoparasitic nematode, Bursaphelenchus xylophilus, (pinewood nematode) is a quarantine pathogen that infects pine trees and has a hugely detrimental economic impact on the forestry industry. Under certain environmental conditions large areas of infected trees can be destroyed, leading to damage on an ecological scale. The interactions of B. xylophilus with plants are mediated by secreted effector proteins produced in the pharyngeal gland cells. Identification of effectors is important to understand mechanisms of parasitism and to develop new control measures for the pathogens. RESULTS: Using an approach pioneered in cyst nematodes, we have analysed the promoter regions of a small panel of previously validated pharyngeal gland cell effectors from B. xylophilus to identify an associated putative regulatory promoter motif: STATAWAARS. The presence of STATAWAARS in the promoter region of an uncharacterized gene is a predictor that the corresponding gene encodes a putatively secreted protein, consistent with effector function. Furthermore, we are able to experimentally validate that a subset of STATAWAARS-containing genes are specifically expressed in the pharyngeal glands. Finally, we independently validate the association of STATAWAARS with tissue-specific expression by directly sequencing the mRNA of pharyngeal gland cells. We combine a series of criteria, including STATAWAARS predictions and abundance in the gland cell transcriptome, to generate a comprehensive effector repertoire for B. xylophilus. The genes highlighted by this approach include many previously described effectors and a series of novel "pioneer" effectors. CONCLUSIONS: We provide a major scientific advance in the area of effector regulation. We identify a novel promoter motif (STATAWAARS) associated with expression in the pharyngeal gland cells. Our data, coupled with those from previous studies, suggest that lineage-specific promoter motifs are a theme of effector regulation in the phylum Nematoda.SE-vdA is supported by BBSRC grant BB/M014207/1 and BB/R011311/

Economic Issues in the Management of Plants Invading Natural Environments: Scotch Broom in Barrington Tops National Park

Scotch broom (Cytisus scoparius, L.), is an exotic leguminous shrub, native to Europe, which invades pastoral and woodland ecosystems and adjoining river systems in cool, high rainfall regions of southeastern Australia. Broom has invaded 10,000 hectares of eucalypt woodland at Barrington Tops National Park in New South Wales, and is having a major impact on the natural ecology of the sub-alpine environment. It is extremely competitive with the native flora, retarding their growth and in many areas blanketing the ground and preventing growth of many understorey species in open forest areas. An active program to manage this invasion is being implemented by the National Parks and Wildlife Service. The management issues include whether eradication or containment is economically desirable, and when biological control is economically desirable. Management choices depend on the marginal costs of increments of government intervention, effects of uncertain budgets on the control of broom, choice of control measures and effects of uncertain values of biodiversity. These issues are addressed through the application of a detailed bioeconomic model of broom management.Scotch broom, economic issues, management issues, natural environments, bioeconomic model, Environmental Economics and Policy,

Variations in flanking residues of thyroid hormone receptor alpha1 isoform

The thyroid hormone receptor (TR) is a transcription factor that represses or activates genes whose regulatory regions bear thyroid hormone (T3) response elements (TREs). Modulation of this function requires shuttling into and out of the nucleus. This is mediated by the binding of importins and exportins to amino acid sequences within TR, called nuclear localization signals (NLSs) and nuclear export signals (NESs), respectively. The fine balance between nuclear import, retention, and export of TR has emerged as a critical control point for modulating T3-responsive gene expression, including genes that control cell proliferation, differentiation, and apoptosis. NLS-2 is a specific NLS located in the N-terminal A/B domain of TR-alpha1 and and its oncogenic form v-ErbA. NLS-2 minimally consists of the basic residue-rich motif KRKRK in TR-alpha1 of rats and humans. However, the flanking residues differ widely among vertebrates, and are likely responsible for lack of a functional NLS-2 in TR-alpha1 in some species. Yet, systemic consequences of NLS-2 flanking sequence variation remain unexplored. Further investigation of the NLS-2 variants and how these motifs synergize with protein function will enhance understanding of how even a single amino acid change can lead to TR mislocalization and potentially trigger oncogenesis

Nitrogen metabolism in iron-respiring bacteria

Nitrogen metabolism was investigated in three species of iron respiring bacteria Geobacter metallireducens, Geobacter sulfurreducens, and Sulfurospirillum barnesii. G. metallireducens and G. sulfurreducens were shown to grow in medium containing N2, NO3-, or NH4 but nitrite inhibited growth. BLAST analysis of the draft genomic sequence of G. metallireducens revealed only Nar, Nrf, and Nif homologues but no assimilatory genes (i.e., Nas). Two nar operons were identified, narCGHJIK1K2, and narCGHI. A putative CAP/CRP recognition binding site was found upstream and two molybdenum biosynthetic genes were found downstream of the large operon. Both operons contain a novel cytochrome c (NarC) that has only been described in Thermus thermophilus strain HB8. An attempt was made to purify the periplasmic nitrate reductase (Nap) from S. barnesii without success. However, a method using lysozyme to solubilize Nap and a rapid activity assay for nitrate reductase using 96 well plates were developed

MOLECULAR AND PHYSIOLOGICAL STUDIES OF SALT TOLERANCE IN THE SALT-SECRETOR MANGROVE AVICENNIA OFFICINALIS

Ph.DDOCTOR OF PHILOSOPH

Bio-Separation Process Improvement via Genomic Manipulation: Development of Novel Strains for Use in Immobilized Metal Affinity Chromatography (IMAC)

The dissertation is comprised of three parts. Part I describes proteomic analysis of native bacterial proteins from Escherichia coli (E.coli) that bind during Immobilized Metal Affinity Chromatography (IMAC). Part II describes the value in exploiting proteome based data as a tool toward the design an E. coli expression strain that is particularly useful when Immobilized Metal Affinity Chromatography is employed as the initial capture step of a homologous protein purification process. Part III describes a methodology of chromosomal mapping of all contaminant gene products. The objective of Part I was to identify all E. coli proteins that bind to Co(II), Ni(II), and Zn(II) IMAC columns, describing the isoelectric point, molecular weight, and metabolic essentiality of the characterized proteins were considered. Information regarding this group of proteins is presented and used to define the IMAC bioseparation-specific metalloproteome of E. coli. Such data concerning the potential contaminant pool is useful for the design of separation schemes, as well as designing optimized affinity tails and strains for IMAC purification. Part II examined proteins known to co-elute during Co(II), Ni(II), and Zn(II) IMAC purifications. Methods to circumvent the effects of punitive protein removal were proposed and carried out. Specifically, triosephosphate isomerase (TIM; tpiA gene product), a protein known to bind during IMAC, was redesigned through site directed mutagenesis to eliminate surface exposed histidine. By extension of this rational, Part III provides a theoretical model of using in silico mapping (Circos diagrams) to create a practical system of applying data described Part I. Such a tool has potential to allow future investigators the possibility of mapping large scale genomic deletions; significantly streamlining cell line development when compared to the individual targeting methodologies seen in Part II

Helminth infections of the European eel Anguilla Anguilla in the United Kingdom

The European eel, Anguilla anguilla, is designated a threatened species and parasites are considered to be one of the contributory factors associated with the species decline. To address the lack of knowledge concerning parasite infections in the UK European eel population, a total of 140 specimens were obtained from the Environment Agency at 14 river sites across England and Wales. Each specimen was dissected and infection status determined for the different classes of helminth using a combination of morphological and molecular approaches, including a PCR-based restriction digestion approach to discriminate between the gill monogeneans Pseudodactylogyrus anguillae and P. bini. Overall, 101 eels (72.1 %) were infected with a total of 1504 helminths and these included gill monogeneans (prevalence = 35.7%), gastrointestinal nematodes (prevalence = 33.6%), the swim bladder nematode Anguillicoloides crassus (prevalence = 25.0%), acanthocephalans (prevalence = 30.0%) and cestodes (prevalence = 9.3%). Seven identified helminth species; the pathogenic pseudodactylids P. anguillae and P. bini, the pathogenic swim bladder nematode A. crassus, the gastrointestinal nematodes Spinitectus inermis and Paraquimperia tenerrima, and the tapeworms Proteocephalus macrocephalus and Bothriocephalus claviceps, are known eel specialists. A further 4 non-specialist eel helminths; the acanthocephalans Acanthocephalus clavula, A. lucii and the category 2 parasite Pomphorhynchus laevis, plus the gastrointestinal nematode Raphidascaris acus, were also identified.Helminth infection appeared to have a significant association with the larger eels and this was particularly evident for infections involving acanthocephalans. This presumably reflects the age and hence prolonged exposure to infection risk, as well as a more diverse diet of the larger, compared to the smaller, eels. Interestingly, the condition factor of the eels infected with helminths was also significantly greater than that of the uninfected eels. Helminth co-infections were commonly observed (54.5% of the infected eels) and the majority of these co-infections (75%) involved one, or both, of the pathogenic A. crassus and pseudodactylids. With regard to catchment sites, interesting differences were noted in the primary helminth infection data. Eel specimens analysed from the rivers in South East England contained the most numerous and diverse range of helminths. Indeed, helminth prevalence was 100% and co-infections, including up to 5 taxa, were common; pseudodactylids and acanthocephalans were present in all the observed co-infections. Eels sampled from rivers in North West England also contained a rich diversity of helminths and although pseudodactytlids were common, the gastrointestinal nematodes were the dominant taxa observed in co-infections. A. crassus and the pseudodactylids were the dominant helminths in co-infected eels sampled from South Wales and interestingly, even though this region had the second highest helminth prevalence, tapeworm infections were not observed. Only 50% of eels sampled from North Wales were infected with helminths; specifically, acanthocephalans and A. crassus were not observed in eels from this region and gastrointestinal nematodes were present in all the co-infections. Taken together, the data in this thesis confirms that European eels in the UK are commonly infected with a plethora of helminths. Some of the eel specimens exhibited known pathologies associated with their infections. However, the precise impacts that these helminths have upon eel health, and importantly the migratory capacity of the host, remain unknown and are worthy of further investigation