Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

BACKGROUND: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. METHODOLOGY/PRINCIPAL FINDINGS: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported. CONCLUSION/SIGNIFICANCE: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances

Comparative genomics reveals 104 candidate structured RNAs from bacteria, archaea, and their metagenomes

Novel motifs identified in a comparative genomic analysis of bacterial, archaeal and metagenomic data reveals over 100 candidate structured RNAs

Am I repeating myself? Determining the repetitive landscape of the pig X chromosome

Mammalian sex chromosomes evolved from a homologous pair of autosomes via the acquisition of a major sex determining gene. This event led to the suppression of recombination between the chromosomes and consequently their independent evolution. Both X and Y have been observed to accumulate amplified gene families in mice and humans, but it remains unclear to what extent this is true in other mammalian species. The pig X chromosome was recently sequenced to a high quality and in this study, it was investigated to determine the presence and extent of potential amplicons. LASTZ was used to align the pig X sequence to itself and identify regions of similarity that could represent ampliconic sequences. Further analysis revealed many of the similarities to be interspersed along the chromosome, and in some cases particularly clustered around the centromere. This distribution of hits suggests many of the similarities to be the result of known repetitive elements which commonly cluster within centromeric and pericentromeric regions. There were also some regions showing segmental duplications and gene duplications. Further investigation of the hits through NCBI BLAST revealed them to be fragments of LINE-1 (Long Interspersed Nuclear Element 1) retrotransposons, orthologues of duplicated genes, and uncharacterised loci. Mammalian X chromosomes are often enriched with LINE-1s which are thought to play a role in X-inactivation and controlling gene expression. Some of the duplicated genes are of potential interest for further analysis as their function is unknown. At this stage, no independent amplicons have been found in the pig X chromosome, in contrast to what has been observed in humans and mice. However, the current assembly contains gaps between contigs and unmasked repeats which may obscure where potential amplicons might be found. The data from this study can be used to improve the current annotation of the pig X chromosome

Computational Biology in Acute Myeloid Leukemia with CEBPA Abnormalities

__Abstract__ In the last decade, tiling-array and next-generation sequencing technologies allowed quantitative measurements of different cellular processes, such as mRNA expression, genomic changes including deletions or amplifications, DNA-methylation, chromatin modifications or Protein-DNA-binding interactions. Using these technologies, thousands of features can now be measured simultaneously in a patient cell sample. The use of for instance mRNA expression profiles or DNA-methylation profiles have already provided new insight into the molecular biology of patients with Acute Myeloid Leukemia (AML). AML is a blood cell malignancy, in which primitive myeloid cells have been transformed and accumulate in the bone marrow and blood. Different forms of AML exist with different molecular abnormalities that associate with distinct responses to therapy. Many subgroups with comparable mRNA expression or DNA-methylation patterns were identified. These studies also revealed the existence of novel previously undefined AML subtypes. Among those was a group of patients with a mutation in a gene called CEBPA. CEBPA is a gene that encodes the transcription factor CCAAT Enhancer Binding Protein Alpha (C/EBPα), which controls the expression of genes in myeloid progenitor cells. Mutated CEBPA encodes a dysfunctional C/EBPα-protein, which consequently results in aberrant control of “target genes”. In this thesis we focus particularly on the role of CEBPA. We studied the predictive and prognostic relevance of mutated CEBPA, and analyzed in a genome wide fashion the mRNA expression, DNA-methylation and the protein-DNA-binding levels corresponding to (mutated) CEBPA in AML. For the analysis of protein-DNA-binding, we developed a novel statistical methodology. With this statistical methodology we studied the fundamental role of (mutant) C/EBPα binding and the effect on gene expression levels. We also integrated gene expression with DNA-methylation profiles of hundreds of AML patients and revealed the existence of two previously unidentified AML subtypes

Front Matter - Soft Computing for Data Mining Applications

Efficient tools and algorithms for knowledge discovery in large data sets have been devised during the recent years. These methods exploit the capability of computers to search huge amounts of data in a fast and effective manner. However, the data to be analyzed is imprecise and afflicted with uncertainty. In the case of heterogeneous data sources such as text, audio and video, the data might moreover be ambiguous and partly conflicting. Besides, patterns and relationships of interest are usually vague and approximate. Thus, in order to make the information mining process more robust or say, human-like methods for searching and learning it requires tolerance towards imprecision, uncertainty and exceptions. Thus, they have approximate reasoning capabilities and are capable of handling partial truth. Properties of the aforementioned kind are typical soft computing. Soft computing techniques like Genetic

INVESTIGATION OF POXVIRUS HOST-RANGE AND GENE EXPRESSION IN MAMMALIAN CELLS.

Members of the Poxviridae family have been known as human pathogens for centuries. Their impact in society included several epidemics that decimated the population. In the last few centuries, Smallpox was of great concern that led to the development of our modern vaccines. The systematic study of Poxvirus host-range and immunogenicity provided the knowledge to translate those observations into practice. After the global vaccination campaign by the World Health Organization, Smallpox was the first infectious disease to be eradicated. Nevertheless, diseases such as Monkeypox, Molluscum contagiosum, new bioterrorist threads, and the use of poxviruses as vaccines or vectors provided the necessity to further understand the host-range from a molecular level. Here, we take advantage of the newly developed technologies such as 454 pyrosequencing and RNA-Seq to address previously unresolved questions for the field. First, we were able to identify the Erytrhomelagia-related poxvirus (ERPV) 25 years after its isolation in Hubei, China. Whole-genome sequencing and bioinformatics identified ERPV as an Ectromelia strain closely related to the Ectromelia Naval strain. Second, by using RNA-Seq, the first MOCV in vivo and in vitro transcriptome was generated. New tools have been developed to support future research and for this human pathogen. Finally, deep-sequencing and comparative genomes of several recombinant MVAs (rMVAs) in conjunction with classical virology allowed us to confirm several genes (O1, F5, C17, F11) association to plaque formation in mammalian cell lines. We also provided additional evidence that plaque formation and virus replication can be independent. More importantly, we identified a gene as the first gene outside MVA's deletion that explains its host-restriction. Replacement of this region with a cassette containing that gene derived from a replication-competent virus demonstrated to be sufficient to increase viral yield in all mammalian cell lines tested. Several research and clinical applications can be envisioned derived from this work

The d-arabitol operon of Klebsiella aerogenes

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The Life of Prion: an investigation into the physiological role of a prion-like protein in the nematode Caenorhabditis elegans

For centuries, the threat of prion disease has plagued populations – whether it be in the form of scrapie ravaging through the sheep populations of Spain in the eighteenth century, fatal familial insomnia afflicting families in Italy, or an outbreak of Creutzfeldt-Jakob disease in the UK triggered by the consumption of contaminated beef. The ability of prions to spread with such pathogenic intent whilst remaining incurable fuels the fire of public concern - yet delving beyond that classical view of prions has led to an enlightened appreciation of the role of such proteins throughout the biota of life. Prion-like proteins subvert the dogma of the one-to-one structure-function relationship traditionally held onto when considering the physiology of proteins. Instead, these proteins display structural polymorphism, capable of adopting different conformations under different conditions; this expands the capabilities of protein function and represents a mechanism for epigenetic coding, with protein conformation possessing the ability convey and propagate specific states for biological advantage without the need for DNA modification. The properties observed in many of these proteins, including those of multivalency and low complexity, predispose them towards phase separation, whereby localised concentration of biomolecules compartmentalise cellular activity. This ability confers upon an organism the ability to respond to stimuli in dynamic and transient fashions without needing to use cumbersome membrane-bound organelles. Using the Prion-Like Amino Acid Composition program, we have identified the stress-responsive protein ABU-13 as a potential prion in the nematode species, Caenorhabditis elegans. Phenotypic analysis of ABU-13 knockout animals has identified a non-redundant role for this protein in ER stress and innate immune responses – in keeping with previous evidence that this family of proteins is involved with a non-canonical unfolded protein response pathway of the endoplasmic reticulum. These knockout animals do not display defects in the activation of the stress-response gene hsp-4 upon tunicamycin treatment, suggesting that these effects occur independently of the canonical UPR pathway. In vivo and in vitro characterisation of ABU-13 has demonstrated a propensity for coalescence, reminiscent of a phase-separated organelle. This is further supported by the ability of this protein to bind RNA – a common feature of prion-like proteins involved with such transitions. Co-immunoprecipitation of ABU-13 confirmed an enrichment of ER and RNA-related protein interactions, supporting a role for this protein in ER stress responses. In addition to this, a number of glycosylation related terms were identified, pointing towards a role for ABU-13 in the folding quality control of such proteins. These punctated structures do, however, appear less mobile than traditional liquid-like condensates, as demonstrated by a slow FRAP recovery, indicative of a more hydrogel-like structure. To further investigate this, we extracted hydrogel-forming proteins from N2 animals using a biotinylated isoxazole precipitation, identifying a number of novel proteins that may be involved with in the formation of such solid-like structures. ABU-13, however, was not amongst this dataset, suggesting that if it were indeed involved in a physiological hydrogel, this transition is driven by another protein component. Overall, we propose that ABU-13 represents a phase-separating prion-like protein capable of modulating ER stress and immune responses, potentially via the regulation of RNA processing

Using functional genetics and epigenetics to dissect the molecular architecture of schizophrenia

Schizophrenia is a severe neuropsychiatric disorder which is within the top ten causes of disability in the developed world. While we currently do not fully understand the aetiology of schizophrenia, a wealth of genetic, epigenetic, and epidemiological evidence suggests a neurodevelopmental origin, and that dysregulation of the immune system and infection can play a role in aetiology. This has been supported throughout the last decade and the “big data” revolution. Genetic and epigenetic data gathered through genome and epigenome wide association studies, have identified over 100 genetic risk loci for schizophrenia. Further to this there have been a number of epidemiological studies examining the effect of environmental risk factors to schizophrenia. However, many of these genetic, epigenetic, and environmental risk factors have no clear mechanism by which they cause disease. Many of these studies published present evidence for the “risk factor” involvement but fail to validate their findings in model systems, to establish how these risk factors cause disease. The main aim of this thesis was to develop functional assays and methods to validate genetic, epigenetic, and environmental risk factors in human cellular based models to elucidate how these risks can contribute to disease. From this work I have developed a protocol for knocking out risk genes, and have identified a role for the schizophrenia risk gene AS3MT in neuronal development. I have also developed several pipelines to validate epigenome wide association studies, and link how changes in DNA methylation can affect gene expression, at both the regional level and at individual genomic loci. And finally, I have examined what effect, if any, the active component of cannabis, which is an established environmental risk factor for schizophrenia, has on the DNA methylome, to understand how environmental risk can cause disease. In summary, the work presented in this thesis represents the movement from large scale (epi)genetic analysis and statistics to functional lab validation. This thesis contains several methods for the functional validation of identified (epi)genetic risk loci and risk factors for schizophrenia in a cellular model. From this we can now begin to explore how these changes in genetics, epigenetics, and environmental risk factors biologically link to the development and progression of schizophrenia and other neuropsychiatric disorders