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1.  WormBase 2007 
Nucleic Acids Research  2007;36(Database issue):D612-D617.
WormBase (www.wormbase.org) is the major publicly available database of information about Caenorhabditis elegans, an important system for basic biological and biomedical research. Derived from the initial ACeDB database of C. elegans genetic and sequence information, WormBase now includes the genomic, anatomical and functional information about C. elegans, other Caenorhabditis species and other nematodes. As such, it is a crucial resource not only for C. elegans biologists but the larger biomedical and bioinformatics communities. Coverage of core areas of C. elegans biology will allow the biomedical community to make full use of the results of intensive molecular genetic analysis and functional genomic studies of this organism. Improved search and display tools, wider cross-species comparisons and extended ontologies are some of the features that will help scientists extend their research and take advantage of other nematode species genome sequences.
doi:10.1093/nar/gkm975
PMCID: PMC2238927  PMID: 17991679
2.  Gene discovery, evolutionary affinity and molecular detection of Oxyspirura petrowi, an eye worm parasite of game birds 
BMC Microbiology  2013;13:233.
Background
Oxyspirura petrowi appears to be emerging as a nematode parasite that could negatively impact Northern Bobwhite quail individuals and populations within Texas and other regions of the United States. Despite this eye worm's potential importance in the conservation of wild quail, little is known about the general biology and genome composition of O. petrowi. To fill the knowledge gap, we performed a small scale random genome sequence survey, sequenced its 18S rRNA and the intergenic region between the 18S and 28S rRNA genes, studied its phylogenetic affinity, and developed a PCR protocol for the detection of this eye worm.
Results
We have generated ~240 kb of genome sequence data derived from 348 clones by a random genome survey of an O. petrowi genomic library. The eye worm genome is AT-rich (i.e., 62.2% AT-content), and contains a high number of microsatellite sequences. The discovered genes encode a wide-range of proteins including hypothetical proteins, enzymes, nematode-specific proteins. Phylogenetic analysis based on 18S rRNA sequences indicate that the Spiruroidea is paraphyletic, in which Oxyspirura and its closely related species are sisters to the filarial nematodes. We have also developed a PCR protocol based on the ITS2 sequence that allows sensitive and specific detection of eye worm DNA in feces. Using this newly developed protocol, we have determined that ~28% to 33% of the fecal samples collected from Northern Bobwhites and Scaled Quail in Texas in the spring of 2013 are O. petrowi positive.
Conclusions
The O. petrowi genome is rich in microsatellite sequences that may be used in future genotyping and molecular fingerprinting analysis. This eye worm is evolutionarily close to the filarial nematodes, implying that therapeutic strategies for filariasis such as Loa loa would be referential in developing treatments for the Thelazoidea parasites. Our qPCR-based survey has confirmed that O. petrowi infection is of potential concern to quail managers in Texas.
doi:10.1186/1471-2180-13-233
PMCID: PMC4015439  PMID: 24144118
3.  Comparative genomics of brain size evolution 
Which genetic changes took place during mammalian, primate and human evolution to build a larger brain? To answer this question, one has to correlate genetic changes with brain size changes across a phylogeny. Such a comparative genomics approach provides unique information to better understand brain evolution and brain development. However, its statistical power is limited for example due to the limited number of species, the presumably complex genetics of brain size evolution and the large search space of mammalian genomes. Hence, it is crucial to add functional information, for example by limiting the search space to genes and regulatory elements known to play a role in the relevant cell types during brain development. Similarly, it is crucial to experimentally follow up on hypotheses generated by such a comparative approach. Recent progress in understanding the molecular and cellular mechanisms of mammalian brain development, in genome sequencing and in genome editing, promises to make a close integration of evolutionary and experimental methods a fruitful approach to better understand the genetics of mammalian brain size evolution.
doi:10.3389/fnhum.2014.00345
PMCID: PMC4033227  PMID: 24904382
brain size evolution; comparative method; comparative genomics; brain development; microcephaly
4.  Revealing Posttranscriptional Regulatory Elements Through Network-Level Conservation 
PLoS Computational Biology  2005;1(7):e69.
We used network-level conservation between pairs of fly (Drosophila melanogaster/D. pseudoobscura) and worm (Caenorhabditis elegans/C. briggsae) genomes to detect highly conserved mRNA motifs in 3′ untranslated regions. Many of these elements are complementary to the 5′ extremity of known microRNAs (miRNAs), and likely correspond to their target sites. We also identify known targets of RNA-binding proteins, and many novel sites not yet known to be functional. Coherent sets of genes with similar function often bear the same conserved elements, providing new insights into their cellular functions. We also show that target sites for distinct miRNAs are often simultaneously conserved, suggesting combinatorial regulation by multiple miRNAs. A genome-wide search for conserved stem-loops, containing complementary sequences to the novel sites, revealed many new candidate miRNAs that likely target them. We also provide evidence that posttranscriptional networks have undergone extensive rewiring across distant phyla, despite strong conservation of regulatory elements themselves.
Synopsis
Organisms have evolved extensive regulatory mechanisms for the appropriate expression of genes within precise spatiotemporal contexts. Until recently most of this regulation was thought to be implemented by processes that operate at the “transcriptional” level, that is, by modifying the rate at which mRNA is synthesized. The discovery of short RNAs, termed microRNAs (miRNAs), which can affect gene expression either by degradation of target mRNAs or by inhibiting their translation, has focused much recent effort on determining their specific functional roles and the extent to which they contribute to establishing protein repertoires within individual cells. Chan and colleagues have applied a computational comparative genomic approach for identifying the targets of these miRNAs within 3′ untranslated regions of mRNAs in closely related flies and worms. Their approach identifies a large number of target genes for most of the known miRNAs in these species, providing evidence that these regulators have a much more extensive role than previously thought. The sets of genes targeted by each miRNA are enriched in various known functional classes, providing strong clues for their role in physiology and development. The authors went on to identify many novel miRNAs based on the sequence of highly conserved target sites. They also found a large number of targets that do not correspond to miRNAs, some of which match the targets of known RNA-binding proteins. By comparing the large catalog of putative regulatory elements between flies and worms, they show that, although a large fraction of these elements are conserved, they are targeting, by and large, different sets of genes.
doi:10.1371/journal.pcbi.0010069
PMCID: PMC1309705  PMID: 16355253
5.  Revealing Posttranscriptional Regulatory Elements Through Network-Level Conservation 
PLoS Computational Biology  2005;1(7):e69.
We used network-level conservation between pairs of fly (Drosophila melanogaster/D. pseudoobscura) and worm (Caenorhabditis elegans/C. briggsae) genomes to detect highly conserved mRNA motifs in 3′ untranslated regions. Many of these elements are complementary to the 5′ extremity of known microRNAs (miRNAs), and likely correspond to their target sites. We also identify known targets of RNA-binding proteins, and many novel sites not yet known to be functional. Coherent sets of genes with similar function often bear the same conserved elements, providing new insights into their cellular functions. We also show that target sites for distinct miRNAs are often simultaneously conserved, suggesting combinatorial regulation by multiple miRNAs. A genome-wide search for conserved stem-loops, containing complementary sequences to the novel sites, revealed many new candidate miRNAs that likely target them. We also provide evidence that posttranscriptional networks have undergone extensive rewiring across distant phyla, despite strong conservation of regulatory elements themselves.
Synopsis
Organisms have evolved extensive regulatory mechanisms for the appropriate expression of genes within precise spatiotemporal contexts. Until recently most of this regulation was thought to be implemented by processes that operate at the “transcriptional” level, that is, by modifying the rate at which mRNA is synthesized. The discovery of short RNAs, termed microRNAs (miRNAs), which can affect gene expression either by degradation of target mRNAs or by inhibiting their translation, has focused much recent effort on determining their specific functional roles and the extent to which they contribute to establishing protein repertoires within individual cells. Chan and colleagues have applied a computational comparative genomic approach for identifying the targets of these miRNAs within 3′ untranslated regions of mRNAs in closely related flies and worms. Their approach identifies a large number of target genes for most of the known miRNAs in these species, providing evidence that these regulators have a much more extensive role than previously thought. The sets of genes targeted by each miRNA are enriched in various known functional classes, providing strong clues for their role in physiology and development. The authors went on to identify many novel miRNAs based on the sequence of highly conserved target sites. They also found a large number of targets that do not correspond to miRNAs, some of which match the targets of known RNA-binding proteins. By comparing the large catalog of putative regulatory elements between flies and worms, they show that, although a large fraction of these elements are conserved, they are targeting, by and large, different sets of genes.
doi:10.1371/journal.pcbi.0010069
PMCID: PMC1309705  PMID: 16355253
6.  Sand fly-Leishmania interactions: long relationships are not necessarily easy 
Sand fly and Leishmania are one of the best studied vector-parasite models. Much is known about the development of these parasites within the sand fly, and how transmission to a suitable vertebrate host takes place. Various molecules secreted by the vector assist the establishment of the infection in a vertebrate, and changes to the vector are promoted by the parasites in order to facilitate or enhance transmission. Despite a generally accepted view that sand flies and Leishmania are also one of the oldest vector-pathogen pairs known, such long history has not been translated into a harmonic relationship. Leishmania are faced with many barriers to the establishment of a successful infection within the sand fly vector, and specific associations have been developed which are thought to represent aspects of a co-evolution between the parasite and its vectors. In this review, we highlight the journey taken by Leishmania during its development within the vector, and describe the issues associated with the natural barriers encountered by the parasite. Recent data revealed sexual replication of Leishmania within the sand fly, but it is yet unknown if such reproduction affects disease outcome. New approaches targeting sand fly molecules to prevent parasite transmission are being sought, and various techniques related to genetic manipulation of sand flies are being utilized.
doi:10.2174/1874421401004010195
PMCID: PMC3805382  PMID: 24159365
7.  A question of size: the eukaryotic proteome and the problems in defining it 
Nucleic Acids Research  2002;30(5):1083-1090.
We discuss the problems in defining the extent of the proteomes for completely sequenced eukaryotic organisms (i.e. the total number of protein-coding sequences), focusing on yeast, worm, fly and human. (i) Six years after completion of its genome sequence, the true size of the yeast proteome is still not defined. New small genes are still being discovered, and a large number of existing annotations are being called into question, with these questionable ORFs (qORFs) comprising up to one-fifth of the ‘current’ proteome. We discuss these in the context of an ideal genome-annotation strategy that considers the proteome as a rigorously defined subset of all possible coding sequences (‘the orfome’). (ii) Despite the greater apparent complexity of the fly (more cells, more complex physiology, longer lifespan), the nematode worm appears to have more genes. To explain this, we compare the annotated proteomes of worm and fly, relating to both genome-annotation and genome evolution issues. (iii) The unexpectedly small size of the gene complement estimated for the complete human genome provoked much public debate about the nature of biological complexity. However, in the first instance, for the human genome, the relationship between gene number and proteome size is far from simple. We survey the current estimates for the numbers of human genes and, from this, we estimate a range for the size of the human proteome. The determination of this is substantially hampered by the unknown extent of the cohort of pseudogenes (‘dead’ genes), in combination with the prevalence of alternative splicing. (Further information relating to yeast is available at http://genecensus.org/yeast/orfome)
PMCID: PMC101239  PMID: 11861898
8.  euGenes: a eukaryote genome information system 
Nucleic Acids Research  2002;30(1):145-148.
euGenes is a genome information system and database that provides a common summary of eukaryote genes and genomes, at http://iubio.bio.indiana.edu/eugenes/. Seven popular genomes are included: human, mouse, fruitfly, Caenorhabditis elegans worm, Saccharomyces yeast, Arabidopsis mustard weed and zebrafish, with more planned. This information, automatically extracted and updated from several source databases, offers features not readily available through other genome databases to bioscientists looking for gene relationships across organisms. The database describes 150 000 known, predicted and orphan genes, using consistent gene names along with their homologies and associations with a standard vocabulary of molecular functions, cell locations and biological processes. Usable whole-genome maps including features, chromosome locations and molecular data integration are available, as are options to retrieve sequences from these genomes. Search and retrieval methods for these data are easy to use and efficient, allowing one to ask combined questions of sequence features, protein functions and other gene attributes, and fetch results in reports, computable tabular outputs or bulk database forms. These summarized data are useful for integration in other projects, such as gene expression databases. euGenes provides an extensible, flexible genome information system for many organisms.
PMCID: PMC99146  PMID: 11752277
9.  i-Genome: A database to summarize oligonucleotide data in genomes 
BMC Genomics  2004;5:78.
Background
Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming.
Results
The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes.
Conclusions
This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.
doi:10.1186/1471-2164-5-78
PMCID: PMC526275  PMID: 15473908
repeat; genome index; oligonucleotide; database
10.  Identification and Characterization of a Mef2 Transcriptional Activator in Schistosome Parasites 
Myocyte enhancer factor 2 protein (Mef2) is an evolutionarily conserved activator of transcription that is critical to induce and control complex processes in myogenesis and neurogenesis in vertebrates and insects, and osteogenesis in vertebrates. In Drosophila, Mef2 null mutants are unable to produce differentiated muscle cells, and in vertebrates, Mef2 mutants are embryonic lethal. Schistosome worms are responsible for over 200 million cases of schistosomiasis globally, but little is known about early development of schistosome parasites after infecting a vertebrate host. Understanding basic schistosome development could be crucial to delineating potential drug targets. Here, we identify and characterize Mef2 from the schistosome worm Schistosoma mansoni (SmMef2). We initially identified SmMef2 as a homolog to the yeast Mef2 homolog, Resistance to Lethality of MKK1P386 overexpression (Rlm1), and we show that SmMef2 is homologous to conserved Mef2 family proteins. Using a genetics approach, we demonstrate that SmMef2 is a transactivator that can induce transcription of four separate heterologous reporter genes by yeast one-hybrid analysis. We also show that Mef2 is expressed during several stages of schistosome development by quantitative PCR and that it can bind to conserved Mef2 DNA consensus binding sequences.
Author Summary
Schistosome parasites infect more than 200 million people worldwide and cause human schistosomiasis. Free-swimming schistosome larvae are highly mobile and invade and penetrate the host's skin to perpetuate their lifecycle in their human host, growing from 90–215 micrometers in length as a schistosomulum to a 7–20 millimeter long adult worm. Few molecular pathways have been identified in schistosome worms that are important for parasite early development. The myocyte enhancer factor protein 2 is a major regulator of muscle and nerve development in mammals and insects and is highly conserved from bread yeast to vertebrates. Here we identify and characterize the Mef2 activator from parasitic schistosome worms, the first described in any parasitic worm, and delineation of its function may be important to further understanding the basic biology of schistosome early development. Additionally, since schistosomes developed early evolutionarily, an investigation of schistosome Mef2 regulatory mechanisms could lead to a greater understanding of the development of early muscle and neurogenic development in animals.
doi:10.1371/journal.pntd.0001443
PMCID: PMC3250504  PMID: 22235355
11.  Inference of Gene-Phenotype Associations via Protein-Protein Interaction and Orthology 
PLoS ONE  2013;8(10):e77478.
One of the fundamental goals of genetics is to understand gene functions and their associated phenotypes. To achieve this goal, in this study we developed a computational algorithm that uses orthology and protein-protein interaction information to infer gene-phenotype associations for multiple species. Furthermore, we developed a web server that provides genome-wide phenotype inference for six species: fly, human, mouse, worm, yeast, and zebrafish. We evaluated our inference method by comparing the inferred results with known gene-phenotype associations. The high Area Under the Curve values suggest a significant performance of our method. By applying our method to two human representative diseases, Type 2 Diabetes and Breast Cancer, we demonstrated that our method is able to identify related Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. The web server can be used to infer functions and putative phenotypes of a gene along with the candidate genes of a phenotype, and thus aids in disease candidate gene discovery. Our web server is available at http://jjwanglab.org/PhenoPPIOrth.
doi:10.1371/journal.pone.0077478
PMCID: PMC3806783  PMID: 24194887
12.  Opportunities for text mining in the FlyBase genetic literature curation workflow 
FlyBase is the model organism database for Drosophila genetic and genomic information. Over the last 20 years, FlyBase has had to adapt and change to keep abreast of advances in biology and database design. We are continually looking for ways to improve curation efficiency and efficacy. Genetic literature curation focuses on the extraction of genetic entities (e.g. genes, mutant alleles, transgenic constructs) and their associated phenotypes and Gene Ontology terms from the published literature. Over 2000 Drosophila research articles are now published every year. These articles are becoming ever more data-rich and there is a growing need for text mining to shoulder some of the burden of paper triage and data extraction. In this article, we describe our curation workflow, along with some of the problems and bottlenecks therein, and highlight the opportunities for text mining. We do so in the hope of encouraging the BioCreative community to help us to develop effective methods to mine this torrent of information.
Database URL: http://flybase.org
doi:10.1093/database/bas039
PMCID: PMC3500518  PMID: 23160412
13.  Genetic Basis for Spontaneous Hybrid Genome Doubling during Allopolyploid Speciation of Common Wheat Shown by Natural Variation Analyses of the Paternal Species 
PLoS ONE  2013;8(8):e68310.
The complex process of allopolyploid speciation includes various mechanisms ranging from species crosses and hybrid genome doubling to genome alterations and the establishment of new allopolyploids as persisting natural entities. Currently, little is known about the genetic mechanisms that underlie hybrid genome doubling, despite the fact that natural allopolyploid formation is highly dependent on this phenomenon. We examined the genetic basis for the spontaneous genome doubling of triploid F1 hybrids between the direct ancestors of allohexaploid common wheat (Triticum aestivum L., AABBDD genome), namely Triticumturgidum L. (AABB genome) and Aegilopstauschii Coss. (DD genome). An Ae. tauschii intraspecific lineage that is closely related to the D genome of common wheat was identified by population-based analysis. Two representative accessions, one that produces a high-genome-doubling-frequency hybrid when crossed with a T. turgidum cultivar and the other that produces a low-genome-doubling-frequency hybrid with the same cultivar, were chosen from that lineage for further analyses. A series of investigations including fertility analysis, immunostaining, and quantitative trait locus (QTL) analysis showed that (1) production of functional unreduced gametes through nonreductional meiosis is an early step key to successful hybrid genome doubling, (2) first division restitution is one of the cytological mechanisms that cause meiotic nonreduction during the production of functional male unreduced gametes, and (3) six QTLs in the Ae. tauschii genome, most of which likely regulate nonreductional meiosis and its subsequent gamete production processes, are involved in hybrid genome doubling. Interlineage comparisons of Ae. tauschii’s ability to cause hybrid genome doubling suggested an evolutionary model for the natural variation pattern of the trait in which non-deleterious mutations in six QTLs may have important roles. The findings of this study demonstrated that the genetic mechanisms for hybrid genome doubling could be studied based on the intrinsic natural variation that exists in the parental species.
doi:10.1371/journal.pone.0068310
PMCID: PMC3738567  PMID: 23950867
14.  Revealing Time-Unlocked Brain Activity from MEG Measurements by Common Waveform Estimation 
PLoS ONE  2014;9(5):e98014.
Brain activities related to cognitive functions, such as attention, occur with unknown and variable delays after stimulus onsets. Recently, we proposed a method (Common Waveform Estimation, CWE) that could extract such brain activities from magnetoencephalography (MEG) or electroencephalography (EEG) measurements. CWE estimates spatiotemporal MEG/EEG patterns occurring with unknown and variable delays, referred to here as unlocked waveforms, without hypotheses about their shapes. The purpose of this study is to demonstrate the usefulness of CWE for cognitive neuroscience. For this purpose, we show procedures to estimate unlocked waveforms using CWE and to examine their role. We applied CWE to the MEG epochs during Go trials of a visual Go/NoGo task. This revealed unlocked waveforms with interesting properties, specifically large alpha oscillations around the temporal areas. To examine the role of the unlocked waveform, we attempted to estimate the strength of the brain activity of the unlocked waveform in various conditions. We made a spatial filter to extract the component reflecting the brain activity of the unlocked waveform, applied this spatial filter to MEG data under different conditions (a passive viewing, a simple reaction time, and Go/NoGo tasks), and calculated the powers of the extracted components. Comparing the powers across these conditions suggests that the unlocked waveforms may reflect the inhibition of the task-irrelevant activities in the temporal regions while the subject attends to the visual stimulus. Our results demonstrate that CWE is a potential tool for revealing new findings of cognitive brain functions without any hypothesis in advance.
doi:10.1371/journal.pone.0098014
PMCID: PMC4039443  PMID: 24879410
15.  WormBase: better software, richer content 
Nucleic Acids Research  2005;34(Database issue):D475-D478.
WormBase (), the public database for genomics and biology of Caenorhabditis elegans, has been restructured for stronger performance and expanded for richer biological content. Performance was improved by accelerating the loading of central data pages such as the omnibus Gene page, by rationalizing internal data structures and software for greater portability, and by making the Genome Browser highly customizable in how it views and exports genomic subsequences. Arbitrarily complex, user-specified queries are now possible through Textpresso (for all available literature) and through WormMart (for most genomic data). Biological content was enriched by reconciling all available cDNA and expressed sequence tag data with gene predictions, clarifying single nucleotide polymorphism and RNAi sites, and summarizing known functions for most genes studied in this organism.
doi:10.1093/nar/gkj061
PMCID: PMC1347424  PMID: 16381915
16.  Computational detection of genomic cis-regulatory modules applied to body patterning in the early Drosophila embryo 
BMC Bioinformatics  2002;3:30.
Background
Regulation of gene transcription is crucial for the function and development of all organisms. While gene prediction programs that identify protein coding sequence are used with remarkable success in the annotation of genomes, the development of computational methods to analyze noncoding regions and to delineate transcriptional control elements is still in its infancy.
Results
Here we present novel algorithms to detect cis-regulatory modules through genome wide scans for clusters of transcription factor binding sites using three levels of prior information. When binding sites for the factors are known, our statistical segmentation algorithm, Ahab, yields about 150 putative gap gene regulated modules, with no adjustable parameters other than a window size. If one or more related modules are known, but no binding sites, repeated motifs can be found by a customized Gibbs sampler and input to Ahab, to predict genes with similar regulation. Finally using only the genome, we developed a third algorithm, Argos, that counts and scores clusters of overrepresented motifs in a window of sequence. Argos recovers many of the known modules, upstream of the segmentation genes, with no training data.
Conclusions
We have demonstrated, in the case of body patterning in the Drosophila embryo, that our algorithms allow the genome-wide identification of regulatory modules. We believe that Ahab overcomes many problems of recent approaches and we estimated the false positive rate to be about 50%. Argos is the first successful attempt to predict regulatory modules using only the genome without training data. Complete results and module predictions across the Drosophila genome are available at http://uqbar.rockefeller.edu/~siggia/.
doi:10.1186/1471-2105-3-30
PMCID: PMC139975  PMID: 12398796
17.  Multiple genetic switches spontaneously modulating bacterial mutability 
Background
All life forms need both high genetic stability to survive as species and a degree of mutability to evolve for adaptation, but little is known about how the organisms balance the two seemingly conflicting aspects of life: genetic stability and mutability. The DNA mismatch repair (MMR) system is essential for maintaining genetic stability and defects in MMR lead to high mutability. Evolution is driven by genetic novelty, such as point mutation and lateral gene transfer, both of which require genetic mutability. However, normally a functional MMR system would strongly inhibit such genomic changes. Our previous work indicated that MMR gene allele conversion between functional and non-functional states through copy number changes of small tandem repeats could occur spontaneously via slipped-strand mis-pairing during DNA replication and therefore may play a role of genetic switches to modulate the bacterial mutability at the population level. The open question was: when the conversion from functional to defective MMR is prohibited, will bacteria still be able to evolve by accepting laterally transferred DNA or accumulating mutations?
Results
To prohibit allele conversion, we "locked" the MMR genes through nucleotide replacements. We then scored changes in bacterial mutability and found that Salmonella strains with MMR locked at the functional state had significantly decreased mutability. To determine the generalizability of this kind of mutability 'switching' among a wider range of bacteria, we examined the distribution of tandem repeats within MMR genes in over 100 bacterial species and found that multiple genetic switches might exist in these bacteria and may spontaneously modulate bacterial mutability during evolution.
Conclusions
MMR allele conversion through repeats-mediated slipped-strand mis-pairing may function as a spontaneous mechanism to switch between high genetic stability and mutability during bacterial evolution.
doi:10.1186/1471-2148-10-277
PMCID: PMC2955026  PMID: 20836863
18.  Biophysical Detection of Diversity and Bias in GPCR Function 
Guanine nucleotide binding protein (G protein)-coupled receptors (GPCRs) function in complexes with a range of molecules and proteins including ligands, G proteins, arrestins, ubiquitin, and other receptors. Elements of these complexes may interact constitutively or dynamically, dependent upon factors such as ligand binding, phosphorylation, and dephosphorylation. They may also be allosterically modulated by other proteins in a manner that changes temporally and spatially within the cell. Elucidating how these complexes function has been greatly enhanced by biophysical technologies that are able to monitor proximity and/or binding, often in real time and in live cells. These include resonance energy transfer approaches such as bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET). Furthermore, the use of fluorescent ligands has enabled novel insights into allosteric interactions between GPCRs. Consequently, biophysical approaches are helping to unlock the amazing diversity and bias in G protein-coupled receptor signaling.
doi:10.3389/fendo.2014.00026
PMCID: PMC3943086  PMID: 24634666
bioluminescence resonance energy transfer; BRET; fluorescence resonance energy transfer; FRET; GPCR; GPCR-HIT; heteromer; Receptor-HIT
19.  From remote enhancers to gene regulation: charting the genome's regulatory landscapes 
Vertebrate genes are characterized by the presence of cis-regulatory elements located at great distances from the genes they control. Alterations of these elements have been implicated in human diseases and evolution, yet little is known about how these elements interact with their surrounding sequences. A recent survey of the mouse genome with a regulatory sensor showed that the regulatory activities of these elements are not organized in a gene-centric manner, but instead are broadly distributed along chromosomes, forming large regulatory landscapes with distinct tissue-specific activities. A large genome-wide collection of expression data from this regulatory sensor revealed some basic principles of this complex genome regulatory architecture, including a substantial interplay between enhancers and other types of activities to modulate gene expression. We discuss the implications of these findings for our understanding of non-coding transcription, and of the possible consequences of structural genomic variations in disease and evolution.
doi:10.1098/rstb.2012.0358
PMCID: PMC3682723  PMID: 23650632
gene regulation; regulatory landscapes and remote enhancers; repressors and latent regulatory activity; specificity of gene–enhancer regulatory interactions
20.  To understand the whole, you must know the parts: Unraveling the roles of protein-DNA interactions in genome regulation 
The Analyst  2011;136(15):3060-3065.
The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. This process dictates which genes are expressed in which tissues, and controls how various cell types grow, differentiate, and respond to their environments. Although the deciphering of the human genome sequence has given us the “source code” for life, we still know far too little about the mechanisms that control which sets of genes are active in which tissues, and how their expression is regulated. It is clear, however, that much of this system depends upon the sequence-specific interactions of regulatory proteins with particular genetic loci. To be able to unravel the details of these interactions on a genome-wide basis, it is necessary to know what proteins are bound to the DNA where in the genome, and to be able to monitor how those proteins change over time and in response to external stimuli. Developing a new technology to provide this information constitutes a “Grand Challenge” for Analytical Chemistry. In this brief article we outline the nature of this challenge, and propose one strategy to address it.
doi:10.1039/c1an15037e
PMCID: PMC3672230  PMID: 21629937
21.  Cyclic nucleotide specific phosphodiesterases of Leishmania major 
BMC Microbiology  2006;6:25.
Background
Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major.
Results
This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation.
Conclusion
The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.
doi:10.1186/1471-2180-6-25
PMCID: PMC1431542  PMID: 16522215
22.  Dynamic Nucleosome Organization at hox Promoters during Zebrafish Embryogenesis 
PLoS ONE  2013;8(5):e63175.
Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.
doi:10.1371/journal.pone.0063175
PMCID: PMC3650070  PMID: 23671670
23.  Functional Characterization of the Human Mariner Transposon Hsmar2 
PLoS ONE  2013;8(9):e73227.
DNA transposons are mobile elements with the ability to mobilize and transport genetic information between different chromosomal loci. Unfortunately, most transposons copies are currently inactivated, little is known about mariner elements in humans despite their role in the evolution of the human genome, even though the Hsmar2 transposon is associated to hotspots for homologous recombination involved in human genetic disorders as Charcot–Marie–Tooth, Prader-Willi/Angelman, and Williams syndromes. This manuscript describes the functional characterization of the human HSMAR2 transposase generated from fossil sequences and shows that the native HSMAR2 is active in human cells, but also in bacteria, with an efficiency similar to other mariner elements. We observe that the sub-cellular localization of HSMAR2 is dependent on the host cell type, and is cytotoxic when overexpressed in HeLa cells. Finally, we also demonstrate that the binding of HSMAR2 to its own ITRs is specific, and that the excision reaction leaves non-canonical footprints both in bacteria and eukaryotic cells.
doi:10.1371/journal.pone.0073227
PMCID: PMC3770610  PMID: 24039890
24.  Heterologous Stop Codon Readthrough of Metazoan Readthrough Candidates in Yeast 
PLoS ONE  2013;8(3):e59450.
Recent analysis of genomic signatures in mammals, flies, and worms indicates that functional translational stop codon readthrough is considerably more abundant in metazoa than previously recognized, but this analysis provides only limited clues about the function or mechanism of readthrough. If an mRNA known to be read through in one species is also read through in another, perhaps these questions can be studied in a simpler setting. With this end in mind, we have investigated whether some of the readthrough genes in human, fly, and worm also exhibit readthrough when expressed in S. cerevisiae. We found that readthrough was highest in a gene with a post-stop hexamer known to trigger readthrough, while other metazoan readthrough genes exhibit borderline readthrough in S. cerevisiae.
doi:10.1371/journal.pone.0059450
PMCID: PMC3609751  PMID: 23544069
25.  WormBase: a cross-species database for comparative genomics 
Nucleic Acids Research  2003;31(1):133-137.
WormBase (http://www.wormbase.org/) is a web-accessible central data repository for information about Caenorhabditis elegans and related nematodes. The past two years have seen a significant expansion in the biological scope of WormBase, including the integration of large-scale, genome-wide data sets, the inclusion of genome sequence and gene predictions from related species and active literature curation. This expansion of data has also driven the development and refinement of user interfaces and operability, including a new Genome Browser, new searches and facilities for data access and the inclusion of extensive documentation. These advances have expanded WormBase beyond the obvious target audience of C. elegans researchers, to include researchers wishing to explore problems in functional and comparative genomics within the context of a powerful genetic system.
PMCID: PMC165500  PMID: 12519966

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