Molecular Biology and Biochemistry, Department of

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Combinatorial RNA Interference In Caenorhabditis elegans Reveals That Redundancy Between Gene Duplicates Can Be Maintained For More Than 80 Million Years of Evolution

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2006
Abstract: 

Background: Systematic analyses of loss-of-function phenotypes have been carried out for mostgenes in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster. Although suchstudies vastly expand our knowledge of single gene function, they do not address redundancy ingenetic networks. Developing tools for the systematic mapping of genetic interactions is thus a keystep in exploring the relationship between genotype and phenotype.Results: We established conditions for RNA interference (RNAi) in C. elegans to target multiplegenes simultaneously in a high-throughput setting. Using this approach, we can detect the greatmajority of previously known synthetic genetic interactions. We used this assay to examine theredundancy of duplicated genes in the genome of C. elegans that correspond to single orthologs inS. cerevisiae or D. melanogaster and identified 16 pairs of duplicated genes that have redundantfunctions. Remarkably, 14 of these redundant gene pairs were duplicated before the divergence ofC. elegans and C. briggsae 80-110 million years ago, suggesting that there has been selective pressureto maintain the overlap in function between some gene duplicates.Conclusion: We established a high throughput method for examining genetic interactions usingcombinatorial RNAi in C. elegans. Using this technique, we demonstrated that many duplicatedgenes can retain redundant functions for more than 80 million years of evolution. This providesstrong support for evolutionary models that predict that genetic redundancy between duplicatedgenes can be actively maintained by natural selection and is not just a transient side effect of recentgene duplication events.

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Article
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A Highly Redundant BAC Library of Atlantic salmon (Salmo salar): An Important Tool for Salmon Projects

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2005
Abstract: 

Background: As farming of Atlantic salmon is growing as an aquaculture enterprise, the need to identify thegenomic mechanisms for specific traits is becoming more important in breeding and management of the animal.Traits of importance might be related to growth, disease resistance, food conversion efficiency, color or taste.To identify genomic regions responsible for specific traits, genomic large insert libraries have previously provento be of crucial importance. These large insert libraries can be screened using gene or genetic markers in orderto identify and map regions of interest. Furthermore, large-scale mapping can utilize highly redundant libraries ingenome projects, and hence provide valuable data on the genome structure.Results: Here we report the construction and characterization of a highly redundant bacterial artificialchromosome (BAC) library constructed from a Norwegian aquaculture strain male of Atlantic salmon (Salmosalar). The library consists of a total number of 305 557 clones, in which approximately 299 000 are recombinants.The average insert size of the library is 188 kbp, representing 18-fold genome coverage. High-density filters eachconsisting of 18 432 clones spotted in duplicates have been produced for hybridization screening, and are publiclyavailable [1]. To characterize the library, 15 expressed sequence tags (ESTs) derived overgos and 12 oligosequences derived from microsatellite markers were used in hybridization screening of the complete BAC library.Secondary hybridizations with individual probes were performed for the clones detected. The BACs positive forthe EST probes were fingerprinted and mapped into contigs, yielding an average of 3 contigs for each probe.Clones identified using genomic probes were PCR verified using microsatellite specific primers.Conclusion: Identification of genes and genomic regions of interest is greatly aided by the availability of theCHORI-214 Atlantic salmon BAC library. We have demonstrated the library's ability to identify specific genes andgenetic markers using hybridization, PCR and fingerprinting experiments. In addition, multiple fingerprintingcontigs indicated a pseudo-tetraploidity of the Atlantic salmon genome. The highly redundant CHORI-214 BAClibrary is expected to be an important resource for mapping and sequencing of the Atlantic salmon genome.

Document type: 
Article

Spindle Assembly Checkpoint Genes Reveal Distinct as well as Overlapping Expression that Implicates MDF-2/Mad2 in Postembryonic Seam Cell Proliferation in Caenorhabditis elegans

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

Background: The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of theanaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to thespindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play,during postembryonic development of a multicellular organism, remain largely unexplored.Results: We have used GFP fusions of 5’ upstream intergenic regulatory sequences to assay spatiotemporalexpression patterns of eight conserved genes implicated in the spindle assembly checkpoint function inCaenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFPexpression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissuespecificexpression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, weshow that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment ofseam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we showthat these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20 bythe SAC component MDF-2 is important for proper postembryonic cell proliferation.Conclusion: Our results indicate that SAC genes display different tissue-specific expression patterns duringpostembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells,suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may ormay not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seamcell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly,our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particularMDF-2 loss, than embryonic cell division.

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A Combined Approach Exploring Gene Function Based on Worm-Human Orthology

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2005
Abstract: 

Background: Many aspects of the nematode Caenorhabditis elegans biology are conservedbetween invertebrates and vertebrates establishing this particular organism as an excellent geneticmodel. Because of its small size, large populations and self-fertilization of the hermaphrodite,functional predictions carried out by genetic modifications as well as RNAi screens, can be rapidlytested.Results: In order to explore the function of a set of C. elegans genes of unknown function, as wellas their potential functional roles in the human genome, we performed a phylogenetic analysis toselect the most probable worm orthologs. A total of 13 C. elegans genes were subjected to downregulationvia RNAi and characterization of expression profiles using GFP strains. Previouslyunknown distinct expression patterns were observed for four of the analyzed genes, as well as fourvisible RNAi phenotypes. In addition, subcellular protein over-expression profiles of the humanorthologs for seven out of the thirteen genes using human cells were also analyzed.Conclusion: By combining a whole-organism approach using C. elegans with complementaryexperimental work done on human cell lines, this analysis extends currently available informationon the selected set of genes.

Document type: 
Article

Gene Expression Profiling of Oxidative Stress Response of C. elegans Aging Defective AMPK Mutants using Massively Parallel Transcriptome Sequencing

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

Background: A strong association between stress resistance and longevity in multicellular organisms has beenestablished as many mutations that extend lifespan also show increased resistance to stress. AAK-2, the C. eleganshomolog of an alpha subunit of AMP-activated protein kinase (AMPK) is an intracellular fuel sensor that regulatescellular energy homeostasis and functions in stress resistance and lifespan extension.Findings: Here, we investigated global transcriptional responses of aak-2 mutants to oxidative stress and in turnidentified potential downstream targets of AAK-2 involved in stress resistance in C. elegans. We employed massivelyparallel Illumina sequencing technology and performed comprehensive comparative transcriptome analysis.Specifically, we compared the transcriptomes of aak-2 and wild type animals under normal conditions andconditions of induced oxidative stress. This research has presented a snapshot of genome-wide transcriptionalactivities that take place in C. elegans in response to oxidative stress both in the presence and absence of AAK-2.Conclusions: The analysis presented in this study has enabled us to identify potential genes involved in stressresistance that may be either directly or indirectly under the control of AAK-2. Furthermore, we have extended ourcurrent knowledge of general defense responses of C. elegans against oxidative stress supporting the function forAAK-2 in inhibition of biosynthetic processes, especially lipid synthesis, under oxidative stress and transcriptionalregulation of genes involved in reproductive processes.

Document type: 
Report

Transcriptome Analysis for Caenorhabditis elegans Based on Novel Expressed Sequence Tags

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2008
Abstract: 

Background: We have applied a high-throughput pyrosequencing technology for transcriptome profiling ofCaenorhabditis elegans in its first larval stage. Using this approach, we have generated a large amount of data forexpressed sequence tags, which provides an opportunity for the discovery of putative novel transcripts andalternative splice variants that could be developmentally specific to the first larval stage. This work alsodemonstrates the successful and efficient application of a next generation sequencing methodology.Results: We have generated over 30 million bases of novel expressed sequence tags from first larval stage wormsutilizing high-throughput sequencing technology. We have shown that approximately 14% of the newly sequencedexpressed sequence tags map completely or partially to genomic regions where there are no annotated genes orsplice variants and therefore, imply that these are novel genetic structures. Expressed sequence tags, which mapto intergenic (around 1000) and intronic regions (around 580), may represent novel transcribed regions, such asunannotated or unrecognized small protein-coding or non-protein-coding genes or splice variants. Expressedsequence tags, which map across intron-exon boundaries (around 300), indicate possible alternative splice sites,while expressed sequence tags, which map near the ends of known transcripts (around 600), suggest extensionof the coding or untranslated regions. We have also discovered that intergenic and intronic expressed sequencetags, which are well conserved across different nematode species, are likely to represent non-coding RNAs.Lastly, we have incorporated available serial analysis of gene expression data generated from first larval stageworms, in order to predict novel transcripts that might be specifically or predominantly expressed in the firstlarval stage.Conclusion: We have demonstrated the use of a high-throughput sequencing methodology to efficientlyproduce a snap-shot of transcriptional activities occurring in the first larval stage of C. elegans development. Suchapplication of this new sequencing technique allows for high-throughput, genome-wide experimental verificationof known and novel transcripts. This study provides a more complete C. elegans transcriptome profile and,furthermore, gives insight into the evolutionary and biological complexity of this organism.

Document type: 
Article

The ABC Transporter Gene Family of Caenorhabditis elegans Has Implications for the Evolutionary Dynamics of Multidrug Resistance in Eukaryotes

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

Background: Many drugs of natural origin are hydrophobic and can pass through cell membranes.Hydrophobic molecules must be susceptible to active efflux systems if they are to be maintained atlower concentrations in cells than in their environment. Multi-drug resistance (MDR), oftenmediated by intrinsic membrane proteins that couple energy to drug efflux, provides this function.All eukaryotic genomes encode several gene families capable of encoding MDR functions, amongwhich the ABC transporters are the largest. The number of candidate MDR genes means that studyof the drug-resistance properties of an organism cannot be effectively carried out without taking agenomic perspective.Results: We have annotated sequences for all 60 ABC transporters from the Caenorhabditiselegans genome, and performed a phylogenetic analysis of these along with the 49 human, 30 yeast,and 57 fly ABC transporters currently available in GenBank. Classification according to a unifiednomenclature is presented. Comparison between genomes reveals much gene duplication and loss,and surprisingly little orthology among analogous genes. Proteins capable of conferring MDR arefound in several distinct subfamilies and are likely to have arisen independently multiple times.Conclusions: ABC transporter evolution fits a pattern expected from a process termed 'dynamiccoherence'.This is an unusual result for such a highly conserved gene family as this one, present inall domains of cellular life. Mechanistically, this may result from the broad substrate specificity ofsome ABC proteins, which both reduces selection against gene loss, and leads to the facile sortingof functions among paralogs following gene duplication.

Document type: 
Article

Target Selection of Soluble Protein Complexes for Structural Proteomics Studies

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2005
Abstract: 

Background: Protein expression in E. coli is the most commonly used system to produce proteinfor structural studies, because it is fast and inexpensive and can produce large quantity of proteins.However, when proteins from other species such as mammalian are produced in this system,problems of protein expression and solubility arise [1]. Structural genomics project are currentlyinvestigating proteomics pipelines that would produce sufficient quantities of recombinant proteinsfor structural studies of protein complexes. To investigate how the E. coli protein expressionsystem could be used for this purpose, we purified apoptotic binary protein complexes formedbetween members of the Caspase Associated Recruitment Domain (CARD) family.Results: A combinatorial approach to the generation of protein complexes was performedbetween members of the CARD domain protein family that have the ability to form hetero-dimersbetween each other. In our method, each gene coding for a specific protein partner is cloned inpET-28b (Novagen) and PGEX2T (Amersham) expression vectors. All combinations of proteincomplexes are then obtained by reconstituting complexes from purified components in nativeconditions, after denaturation-renaturation or co-expression. Our study applied to 14 solubleCARD domain proteins revealed that co-expression studies perform better than native anddenaturation-renaturation methods. In this study, we confirm existing interactions obtained invivoin mammalian cells and also predict new interactions.Conclusion: The simplicity of this screening method could be easily scaled up to identify solubleprotein complexes for structural genomic projects. This study reports informative statistics on thesolubility of human protein complexes expressed in E.coli belonging to the human CARD proteinfamily.

Document type: 
Article

Genomic Sequence of a Mutant Strain of Caenorhabditis elegans with an Altered Recombination Pattern

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

Background: The original sequencing and annotation of the Caenorhabditis elegans genome along with recentadvances in sequencing technology provide an exceptional opportunity for the genomic analysis of wild-type andmutant strains. Using the Illumina Genome Analyzer, we sequenced the entire genome of Rec-1, a strain that altersthe distribution of meiotic crossovers without changing the overall frequency. Rec-1 was derived fromethylmethane sulfonate (EMS)-treated strains, one of which had a high level of transposable element mobility.Sequencing of this strain provides an opportunity to examine the consequences on the genome of altering thedistribution of meiotic recombination events.Results: Using Illumina sequencing and MAQ software, 83% of the base pair sequence reads were aligned to thereference genome available at Wormbase, providing a 21-fold coverage of the genome. Using the softwareprograms MAQ and Slider, we observed 1124 base pair differences between Rec-1 and the reference genome inWormbase (WS190), and 441 between the mutagenized Rec-1 (BC313) and the wild-type N2 strain (VC2010). Themost frequent base-substitution was G:C to A:T, 141 for the entire genome most of which were on chromosomes Ior X, 55 and 31 respectively. With this data removed, no obvious pattern in the distribution of the base differencesalong the chromosomes was apparent. No major chromosomal rearrangements were observed, but additionalinsertions of transposable elements were detected. There are 11 extra copies of Tc1, and 8 of Tc2 in the Rec-1genome, most likely the remains of past high-hopper activity in a progenitor strain.Conclusion: Our analysis of high-throughput sequencing was able to detect regions of direct repeat sequences,deletions, insertions of transposable elements, and base pair differences. A subset of sequence alterations affectingcoding regions were confirmed by an independent approach using oligo array comparative genome hybridization.The major phenotype of the Rec-1 strain is an alteration in the preferred position of the meiotic recombinationevent with no other significant phenotypic consequences. In this study, we observed no evidence of a mutatoreffect at the nucleotide level attributable to the Rec-1 mutation.

Document type: 
Article

Assessing the Precision of High-Throughput Computational and Laboratory Approaches for the Genome-Wide Identification of Protein Subcellular Localization in Bacteria

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2005
Abstract: 

AbstractBackground: Identification of a bacterial protein's subcellular localization (SCL) is important forgenome annotation, function prediction and drug or vaccine target identification. Subcellularfractionation techniques combined with recent proteomics technology permits the identification oflarge numbers of proteins from distinct bacterial compartments. However, the fractionation of acomplex structure like the cell into several subcellular compartments is not a trivial task.Contamination from other compartments may occur, and some proteins may reside in multiplelocalizations. New computational methods have been reported over the past few years that nowpermit much more accurate, genome-wide analysis of the SCL of protein sequences deduced fromgenomes. There is a need to compare such computational methods with laboratory proteomicsapproaches to identify the most effective current approach for genome-wide localizationcharacterization and annotation.Results: In this study, ten subcellular proteome analyses of bacterial compartments werereviewed. PSORTb version 2.0 was used to computationally predict the localization of proteinsreported in these publications, and these computational predictions were then compared to thelocalizations determined by the proteomics study. By using a combined approach, we were able toidentify a number of contaminants and proteins with dual localizations, and were able to moreaccurately identify membrane subproteomes. Our results allowed us to estimate the precision levelof laboratory subproteome studies and we show here that, on average, recent high-precisioncomputational methods such as PSORTb now have a lower error rate than laboratory methods.Conclusion: We have performed the first focused comparison of genome-wide proteomic andcomputational methods for subcellular localization identification, and show that computationalmethods have now attained a level of precision that is exceeding that of high-throughput laboratoryapproaches. We note that analysis of all cellular fractions collectively is required to effectivelyprovide localization information from laboratory studies, and we propose an overall approach togenome-wide subcellular localization characterization that capitalizes on the complementary natureof current laboratory and computational methods.

Document type: 
Article