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Annotation of Two Large Contiguous Regions from the Haemonchus contortus Genome Using RNA-seq and Comparative Analysis with Caenorhabditis elegans  [PDF]
Roz Laing, Martin Hunt, Anna V. Protasio, Gary Saunders, Karen Mungall, Steven Laing, Frank Jackson, Michael Quail, Robin Beech, Matthew Berriman, John S. Gilleard
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023216
Abstract: The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes.
Transcriptome analysis for Caenorhabditis elegans based on novel expressed sequence tags
Heesun Shin, Martin Hirst, Matthew N Bainbridge, Vincent Magrini, Elaine Mardis, Donald G Moerman, Marco A Marra, David L Baillie, Steven JM Jones
BMC Biology , 2008, DOI: 10.1186/1741-7007-6-30
Abstract: We have generated over 30 million bases of novel expressed sequence tags from first larval stage worms utilizing high-throughput sequencing technology. We have shown that approximately 14% of the newly sequenced expressed sequence tags map completely or partially to genomic regions where there are no annotated genes or splice variants and therefore, imply that these are novel genetic structures. Expressed sequence tags, which map to intergenic (around 1000) and intronic regions (around 580), may represent novel transcribed regions, such as unannotated or unrecognized small protein-coding or non-protein-coding genes or splice variants. Expressed sequence 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 extension of the coding or untranslated regions. We have also discovered that intergenic and intronic expressed sequence tags, 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 stage worms, in order to predict novel transcripts that might be specifically or predominantly expressed in the first larval stage.We have demonstrated the use of a high-throughput sequencing methodology to efficiently produce a snap-shot of transcriptional activities occurring in the first larval stage of C. elegans development. Such application of this new sequencing technique allows for high-throughput, genome-wide experimental verification of 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.Computationally based genomic analyses have been able to accomplish interpretation of the genome of Caenorhabditis elegans on a global scale. The aims of s
Chromosome-Biased Binding and Gene Regulation by the Caenorhabditis elegans DRM Complex  [PDF]
Tomoko M. Tabuchi equal contributor,Bart Deplancke equal contributor,Naoki Osato,Lihua J. Zhu,M. Inmaculada Barrasa,Melissa M. Harrison,H. Robert Horvitz,Albertha J. M. Walhout ,Kirsten A. Hagstrom
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002074
Abstract: DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we describe new aspects of DRM binding and function revealed through genome-wide analyses of the Caenorhabditis elegans DRM subunit LIN-54. We show that LIN-54 DNA-binding activity recruits DRM to promoters enriched for adjacent putative E2F/DP and LIN-54 binding sites, suggesting that these two DNA–binding moieties together direct DRM to its target genes. Chromatin immunoprecipitation and gene expression profiling reveals conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find that LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, C. elegans DRM does not act uniformly throughout the genome: the DRM recruitment motif, DRM binding, and DRM-regulated embryonic genes are all under-represented on the X chromosome. However, germline genes down-regulated in lin-54 mutants are over-represented on the X chromosome. We discuss models for how loss of autosome-bound DRM may enhance germline X chromosome silencing. We propose that autosome-enriched binding of DRM arose in C. elegans as a consequence of germline X chromosome silencing and the evolutionary redistribution of germline-expressed and essential target genes to autosomes. Sex chromosome gene regulation may thus have profound evolutionary effects on genome organization and transcriptional regulatory networks.
The draft genome sequence of the nematode Caenorhabditis briggsae, a companion to C. elegans
Bhagwati P Gupta, Paul W Sternberg
Genome Biology , 2003, DOI: 10.1186/gb-2003-4-12-238
Abstract: Each genome is fascinating in its own right, but some genomes have been chosen for study because of the added advantages of understanding a closely related genome. Caenorhabditis briggsae (Figure 1a) is just such a companion to Caenorhabditis elegans, a model system that has been widely used to study the genetic basis of animal development, behavior and physiology. The sequencing of the genome of C. elegans [1] has led to rapid advances in our understanding of gene function, particularly through the use of techniques such as RNA interference (RNAi) [2]. Stein et al. [3] now report a draft sequence of the C. briggsae genome.C. briggsae is closely related to C. elegans (Figure 2) and has almost identical morphology (Figure 1b,c) [4-6]. Given the lack of fossil records, the evolutionary distance between C. briggsae and C. elegans has been estimated using a molecular clock, which gives a divergence time of between 20 and 120 million years ago (Mya) [7-11]. By analyzing the 338 sets of orthologous genes found in the C. briggsae, C. elegans, Anopheles and human genomes and using a molecular clock calibrated by the known date of divergence of nematodes and arthropods, Stein et al. [3] now report a much tighter estimate of the divergence time of C. briggsae and C. elegans, between 80 and 110 Mya. This divergence is slightly greater than the estimate of the human-mouse divergence time (65-75 Mya) [12]. The draft covers 98% of the 104 Mb genome; the slightly larger size of the C. briggsae genome compared with that of C. elegans (100.3 Mb according to the WS108 release of September 2003 [13]) is primarily due to additional repetitive DNA.The C. briggsae genome was annotated using various gene-finding programs (such as Genefinder [14]) and by comparison with C. elegans. As different programs often disagree with each other in predicting genes, Stein et al. [3] adopted a 'hybrid' approach by combining the predictions made by multiple gene-finding programs and selecting the consen
Genomic sequence of a mutant strain of Caenorhabditis elegans with an altered recombination pattern
Ann M Rose, Nigel J O'Neil, Mikhail Bilenky, Yaron S Butterfield, Nawar Malhis, Stephane Flibotte, Martin R Jones, Marco Marra, David L Baillie, Steven JM Jones
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-131
Abstract: Using Illumina sequencing and MAQ software, 83% of the base pair sequence reads were aligned to the reference genome available at Wormbase, providing a 21-fold coverage of the genome. Using the software programs MAQ and Slider, we observed 1124 base pair differences between Rec-1 and the reference genome in Wormbase (WS190), and 441 between the mutagenized Rec-1 (BC313) and the wild-type N2 strain (VC2010). The most frequent base-substitution was G:C to A:T, 141 for the entire genome most of which were on chromosomes I or X, 55 and 31 respectively. With this data removed, no obvious pattern in the distribution of the base differences along the chromosomes was apparent. No major chromosomal rearrangements were observed, but additional insertions of transposable elements were detected. There are 11 extra copies of Tc1, and 8 of Tc2 in the Rec-1 genome, most likely the remains of past high-hopper activity in a progenitor strain.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 affecting coding 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 recombination event with no other significant phenotypic consequences. In this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the Rec-1 mutation.Caenorhabditis elegans is an animal model widely used in biomedical and biological research. C. elegans was the first animal to have its genome completely sequenced [1] and the compiled and annotated sequence is available at WormBase http://www.wormbase.org webcite. The ready availability of genomic sequence information along with an extensive body of knowledge about gene function in this species provides an exceptional opportunity to examine th
Leptotene/Zygotene Chromosome Movement Via the SUN/KASH Protein Bridge in Caenorhabditis elegans  [PDF]
Antoine Baudrimont,Alexandra Penkner,Alexander Woglar,Thomas Machacek,Christina Wegrostek,Jiradet Gloggnitzer,Alexandra Fridkin,Franz Klein,Yosef Gruenbaum,Pawel Pasierbek,Verena Jantsch
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1001219
Abstract: The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2–dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.
Chromosome Painting Reveals Asynaptic Full Alignment of Homologs and HIM-8–Dependent Remodeling of X Chromosome Territories during Caenorhabditis elegans Meiosis  [PDF]
Kentaro Nabeshima ,Susanna Mlynarczyk-Evans,Anne M. Villeneuve
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002231
Abstract: During early meiotic prophase, a nucleus-wide reorganization leads to sorting of chromosomes into homologous pairs and to establishing associations between homologous chromosomes along their entire lengths. Here, we investigate global features of chromosome organization during this process, using a chromosome painting method in whole-mount Caenorhabditis elegans gonads that enables visualization of whole chromosomes along their entire lengths in the context of preserved 3D nuclear architecture. First, we show that neither spatial proximity of premeiotic chromosome territories nor chromosome-specific timing is a major factor driving homolog pairing. Second, we show that synaptonemal complex-independent associations can support full lengthwise juxtaposition of homologous chromosomes. Third, we reveal a prominent elongation of chromosome territories during meiotic prophase that initiates prior to homolog association and alignment. Mutant analysis indicates that chromosome movement mediated by association of chromosome pairing centers (PCs) with mobile patches of the nuclear envelope (NE)–spanning SUN-1/ZYG-12 protein complexes is not the primary driver of territory elongation. Moreover, we identify new roles for the X chromosome PC (X-PC) and X-PC binding protein HIM-8 in promoting elongation of X chromosome territories, separable from their role(s) in mediating local stabilization of pairing and association of X chromosomes with mobile SUN-1/ZYG-12 patches. Further, we present evidence that HIM-8 functions both at and outside of PCs to mediate chromosome territory elongation. These and other data support a model in which synapsis-independent elongation of chromosome territories, driven by PC binding proteins, enables lengthwise juxtaposition of chromosomes, thereby facilitating assessment of their suitability as potential pairing partners.
A Caenorhabditis elegans Locomotion Phenotype Caused by Transgenic Repeats of the hlh-17 Promoter Sequence  [PDF]
Randy F. Stout Jr, Vladimir Grubi?i?, Vladimir Parpura
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081771
Abstract: Transgene technology is one of the most heavily relied upon tools in modern biological research. Expression of an exogenous gene within cells, for research and therapeutic applications, nearly always includes promoters and other regulatory sequences. We found that repeats of a non-protein coding transgenic sequence produced profound changes to the behavior of the nematode Caenorhabditis elegans. These changes were produced by a glial promoter sequence but, unexpectedly, major deficits were observed specifically in backward locomotion, a neuron-driven behavior. We also present evidence that this behavioral phenotype is transpromoter copy number-dependent and manifests early in development and is maintained into adulthood of the worm.
Caenorhabditis elegans Histone Methyltransferase MET-2 Shields the Male X Chromosome from Checkpoint Machinery and Mediates Meiotic Sex Chromosome Inactivation  [PDF]
Paula M. Checchi,JoAnne Engebrecht
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002267
Abstract: Meiosis is a specialized form of cellular division that results in the precise halving of the genome to produce gametes for sexual reproduction. Checkpoints function during meiosis to detect errors and subsequently to activate a signaling cascade that prevents the formation of aneuploid gametes. Indeed, asynapsis of a homologous chromosome pair elicits a checkpoint response that can in turn trigger germline apoptosis. In a heterogametic germ line, however, sex chromosomes proceed through meiosis with unsynapsed regions and are not recognized by checkpoint machinery. We conducted a directed RNAi screen in Caenorhabditis elegans to identify regulatory factors that prevent recognition of heteromorphic sex chromosomes as unpaired and uncovered a role for the SET domain histone H3 lysine 9 histone methyltransferase (HMTase) MET-2 and two additional HMTases in shielding the male X from checkpoint machinery. We found that MET-2 also mediates the transcriptional silencing program of meiotic sex chromosome inactivation (MSCI) but not meiotic silencing of unsynapsed chromatin (MSUC), suggesting that these processes are distinct. Further, MSCI and checkpoint shielding can be uncoupled, as double-strand breaks targeted to an unpaired, transcriptionally silenced extra-chromosomal array induce checkpoint activation in germ lines depleted for met-2. In summary, our data uncover a mechanism by which repressive chromatin architecture enables checkpoint proteins to distinguish between the partnerless male X chromosome and asynapsed chromosomes thereby shielding the lone X from inappropriate activation of an apoptotic program.
The Genome Sequence of Caenorhabditis briggsae: A Platform for Comparative Genomics  [PDF]
Lincoln D. Stein,Zhirong Bao,Darin Blasiar,Thomas Blumenthal,Michael R. Brent,Nansheng Chen,Asif Chinwalla,Laura Clarke,Chris Clee,Avril Coghlan,Alan Coulson,Peter D'Eustachio,David H. A. Fitch,Lucinda A. Fulton,Robert E. Fulton,Sam Griffiths-Jones,Todd W. Harris,LaDeana W. Hillier,Ravi Kamath,Patricia E. Kuwabara,Elaine R. Mardis,Marco A. Marra,Tracie L. Miner,Patrick Minx,James C. Mullikin,Robert W. Plumb,Jane Rogers,Jacqueline E. Schein,Marc Sohrmann,John Spieth,Jason E. Stajich,Chaochun Wei,David Willey,Richard K. Wilson,Richard Durbin,Robert H. Waterston
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.0000045
Abstract: The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.
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