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[
International C. elegans Meeting,
1997]
Bacterial feeding nematodes including Zeldia punctata (Rhabditida: Nematoda), and Caenorhabditis elegans differ profoundly in feeding adaptations and specifically in the rhabdions (cuticular thickenings), and associated cells lining in the buccal capsule. We are carrying out a range of tests to determine what rhabdions are evolutionarly homologous between the two species. Reconstruction of the buccal capsule and procorpus (pharynx) with transmission electron microscopy indicates that the lining of the buccal capsule of Z. punctata includes four sets of muscular radial cells ma, mb, mc, md in contrast the same region in C. elegans which has two sets of epithelial radial cells (
e1,
e3) and two sets of radial muscle cells (
m1,
m2). In Z. punctata ma is a set of three radial muscle cells each with 2 nuclei. In C. elegans
m1 has the identical arrangement. Similarly, in Z. punctata mb is a set of 3 muscle cells each with one nucleus, similar to
m1 in C. elegans. These findings could contradict all previous hypotheses of homology, and suggest instead that ma and mb in Z. punctata are homologous respectively with
m1 and
m2 in C. elegans. Ongoing additional tests of homology include comparative cell lineages and screening mutants to recognize genes involved in buccal capsule expression.
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[
International C. elegans Meeting,
1997]
In order to obtain the maximum information in the shortest time, C. elegans sequencing was initiated within the gene-rich regions of the genome, which are estimated to contain over 80% of genes. Encompassing 60 MB, this region also had the advantage of being well represented in cosmid contigs. Sequencing of this 60 Mb region is now essentially complete. Of the remaining sequence approximately 20 Mb is only represented by YAC clones. Thus it was imperative that new sequencing strategies were developed so that YACs could be utilised as part of the initial sequencing substrate. During 1996, production scale methodologies were established in both labs to allow a shotgun sequencing approach to be efficiently applied to YAC clones. The major difficulties are handling the small amount of DNA that can be recovered from yeast hosts and elimination of yeast contamination. However, by double gel purification of YAC isolates, and the use of yeast 'window' strains where necessary, yeast sequence contamination can be reduced below 10%. pUC is better than M13 for providing adequate libraries from small amounts of DNA. (The Sanger Centre uses exclusively pUC, while the Genome Sequencing Center uses a mixture of pUC and M13). The pUC clones have the additional advantages of bridging inverted repeats (which M13 cannot handle) and of providing an extensive check on assembly from forward and reverse read pairs. Our strategy for sequencing the final 40 Mb will be to shotgun sequence YAC clones in conjunction with any underlying cosmid clones. This strategy is proceeding well and at the time of writing more than 40 YAC clones are being sequenced. We estimate that approximately 150 YACs will need to be shotgunned to complete the genomic sequence.
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Ichikawa, K., Fire, A.Z., Morishita, S., Edgley, M.L., Artiles, K.L., Gabdank, I., Schwarz, E.M., Yoshimura, J., Rougvie, A.E., Smith, C.L., Shoura, M., Wahba, L.
[
International Worm Meeting,
2017]
By 2005, the C. elegans genome was sequenced to apparent completion from a mixture of divergent laboratory strains, all derivatives of Brenner's original "N2". The lack of a single worm strain matching this sequence creates challenges for genomics and systems biology. We thus chose to produce a updated reference genome for C. elegans from the strain VC2010, which was derived from a clonal isolate of N2. After sequencing to a depth of 115x coverage with trimmed, error-corrected PacBio and Oxford Nanopore reads having an average length of 11.8 kb, we assembled a updated reference genome with near-chromosomal contiguity. Half of the assembly falls into 10 blocks of continuous sequence (contigs) ranging from 3.6 Mb to 8.4 Mb in size, and the entire assembly is contained in only 82 contigs. Our assembly is very close to the previous reference assembly's quality (97.6% full-length CEGMA hits, versus 98.4% in previous N2). It is also slightly larger than the previous reference assembly (102.0 Mb versus 100.3 Mb in previous N2, a difference of 1.7%). We have closed some remaining gaps in the updated reference genome assembly with PacBio and Oxford Nanopore single reads, and used Hi-C to estimate gap sizes. This revealed 26 unlinkable regions in the updated reference genome of =130 kb, whose flanks contain highly repetitive DNA. In the official N2 genome, these regions have relatively few copies of repetitive DNA, and are ungapped. We suspect that the large gaps are real, but were lost during propagation and sequencing of cosmid clones during the original N2 genome project, making them a kind of genomic "dark matter". In other words, we think that we have de-completed the C. elegans genome. The apparent 1.7% increase in the updated reference's genome size may reflect other overlooked repeats that exist biologically in N2. Sequences in N2 that are missing from the official genome were recently described by Zhao et. al. (PubMed 26039588), with our observations expanding the discrepancy from 40 kb to 1.7 Mb. Such "dark matter" regions probably exist in other nematode genomes and make them harder to assemble. Since they have persisted in C. elegans, they may have biological functions; possibilities for such functions include generation of ncRNAs and aiding chromosomal segregation. Our consensus VC2010 genome assembly will be made available in Wormbase, with CGC distributing a VC2010-derived strain (PD1074) after July 2017.
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[
International C. elegans Meeting,
1995]
We present here an update on our efforts to obtain the complete nucleotide sequence of the worm genome. Following an initial pilot phase sequencing project of a 2.2 Mb region on chromosome III, the Consortium labs in both Cambridge and St. Louis have scaled up their respective operations with the goal of obtaining the sequence of the genome by 1998. Progress toward this goal has been substantial. The Consortium has concentrated its efforts on those genomic regions that are richest in physical map resources and genes. Sequences have been obtained from 455 cosmids, resulting in 14 Mb of finished sequence, primarily from the central regions of chromosomes II, III and X. Analysis of the sequence data by the program GENEFINDER and other tools has shown that predicted genes occur at an average density of one per five kb. Approximately 40% of these predicted genes show significant similarity to other sequences in the public domain databases.
-
[
International Worm Meeting,
2015]
Cytokinesis consists in the physical separation of the two daughter cells, after mitosis. However, during development of certain tissues, mitotic division is followed by incomplete cytokinesis, giving rise to interconnected cells in a shared cytoplasm, or syncytium. Syncytial structures have been repeatedly described in female and male germlines in species ranging from insects to humans. Nevertheless, the mechanism of syncytium formation is poorly characterized. To understand this, we studied the syncytial C. elegans germline wherein germ cells (GCs) remain connected via stable intercellular bridges, also known as GC bridges. Using live-cell imaging, we found that ANI-1, ANI-2, NMY-2, UNC-59Septin, CYK-7 (a novel midbody component), ZEN-4Mklp1 are all stable components of GC bridges while RHO-1RhoA and ECT-2RhoGEF have a cortical localization throughout germline development from the L1 stage to adulthood. All GCs originate from the germline blastomere P4 that divides into the two primordial germ cells (PGCs) during embryogenesis. To understand the mechanism of syncytium formation, we monitored the dynamics of P4 cytokinesis. We found that P4, similar to its somatic neighbors, goes normally through the first phase of cytokinesis, also known as cytoplasmic isolation. Interestingly however, unlike somatic blastomeres, P4 fails to complete the second phase of cytokinesis wherein the midbody (MB) is released from the cell-cell boundary. While the MBs of somatic cells disappear soon after their cortical release, the MB connecting the two PGCs remains tightly associated to the cortex throughout embryogenesis. Our findings support a model in which incomplete cytokinesis of the P4 blastomere results in persistence of the MB between the two PGCs, thus forming a stable bridge that, in turn, promotes syncytium biogenesis.
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Chandrasekar, Sinduja, Koutsovoulos, Georgios, Yin, Da, Ralston, Edward J., Blaxter, Mark, Stevens, Lewis, Haag, Eric S., Anderson, Erika C., Meyer, Barbara J., Schartner, Caitlin M., Schwarz, Erich M.
[
International Worm Meeting,
2019]
Hermaphroditism has independently evolved at least three times within the Caenorhabditis genus, and six times in Pristionchus. This has often coincided with substantial losses of protein-coding genes, which are often implicated in male reproduction. However, the hermaphrodite C. tropicalis challenges this pattern. Although C. tropicalis has a substantially reduced genome (83 Mb in size, versus ~130 Mb in several male-female Caenorhabditis species), its closest male-female relative (C. wallacei) has an almost equally small genome (85 Mb). One explanation might be that genome shrinkage in C. tropicalis arose independently of hermaphroditism; this would fit the recent discovery of male-female Caenorhabditis with remarkably compact genomes, such as C. sulstoni with 65 Mb. An alternative explanation might be that C. wallacei reverted to male-female sexuality after hermaphroditism had already shrunk the genome of its shared tropicalis/wallacei ancestor. To begin testing these hypotheses, we used PacBio, Illumina, and Hi-C sequencing to produce third-generation genome assemblies for C. tropicalis and C. wallacei, each having six complete chromosomal scaffolds. Both assemblies are 98.6%-98.7% complete as scored by BUSCO, which matches the score for C. elegans (98.6%). In hermaphroditic C. briggsae versus its male-female sister species C. nigoni, ~7,000 genes lost in C. briggsae disproportionately include small genes with male-biased gene expression, such as the male secreted short (mss) gene family; the mss family encodes sperm surface glycoproteins, found only in outcrossing species, that are required for sperm competitiveness in mating. In contrast, C. tropicalis has only ~1,400 fewer protein-coding genes than C. wallacei (19,722 versus 21,017), 20% the disparity of C. briggsae vs. C. nigoni. Two clustered multigene families with male-biased expression conserved widely in male-female species (mss and a CAP-domain family that includes CRE28795) are absent not only in C. tropicalis but also in C. wallacei. More generally, gene families with conserved XO- or XX-biased expression have consistently fewer members in C. wallacei than in male-female species C. nigoni, C. remanei, or C. brenneri, and the diminished gene numbers of C. wallacei approach or equal those seen in hermaphrodites (for XO-biased and XX-biased gene families, respectively). These data suggest that C. wallacei might indeed be an atypical male-female Caenorhabditis species that underwent a temporary period of hermaphroditism, and jettisoned male reproductive genes during that period.
-
[
International Worm Meeting,
2019]
Long-read sequencing-based de novo genome assembly has made advances in discovering candidate genes of phenotypic variations by comparing several species at the genome level. To understand phenotypic variations in different nematode species, we are currently working on two variant forms of a phoretic behavior, nictation and tube-waving. C. elegans dauers can hitchhike their carriers by waving their heads with a rough surface. However, in some Rhabditids, such as Rhabditella axei, dauers can also wave their heads by using their old cuticle after molting. These behaviors are called nictation and tube-waving, respectively. To elucidate the genetic basis of this phenotypic variation, we assembled a draft genome of R. axei (DF5006) using MinION-based long-read sequencing. We obtained 3 Gb data with a single flow cell and assembled a draft genome (394 contigs, N50=0.99 Mb, 144 Mb in total) using the wtdbg2 assembler (Ruan et al., 2019). The quality of the genome will be improved and further analysis such as gene annotation will be carried out. This research will help clarify the genetic basis of the two variant forms of phoretic behavior and establish the basis for studying phenotypic variations of Rhabditella axei (DF5006).
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Dillman, Adler, Rogers, Alicia, Adams, Byron, Williams, Brian, Macchietto, Marissa, Antoshechkin, Igor, Lewis, Edwin, Finlinson, Camille, Lu, Xiaojun, Goodrich-Blair, Heidi, Mortazavi, Ali, Sternberg, Paul, Goodwin, Zane, Stock, Patricia
[
International Worm Meeting,
2013]
Numerous nematode genera are major parasites of plants, animals, and humans, despite sharing a conserved body plan. Steinernema comprise over 70 characterized species that are lethal parasites of insects with differing foraging strategies and host ranges. We have sequenced the genomes and transcriptomes of five key members of Steinernema (S. carpocapsae, S. scapterisci, S. monticolum, S. glaseri, and S. feltiae) for comparative analysis. We find 20 Mb of conserved sequence, which represents about 23% of the S. carpocapsae assembly. This includes 127,282 non-coding elements accounting for about 5 Mb. We explore genomic differences likely to be involved in insect parasitism. We find gene family evolution of proteases, protease inhibitors, proteolytic cascade proteins, and GPCRs, many of which correlate with known differences in host range and specificity. Steinernema RNA-seq data allows for powerful comparisons to Caenorhabditis gene expression at defined stages, which show surprising plasticity of timing across one-to-one orthologous genes when compared to C. elegans. Our analysis of the conserved non-coding regions reveals that a limited number of motifs are associated with conservation of stage-specific ortholog expression, which suggests that key underlying gene regulatory relationships that control development are similar in the two genera.
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[
West Coast Worm Meeting,
2002]
Sensory processing is mediated by interneurons in various animal species. In insect, interneurons of the mushroom bodies (MBs) of the brain are responsible for sensory integration, memory and learning. We have previously identified a honeybee gene, termed Mblk-1, encoding a transcription factor that is expressed preferentially in the MB-interneurons of the honeybee brain. Homology search revealed that the DNA binding motif of Mblk-1 has significant sequence homology with those encoded by genes from various animal species, including the nematoda, fruit fly, mouse and human, suggesting that the functions of these proteins in neural system are conserved among these animals.
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Kratzer, Stella, Wang, Jianbin, Koutsovoulos, Georgios, Blaxter, Mark, Beriman, Matthew, Mitreva, Makedonka, Thorne, Alicia, Kumar, Sujai, Balas, Maggie, Magrini, Vincent, Davis, Richard E
[
International Worm Meeting,
2013]
Chromatin diminution is the programmed elimination of specific DNA sequences during development. It occurs in diverse species, but the function(s) of diminution and the specificity of sequence loss remain largely unknown. Diminution in the nematode Ascaris suum occurs during early embryonic cleavages and leads to the loss of germline genome sequences and the formation of a distinct genome in somatic cells. We found that ~43 Mb (~13%) of genome sequence is eliminated in A. suum somatic cells, including ~12.7 Mb of unique sequence. The eliminated sequences and location of the DNA breaks are the same in all somatic lineages from a single individual, and between different individuals. At least 685 genes are eliminated. These genes are preferentially expressed in the germline and during early embryogenesis. Soma-specific elimination provides a unique mechanism of gene repression and differentiation between germline and soma. We found no temporal or any other correlation of small RNAs with diminution. Preliminary data suggest that a possible mechanism of differential segregation of DNA following the breaks may be due to differential deposition of CenpA (as well as other histone marks) on retained vs eliminated DNA sequences. For comparison, we have also sequenced the germline and somatic genomes of a second nematode with a single large haploid chromosome that undergoes DNA elimination, Parascaris univalens. These data will be discussed. Overall, our studies suggest that diminution is a unique mechanism of germline gene regulation that specifically silences genes involved in gametogenesis and early embryogenesis through their elimination and that this process contributes to the soma-germline differentiation.