Dieterich, Christoph et al. (2009) International Worm Meeting "Transcriptome profiling of 7 Pristionchus species: Horizontal gene transfer, novel gene families and gene turnover."

Herrmann, Matthias, Mayer, Werner, Sommer, Ralf, Schuster, Lisa, Dieterich, Christoph

[International Worm Meeting, 2009]

Horizontal gene transfer (HGT), the acquisition of novel genes and gene turnover are of utmost importance for genome evolution. The analysis of these processes requires genome-level studies of related species in a well-established phylogenetic framework. The nematode Pristionchus pacificus is a model system in evolutionary biology with genetic and transgenic tools that in nature is found in a necromenic association with scarab beetles. We have isolated and keep more than 160 P. pacificus ecotypes and 24 different Pristionchus species from around the world in the lab. In addition, we have isolated strains from 13 other genera of the family of the Diplogasteridae and their phylogenetic relationship has been revealed. P. pacificus constitutes a good starting point for the analysis of HGT. For example, seven members of the glycosyl hydrolase 5 family are found in its genome and cellulase activity has been observed in the supernatant of its cultures. Likewise, thousands of novel protein-coding genes were discovered in the genome sequence, that do not show any sequence similarity to the known protein universe yet are transcribed and cluster into gene families. To study the longevity and evolution of novel genes and those acquired by HGT, we used the 454 next-generation sequencing platform, to index the transcriptomes of 10 species (7 Pristionchus and 3 species of other genera). This approach is a rapid and cost-effective method to study nematode transcriptome complexity and the gene birth/death process within the genus Pristionchus. First, we could verify most of our initial discoveries in the P. pacificus genome sequence. For example, cellulase and diapausin genes are present in Pristionchus, but not in the outgroup species. We assigned HGT events to the most probable source organism and dated them to their respective last common ancestor. The dynamics of gene family evolution (e.g. gene turnover in the Cytochrome P450 family) was assessed in detail. The speed of arrival and departure of truly novel gene inventions could be estimated. We also speculate on the dependency of transcriptome complexity on nematode lifestyle or mode of sexual reproduction.

Holly Sassi et al. (2005) International Worm Meeting "Gene CATCHR Gene Cloning and Tagging for C. elegans using yeast Homologous Recombination: An innovative approach for the analysis of gene expression"

Stephanie Renihan, Andrew Spence, Holly Sassi, Ramona Cooperstock

[International Worm Meeting, 2005]

To facilitate a systematic investigation of C. elegans expression patterns and interactions, we have developed Gene CATCHR (Gene Cloning and Tagging for C. elegans using yeast Homologous Recombination). Gene CATCHR is a novel method of generating reporter constructs that exploits yeast homologous recombination to subclone and tag worm genes. The cloning and tagging steps require PCR, two yeast transformations, and several platings on selective media. Each step is relatively straightforward, and amenable to high throughput techniques. Using Gene CATCHR we can produce functional tagged transgenes that provide accurate information about the spatiotemporal pattern of gene expression and subcellular localization of gene products. Major advantages of Gene CATCHR include:1) Clone size potential: we can capture large genomic regions facilitating the isolation of regulatory sequences that may be important for gene expression. We have used Gene CATCHR to clone 5 operons, up to 20 kb in size. Notably, we have successfully cloned and tagged a 36 kb genomic fragment containing the tra-1 gene.2) Flexibility: any tag can be inserted at any position in a gene without introducing extraneous sequence. Our system can also be exploited to create targeted mutations.3) Scalability: we can clone and tag many genes at once in 96 well format. To date, we have cloned 68 genes, 22 of which have been tagged with GFP. In order to generate a high throughput system, we are currently developing a bioinformatics tool.4) Expression and Function: The expression patterns generated from Gene CATCHR derived transgenes are consistent with those previously reported, and we have documented additional uncharacterized expression patterns. Our transgenes are functional in rescue assays; as proof of principle, we have rescued 5 mutants.All Gene CATCHR related protocols and reagents will be made available to the worm community through our website, www.genecatchr.org (stay tuned!). The expression data generated will also be submitted to WormBase. Gene CATCHR represents an efficient, highly versatile, and powerful system for functional genomic applications.

John Yochem et al. (2001) International C. elegans Meeting "An analysis of gene redundancy based on the mec-8 gene"

John Yochem, Jocelyn Shaw, Robert Herman, Claire Kari

[International C. elegans Meeting, 2001]

A surprise of functional genomics in worms and yeast is the extent to which gene knockouts confer no obvious phenotype. Such genes may contribute to fitness in subtle ways, but they may also contribute to essential functions redundantly. Adding to the confusion, genes with overlapping or redundant functions need not be related by DNA sequence. We are using classical genetics and the mec-8 gene to explore the issue of gene redundancy. MEC-8 is a putative RNA-binding protein that is required for processing of unc-52 mRNA. Null mutations in mec-8 confer mechanosensory and chemosensory defects but not highly-penetrant lethality. The combination of a mec-8 loss-of-function mutation, however, with a viable mutation in any of six sym ( sy nthetic lethal with m ec-8 ) genes (or with a viable allele of unc-52 , the prototype analysis for this study) results in embryonic or early larval lethality. sym-1 X has been previously shown to encode a hypodermally-secreted, leucine-rich repeat protein that, in the absence of MEC-8, is required for attachment of body muscles to the cuticle. Thus, sym-1 and mec-8 compose an example of redundant genes whose products are not related in structure and function, leading to the suggestion that sym-1 overlaps in function with a gene whose mRNA needs to be processed by MEC-8 (Davies et al. 1999; Genetics 153: 117). With the aim of identifying the hypothesized mec-8 target and other genes that overlap functionally with sym-1 , we have been screening for mutations that are s ynthetically l ethal with s y m-1(mn601). Candidate sly mutations are being genetically characterized. We are interested in understanding the nature of the overlap between sym-2 II and mec-8 . Rescue experiments have suggested that ZK1067.6 is sym-2 . DNA sequence analysis has now confirmed this supposition: sym-2(mn617) correlates with a change of a tyrosine to an asparagine codon at position 163 in the 618-codon open reading frame. SYM-2 contains three copies of an RNA recognition motif, suggesting that SYM-2 is an RNA binding protein that overlaps with MEC-8 in the processing of an unknown gene transcript that is essential for viability. The structure of SYM-2 will be compared with MEC-8 and with heterogeneous nuclear ribonucleoproteins (hnRNPs) from mammals.

Williams, Gareth et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "WormBase Gene Curation"

Davis, Paul, Williams, Gareth

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

WormBase is a central data repository for nematode biology. Initially created as a service to the Caenorhabditis elegans research field, WormBase has evolved into a powerful research tool in its own right. In the past 2 years, we expanded WormBase to include the complete genomic sequence, gene predictions and orthology assignments from a range of related nematodes. WormBase continues to utilise other database resources such as miRBase, RFam and modENCODE to aid in the curation of worm gene structures and sequence based data types. As part of a collaboration with modENCODE submitters we have conducted a genome wide review of the WormBase C. elegans Pseudogene set which has resulted in the conversion of some pseudogene loci back to coding loci. One of our newest challenges has come with with the advent of next generation sequencing technologies. The scale of data production has increased enormously, and this has provided challenges for data incorporation but has greatly increased our ability to detect erroneous gene models and new alternate Isoforms. This RNASeq data and other modENCODE data will shortly be available from the WormBase website.

JE Abrahante et al. (1999) International C. elegans Meeting "Characterization of the heterochronic gene lin-57, a gene hypostatic to lin-4"

EA Miller, AE Rougvie, JE Abrahante

[International C. elegans Meeting, 1999]

lin-57 is a novel gene identified in a screen for heterochronic mutants that exploits the adult specific nature of the rol-1 phenotype. rol-1(e61) mutants exhibit a stage-specific locomotion defect: larvae swim in the normal sinusoidal pattern but adults roll. This adult specific rol-1 phenotype depends upon the synthesis of adult cuticle and is temporally altered by mutations in the heterochronic gene pathway. For example, lin-4 and lin-29 mutants never synthesize adult cuticle and consequently lin-4 rol-1 and rol-1 lin-29 double mutants never roll. Conversely, lin-14 and lin-28 mutants synthesize adult cuticle early and rol-1 double mutant combinations with these genes result in animals that roll as larvae. In attempts to identify additional heterochronic genes, we mutagenized rol-1(e91) animals and screened for animals that either roll precociously or fail to roll as adults. Among the mutants identified in this screen was a precocious roller that defined lin-57. In lin-57 mutants, seam cell terminal differentiation and adult cuticle synthesis occur one stage early, during the L3 molt. In this respect lin-57 is similar to the other known precocious mutants lin-14, lin-28, lin-42 and lin-58. However, lin-57 is unique because it is the first known heterochronic gene to which lin-4 is epistatic. In lin-4; lin-57 double mutants the seam cells fail to terminally differentiate. To better understand the role of lin-57 in the heterochronic gene pathway, we initiated its molecular cloning. lin-57 resides on the X chromosome, in a 1.5 mu interval between mec-2 and stP33. A single cosmid from this region, F13D11, fully rescues the lin-57 phenotype. Genefinder predicts four ORFs in F13D11: 1) a tyrosine phosphatase, 2) a zinc finger protein, 3) a histidine phosphatase and 4) a dihydroflavonol 4-reductase (DFR). We narrowed the lin-57 rescuing activity to a 12Kb fragment containing the histidine phosphatase and DFR. To determine which ORF encodes lin-57, we have created additional constructs, including frameshifts in the predicted ORFs. In addition, we have generated a second allele and are sequencing both alleles. A full report on the identity, expression patterns and genetic interactions of lin-57 will be presented.

Kishore, Ranjana et al. (2013) International Worm Meeting "Gene Function (and Gene Dysfunction) Data in WormBase: Where and How to Find It."

Lee, Raymond, Yook, Karen, Schindelman, Gary, Van Auken, Kimberly, Kishore, Ranjana

[International Worm Meeting, 2013]

WormBase curates biological data from the published literature towards presenting a complete picture about the function of a gene. The different categories of data are displayed in different widgets on WormBase gene pages and include: 1. Concise Descriptions. These free-text summaries reside in the Overview widget and are meant to give the user a quick introduction to a gene. They describe a gene's molecular identity and orthology, processes and pathways, genetic and physical interactions, and expression at the tissue and sub-cellular levels. 2. Gene Ontology. Gene Ontology (www.geneontology.org) curation provides computable annotations for a gene's Biological Process, Molecular Function and Cellular Component, using a controlled vocabulary and evidence codes, which are shared across multiple model organism databases. 3. Phenotypes. Phenotypes are curated in great detail for mutations, RNAi knockdown experiments, anatomy function, overexpression studies and interactions, using a Worm Phenotype Ontology, developed by WormBase. Information about strains, conditions, and chemicals used in the experiment are also included. 4. Worm Models of Human Disease. WormBase has begun to highlight data about how worm genes and biology inform the study of human disease. We now include free-text 'disease relevance' descriptions, and tag genes as 'potential' or 'experimental' models, based on orthology to a human disease gene, and/or experimental data, respectively. The details of these data and the different ways in which they can be accessed and mined will be discussed.

Sosnowski BA et al. (1987) International C. elegans Meeting "developmental localization of a sperm-specific gene and isolation of its gene."

Sosnowski BA, Ward S, Hogan E

[International C. elegans Meeting, 1987]

Peter L Barrett et al. (2005) International Worm Meeting "Targeted gene alteration in C. elegans via gene conversion"

Verena Gobel, Peter L Barrett, John T Fleming

[International Worm Meeting, 2005]

Given the completion of whole genome sequences, the ability to direct specific mutations into genomes is particularly desirable. We present here an approach to efficiently create mutations in the C. elegans genome through transgene-directed, transposon-mediated gene conversion. Briefly, a transgene containing an engineered alteration in a gene of interest is introduced into a mutator strain with a transposon in this gene. Homologous sequence in the transgene makes it a suitable template for conversion at the genomic locus in DNA repair following transposon excision. The strain is expanded, and subpopulations are tested by PCR with primers designed such that one will amplify only from the genomic sequence and not the transgene; therefore a product will only be amplified upon successful gene conversion of the alteration from the transgene to the genomic locus. To demonstrate this approach, we have targeted engineered deletions into two genes, tkr-1 and frm-3. We also targeted an engineered green fluorescent protein (GFP) cassette to frm-3, allowing for examination of the genes expression pattern in its native context. These germline alterations were confirmed by structural characterization and phenotypic rescue. These targeted mutations were obtained at high frequencies, comparing favorably to those for deletions obtained through interrupted dsDNA break repair following excision (the standard approach to generate deletions in transposon-tagged genes). Gene conversion events could be detected in as few as 16 starting plates, or in less than 15,000 animals. By this technique, custom alleles of different types can be created in vitro to make the corresponding mutations in vivo. In this way, specific modifications can be introduced to analyze particular aspects of gene function in vivo such as the addition or removal of DNA encoding sequence expression elements, protein domains, heterologous proteins, etc. The approach should also be applicable to heterologous transposons in C. elegans, such as Mos.

R Terranova et al. (1999) International C. elegans Meeting "set-1, a SET domain gene involved in Hox gene regulation?"

N Pujol, R Terranova, M Djabali, JJ Ewbank

[International C. elegans Meeting, 1999]

Hox genes specify cell fates in successive antero-posterior body domains in vertebrates, insects and nematodes (1,2). Polycomb and trithorax group genes are responsible for the initiation and maintenance of Hox gene expression. Nematode homologues of these genes have been discovered and their function is being studied in several labs. We are interested in the function of a domain which is shared between trithorax and polycomb members: the SET domain. This domain which is shared between antagonistic proteins is very conserved and may play an important role in the assembly of either transcriptional activating or repressing protein complexes during development (3). In C. elegans , the 130 amino acid SET motif is present in the polycomb group of proteins (4) including MES-2, a protein similar to E(z) (5). It is also present in SET-1 (T26A5.7), which is predicted to be a 288 residue protein. We have examined the expression pattern of set-1 in worms carrying a GFP reporter transgene. Early in development, set-1 is expressed in the majority of cells, its expression being more and more restricted as development progress. The set-1 gene has been shown to be expressed in seam cells and in vulval cells as well as in the male tail. This expression pattern of set-1 is reminiscent of the expression pattern of the ensemble of Hox genes (6). The change in the pattern with the GFP-fusion construct is consistent with the variation of transcript levels during development. RNA inactivation has revealed a marked Vab phenotype. The weakest reproducible phenotype observed is a progressive paralysis with adult onset. To study further the role of set-1 in Hox gene expression, we are injecting set-1 dsRNA in Hox-LacZ transgenic worms (the kind gifts of C. Kenyon). Also, we are currently screening a bank of UV-TMP mutant worms, using the Barstead protocol, in the hope of obtaining a strain in which set-1 is knocked out. 1/ Kenyon C. (1994). Cell 78, 175-180 2/ Emmons S. W. (1996). Nature 382, 301-302 3/ Laible et al. (1997). EMBO J. 16, 3219-3232 4/ Korf I., Fan Y.A., Strome S. (1998) Development 125, 2469-2478 5/ Holdeman R., Nehrt S., Strome S. (1998) Development 125, 2457-2467 6/ Wang et al., (1993). Cell 74, 29-42

Praitis, Vida et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Characterization of a gene important for C. elegans gastrulation"

Schacht, Angela, Liszewski, Walter, Miller, Michael, Chakravorty, Adityarup, Tyson, Stock, Imlay, Leah, Kniss, Sarah, Praitis, Vida, Mushi, Juliet, Ha, Dae Gon, Yilma, Zelealem, Yohannes, Lensa, Feddersen, Charlotte, Sullivan-Wilson, Alexander

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In C. elegans , gastrulation begins with ingression of E blastomeres at the 26-cell stage and ends with the establishment of a mature vermiform embryo 6 hours later (Sulston et al 1983). While significant research has been done on the initiation of gastrulation, ingression of cells in mid-gastrulation is not as well understood. What is clear is these later cells, derived from very distinct lineages, must acquire specific fates and positional information to ingress properly (Nance, et al 2005; Rohrschneider & Nance 2009). Using a genetic approach, my research group has been characterizing a gene important during mid-gastr ulation. We have identified ru5 , a conditional embryonic lethal mutation that causes terminal phenotypes that include body and head enclosure failure, a detached pharynx, and other body morphogenesis defects. Temperature shift experiments indicate that the gene acts during gastrulation. The ru5 mutation does not cause major changes in cell fate or position, as expression of six tissue-specific markers is normal. We do observe changes in the expression of vab-7/even-skipped homeodomain protein. The defects in vab-7 expression caused by ru5 appear to be distinct from those caused by tbx-8/9 mutants (Pocock, 2004; Andachi, 2004) indicating that the ru5 allele helps to define a second regulatory pathway for vab-7 . We have mapped the ru5 locus to ~19 on L.G.I and are continuing experiments to identify the altered gene. We have also begun a genetic suppressor screen to identify interacting genes, using the mos1 transposon as a mutagen (Bessereau, 2001). We wi ll report progress in these areas at the meeting.