John White (2008) Development & Evolution Meeting "Cell Fate: Embryonic"

John White

[Development & Evolution Meeting, 2008]

No abstract submitted.

John Spieth (2007) International Worm Meeting "Caenorhabditis Genome Projects: An Update."

John Spieth

[International Worm Meeting, 2007]

In 2004 NHGRI approved whole genome, shotgun sequencing (WGS) to a high-quality draft level of three Caenorhabditis genomes; C. remainei, C. japonica and C. sp.4. (PB2801). These genomes, along with the finished C. elegans genome and the high-quality draft assembly of C. briggsae, will bring the number of Caenorhabditis genomes available for comparative genome analysis to five. All three of the new, genome projects will involve sequencing to a WGS coverage of ~9x in plasmid and fosmid end reads. This will be followed by two rounds of primer-directed sequence improvement focused on improving low-quality sequence regions and extending the assembly into, or filling, gaps. Additionally ~20,000 EST reads for each genome are to be done. C. japonica is in the WGS sequencing phase. Currelntly, WGS sequencing has been completed for PB2801 and sequence improvement is in progress. A preliminary assembly of the PB2801 WGS data is available for downloading (http://genome.wustl.edu/pub/organism/Invertebrates/Caenorhabditis_PB2801/assembly/draft) and a BLAST server (http://genome.wustl.edu/tools/blast/) for searching. For C. remanei both WGS sequencing and sequence improvement have been completed with a final assembly expected soon. Progress on completion of the genome projects and preliminary analysis will be presented.

John Spieth et al. (2002) Mid-west Worm Meeting "WormBase; An Update"

WormBase Consortium, John Spieth

[Mid-west Worm Meeting, 2002]

WormBase is an international consortium of biologists and computer scientists dedicated to providing the research community with accurate, current, and easily accessible genetic, sequence and phenotypic information for C. elegans and a few closely-related nematodes. This information is accessed through our website (www.wormbase.org). The database is also freely available with new releases being made every two weeks. The database is constantly improving through the curation of existing data and the addition of new datasets. With the goal of making all the data easily available, improvements to the website and the addition of data mining tools are continuely being made. Recent improvements and ongoing projects will be presented. Feedback, question and contributions from the C. elegans, as well as the broader biomedical, community are welcome and encouraged and can be made by emailing to wormbase-help@wormbase.org

John S Satterlee et al. (2002) East Coast Worm Meeting "CaMKI AND THERMOSENSATION"

Piali Sengupta, John S Satterlee

[East Coast Worm Meeting, 2002]

Thermosensation is perhaps the most poorly understood of the sensory modalities, despite its critical role in modulating the behavior and metabolism of organisms. Worms respond to temperature using the AFD thermosensory and the AIY and AIZ interneurons. To investigate AFD neuron development and function, we isolated mutants with defects in AFD-specific gene expression. We showed that the ttx-1 OTX/OTD-like homeodomain transcription factor is both necessary and sufficient to specify AFD fate (Satterlee et al, 2001). We also found that mutations in the tax-2 and tax-4 cGMP-gated channel genes and the cmk-1 gene result in defects in expression of a subset of AFD-specific markers. cmk-1 encodes the C. elegans ortholog of the calcium/calmodulin-dependent protein kinase I (CaMKI). Although CaMKI has been well characterized biochemically, the in vivo role of CaMKI is poorly understood. Both tax-2/4 and cmk-1 mutants have reduced expression of the AFD-specific genes gcy-8 and nhr-38 encoding a receptor guanylate cyclase and a nuclear hormone receptor homolog respectively, although expression of the ceh-14 LIM-homeobox gene is unaffected. While tax-2/4 mutants exhibit athermotactic behaviors, cmk-1 mutants are thermophilic, preferring temperatures warmer than their cultivation temperature. The cmk-1 promoter drives GFP expression in many neurons, including the AFD neurons, and a GFP-tagged CMK-1 protein is cytosolic. The gene expression and thermotaxis defects of cmk-1 mutants are rescued by AFD-specific expression of cmk-1 indicating that cmk-1 functions in a cell autonomous manner for these phenotypes. In vitro, CaMKI enzymes can be activated by both calcium/calmodulin binding and by phosphorylation of threonine-179 by upstream kinases. To determine the relationship between CMK-1 and TAX-2/4 in the regulation of expression of AFD-specific genes, we generated a truncated CMK-1 mutant protein carrying a T179D substitution. Based on in vitro experiments (Eto et al, 1999), this mutant protein is predicted to be calcium-independent and constitutively active. We found that expression of this gain-of-function CMK-1 protein in the AFD neurons partly rescues the gene expression defects of tax-4 mutants, suggesting that CMK-1 acts downstream of TAX-4. Our working hypothesis is that cmk-1 does not function directly in thermosensory signal transduction, but may instead play a role in modulating both long-term and short-term sensory plasticity. Preliminary experiments indicate that CMK-1 is also involved in adaptation to odorants sensed by the AWC olfactory neurons. We suggest that short-term sensory plasticity such as adaptation may occur via CMK-1 phosphorylation of AFD and AWC signal transduction components, whereas CMK-1 may regulate long-term changes via the modulation of the type and quantity of signaling molecules expressed. Current experiments are aimed at identifying both upstream regulators of CMK-1 function and downstream targets, utilizing genetic, genomic and biochemical methods.

John Yochem (2001) International C. elegans Meeting "From a tetraspanin mutation, hopeful monsters"

John Yochem

[International C. elegans Meeting, 2001]

Mutations have been isolated that disrupt the completion of molting. Among these are three that confer unusual phenotypes. sv3 homozygotes arrest growth in the L3 stage and are constricted by unshed cuticle for most of the middle part of the body. Cuticle is shed from the extremities, producing worms that resemble dumbbells. Genetic mapping has placed sv3 within a few cosmids of lin-2 on the X chromosome. The sv3 phenotype is mimicked by an unlinked mutation, sv9 , suggesting that at least two genes function in a biochemical or signal-transduction pathway that is required for proper synthesis or shedding of part of the cuticle. Located near unc-29 on LGI, sv15 confers dumpiness, molting defects, blistering of the cuticle, and the growth of small protuberances from the cuticle. Present at low penetrance are worms disfigured by large protuberances. The sv15 mutation has been rescued. Subcloning has indicated that sv15 is a mutation in a tetraspanin gene, a supposition proved by DNA sequence analysis. Mosaic analysis indicates that hyp7 is the focus of gene activity. More detailed analysis of the sv15 mutant phenotype and the isolation of interacting mutations have the potential to expand our knowledge of these enigmatic integral membrane proteins.

Rummage, John et al. (2011) International Worm Meeting "A Website to Annotate SNP Data."

Barstead, Robert J., Miller, Kenneth G., Rummage, John

[International Worm Meeting, 2011]

The first step in analyzing whole genome sequence alignments is to detect variant sequences and associate them with genome annotations. Most genome sequence facilities have access to developers who run command line driven scripts to make this association. Our mission is to simplify this and make the annotation of SNP and Indel calls accessible to the average investigator. To this end we have launched Whole Genomes, a website where investigators can upload SNP-called files for custom annotations (http://seqreport.omrf.org/genome: open to all on May 1, 2011). Administratively, the site design is flexible, allowing one to easily create annotation tables based on tab delimitated flat files like the GFF files distributed by Wormbase. Further, the site uses simple forms to establish persistent templates that describe the format for uploaded SNP files. All files are imported into a MySQL database and all comparisons are done via MySQL queries that offer a much more dynamic interaction with the data compared to a simple script driven approach. Users do not need to know anything about annotation files, templates, or MySQL to use the site. Whole Genomes allows users to map SNPs to any combination of features in the annotation database, such as coding sequence, potential splice sites, untranslated regions, or microRNAs. For examples, a coding sequence query would report the specific amino acid changes for SNPs that fall within translatable sequence along with a metric of evolutionary conservation for that particular change. The site also allows the user to compare SNPs in two strains to identify both unique and common variations. In addition, if a user has minimal or no mapping information Whole Genomes can compare two allelic strains to find the gene that is affected in both. Each annotated SNP is associated with a unique "Download" link that lets users download the DNA sequence associated with the annotated feature. Finally, for any given SNP, the site will design PCR primers to isolate DNA for SNP verification.

John E Kratz et al. (2005) International Worm Meeting "A worm Identikit for neuron identification"

John E Kratz, Marty Chalfie

[International Worm Meeting, 2005]

C. elegans neuronal gene expression patterns become much more interesting when they can be correlated with the wealth of anatomic and developmental data available for the worm. Although the expression pattern of a gene is easy to visualize with a fluorescent reporter, identification of the fluorescing cells can be quite difficult. This is particularly true for the majority of neurons whose cell bodies are packed into dense head and tail ganglia. We are addressing this problem by constructing a worm "Identikit" to simplify identification of neurons. The Identikit uses reconstituted GFP (recGFP) to address the problem of neuron identification. recGFP has been split into two halves that can be expressed individually from independent promoters. If a cell expresses both polypeptides, they associate to form a functional fluorescent molecule. Consequently, the expression pattern of an unknown promoter can be determined by comparison with the patterns of series of known promoters. In principle, multiple comparisons should be performed in one worm, because the C-terminal half of CFP (CZCFP) can associate with the N-terminus of GFP, YFP or CFP to produce spectrally distinguishable hybrids. The number of experiments necessary can be further reduced by conjugation of a nuclear localization signal to restrict the products of some promoters to the nucleus, yielding an easily distinguishable signal. We are testing these possibilities by making multiply marked strains. If multiple constructs function in a single strain, we estimate that the Identikit might only require 20 strains to allow identification of all of the 302 neurons in the hermaphrodite. We are also interested in generating strains that can be used to identify male neurons. To test promoters driving the expression of various NZ-fluorescent protein constructs for use in the Identikit, we have made a strain in which CZCFP is driven from the unc-119 promoter. We are grateful to our fellow worm workers for suggesting promoters for use in the Identikit and encourage further suggestions.

John Gladden et al. (2007) International Worm Meeting "Concerted action of X-signal elements."

Barbara Meyer, Behnom Farboud, John Gladden

[International Worm Meeting, 2007]

Development of multicellular organisms requires the specification of distinct cell fates. Often, different concentrations of dose-sensitive signals specify those distinct fates. Sex determination in C. elegans provides an excellent opportunity to dissect quantitative molecular signals. Sex is specified by a dose-sensitive signal that measures the ratio of X chromosomes to sets of autosomes. The X component consists of X-signal elements (XSEs) that act in a synergistic and dose-dependent manner to repress the sex switch gene xol-1. The autosomal component contains autosomal signal elements (ASEs) that oppose XSEs. In XX animals, XSEs overcome ASEs to repress xol-1 and establish the hermaphrodite fate, including the activation of dosage compensation. In XO animals, ASEs overcome the single dose of XSEs, causing xol-1 activation and the male fate. Two XSEs, sex-1 and fox-1, have been characterized previously. FOX-1 represses xol-1 by blocking proper pre-mRNA splicing, while SEX-1, a nuclear hormone receptor (NHR), associates with the xol-1 promoter in vivo to repress its transcription. SEX-1 binding sites have been identified within the xol-1 promoter in vitro, and confirmed in vivo, revealing that SEX-1 association with xol-1 is direct. Two newly defined XSEs, ceh-39 and sex-2, reduce endogenous xol-1 transcript levels. CEH-39, a ONECUT homeobox protein represses xol-1 through binding sites distinct from SEX-1 sites. A fifth XSE, located on left end of X (region 1), also affects xol-1 transcription. Since four of five XSEs regulate xol-1 transcript levels, transcriptional control is an important focus of this dose-sensitive signal. XSEs act cumulatively to repress xol-1, but they do not contribute equally. The relative strength of XSEs is sex-1 &#62;&#62; sex-2 &#62; fox-1 &#62; ceh-39 <font face=symbol>&#179;</font> region 1. A detailed analysis of sex-1 revealed why it is the strongest XSE. A newly obtained sex-1 null allele causes greater XX lethality, and xol-1(-) only partially rescues sex-1(-). Moreover, RNAi of an sdc gene is synergistically lethal with xol-1(-) sex-1(-), suggesting sex-1 acts both upstream and downstream of xol-1. The downstream sex-1 function regulates both sex determination and dosage compensation, like sdc genes. While sex-1s XSE function is that of a transcriptional repressor, its downstream function requires SEX-1-dependent transcriptional activation that is mediated through the NHR ligand-binding domain. On-going genetic and biochemical studies will define how XSEs, particularly sex-1, may carry out multiple roles in development, reproduction, and homeostasis.

John Koreth et al. (2005) International Worm Meeting "he121: a suppressor of cyclin-D1 in C. elegans"

Jerome Korzelius, Mike Boxem, Sander Van Den Heuvel, John Koreth, Abha Chandra, Na Li, Huihong Xu, Stuart H Orkin

[International Worm Meeting, 2005]

Regulation of cell division is a critical aspect of development. In general, the decision to undergo a further round of division or to withdraw from the cell cycle is made during the G1 phase. The cyclin D1/cyclin dependent kinase-4 (CYD-1/CDK-4) complex acts on downstream targets to enable progression through G1 and entry into S phase. Loss-of-function mutations in cyd-1 and cdk-4 abrogate post embryonic cell division and growth in C. elegans, which is rescued in part by inactivation of lin-35 Rb (Boxem, Dev. 2000). These results support not only that inactivation of LIN-35 Rb is an important function of CYD-1/CDK-4, but also that cyclin D kinases must have additional functions. To identify additional targets of cyd-1/cdk-4, we have performed a forward genetic screen for mutations that suppress the sterility of lin-35(RNAi);cyd-1(he112) animals. We have identified several candidate mutations in a substantial screen, including the mutation he121, which restores complete fertility to lin-35; cyd-1 double mutant animals. he121 single mutant animals are viable, without apparent abnormalities or hyperplasia. However, when combined with lin-35 RNAi, he121 dominantly enhances intestinal proliferation. Initial characterizations indicate that he121 defines a regulator of cell-cycle progression, which likely acts in parallel to lin-35 Rb. CDK Inhibitors (CKIs) of the Cip/Kip family, cki-1,2 Cip/Kip, act in a pathway distinct from lin-35 Rb and likely downstream of cyd-1/cdk-4 (Boxem, Dev. 2000). However, SNP mapping has placed the gene affected by he121 within a narrow interval to the right of cki-1,2 Cip/Kip on LG II, indicating that it may represent a novel cell-cycle regulator. Further details on this screen and the he121 mutation will be presented.

John S Satterlee et al. (2003) International Worm Meeting "Calcium, cGMP, and Thermosensory Neurons"

John S Satterlee, Piali Sengupta

[International Worm Meeting, 2003]

Thermosensation is a poorly understood sensory modality, yet critically important for modulating the behavior and metabolism of organisms. In C. elegans, the AFD neurons are required for thermosensation. In order to investigate AFD function, we have identified genes required for expression of the AFD-specific marker gcy-8::gfp. cmk-1 encodes the C. elegans ortholog of the calcium/calmodulin dependent protein kinase I. cmk-1 mutants show reduced expression of gcy-8::gfp, and exhibit defects in the morphology of the specialized microvillar endings of the AFD neurons. cmk-1 animals exhibit a thermophilic behavior in radial temperature gradients. Taken together, these results implicate cmk-1 in the development and function of the AFD neurons. The cmk-1 promoter drives GFP expression in many neurons, including the AFD thermosensory neurons, where it is localized to the cytoplasm. Expression of the cmk-1 cDNA only in the AFD neurons is sufficient to rescue the cmk-1 gene expression defect, indicating that cmk-1 functions cell autonomously to regulate AFD-specific gene expression. Vertebrate CaMKI can be regulated via CaMKK phosphorylation, however we have not found evidence for this regulation in the AFD neurons. The tax-2/4 genes encode cGMP-gated channel subunits believed to be involved in thermosensory signal transduction. tax-2/4 mutants exhibit athermotactic behavior on radial temperature gradients. Similar to cmk-1 mutants, we found that tax-2/4 mutants also exhibit reduced expression of the AFD-specific gcy-8::gfp marker. Furthermore, the AFD microvillar endings are defective in both tax-4 and tax-2 mutants. Double mutant analysis suggests that cmk-1 acts downstream of, or parallel to, tax-4 in the regulation of AFD-specific gene expression. Preliminary evidence suggests that regulation of gene expression by the CMK-1 pathway is temperature sensitive, perhaps providing a mechanism for the AFD neurons to adapt to different temperatures. Little is known about the functions of CaMKI enzymes in any organism, although other CaMKs have been implicated in activity-dependent gene expression and synaptic plasticity. Genetic characterization of CMK-1 regulation of gene expression in the AFD neurons may shed light on these processes. A subset of CMK-1 targets likely participate directly in thermosensory signal transduction. We will utilize both genetic and genomic approaches to identify CMK-1 targets, and anticipate that CMK-1 characterization will reveal much about the neuronal functions of CaMKs.