David Baillie et al. (2003) International Worm Meeting "Evaluation of SAGE for the study of developmental gene expression profiles in C. elegans"

Heide Kai, Adam Warner, Greg Vatcher, Pawan Pandoh, Peter Huang, Richard Varhol, Helen McDonald, Kim Wong, Scott Zuyderduyn, David Baillie

[International Worm Meeting, 2003]

As part of a consortium of laboratories in Canada and Sweden (see McKay, et al, "Gene expression profiles in cells, tissues and development of C. elegans"), we have developed computational and biological approaches to assist in high throughput analysis of gene expression. Biological source material for SAGE libraries was generated using synchronized populations as well as micro-dissected gut tissue. We are also refining GFP-mediated cell sorting techniques to isolate other cell types from disrupted embryos. SAGE data are generated and tracked in an automated pipeline from sequencing of cloned concatamers through to annotation, quality control and mapping of individual tags. Tags are associated with attributes such as cumulative phred scores, source clone, parent ditag and tag-to-gene mapping data to aid in downstream quality filtering and analysis steps. To facilitate tag-to-gene mapping and comparison of SAGE and Affymetrix GeneChip technologies, a virtual C. elegans transcriptome with both confirmed and estimated untranslated regions (UTRs) for all known and predicted transcripts was constructed using the current gene models and expressed sequence tag (EST) data annotated in wormbase. We are also refining tag-to-gene mapping methods in order to detect previously undocumented transcripts and alternative splice variants. Comparison of the Affymetrix C. elegans chip with the current virtual transcriptome showed that about 97% of the confirmed or predicted transcripts are detected by all or part of at least one probe set. At the time of writing, four developmentally staged SAGE libraries to be used in developmental gene expression profiling have been completed. Preliminary SAGE studies and results of a direct comparison of gene expression levels measured from the same biological source material with SAGE, long-SAGE and Affymetrix GeneChip technologies will be presented. This project is funded by Genome British Columbia and Genome Canada.

Ana Vaz Gomes et al. (2003) International Worm Meeting "Gene expression profiles in cells, tissues and development of C. elegans"

Peter Huang, Claes Wahlestedt, Nicholas Dube, Ana Vaz Gomes, Kim Wong, David Baillie, Heide Kai, Adam Warner, Lily Fang, Allan Mah

[International Worm Meeting, 2003]

A new consortium of labs in Canada and Sweden has been established to complement other Caenorhabditis elegans functional genomics efforts by using additional high-throughput approaches to gene expression profiling through the course of development. We are examining expression profiles for 2000 genes, with an emphasis on human orthologs and genes that are currently being knocked out by the C. elegans gene knockout consortium. We are interested in profiling temporal and spatial patterns of gene expression and furthering our understanding of cellular protein network organization in a three dimensional, organismal context. The advanced state of genome science and well-defined developmental program in C. elegans make it an ideal candidate organism. We are using serial analysis of gene expression (SAGE) and high resolution image analysis of expression patterns detected with green fluorescent protein (GFP)-promoter fusion constructs. The initial focus is on developmental SAGE of various life stages from egg to adult and the generation of GFP-promoter fusion strains suitable for high-resolution image analysis of in vivo gene expression patterns. Thus far, we have generated promoter-GFP transgenic strains for 400 genes (see Johnson et al, "Expression of promoter-GFP constructs in C. elegans") and SAGE libraries for various stages of C. elegans development including embryo, the four larval stages and young adult (see McKay et al, "Evaluation of SAGE for the study of developmental gene expression profiles in C. elegans"). To generate tissue-specific SAGE libraries, we are refining techniques to use selected GFP constructs in conjunction with fluorescence activated cell sorting to isolate samples enriched for particular cell types from disrupted embryos. This project is funded by Genome British Columbia and Genome Canada

Eric A Evans et al. (2005) International Worm Meeting "The regulation of innate immunity by the DAF-2 insulin signaling pathway: CBP-1 is a potential DAF-16 cofactor"

Eric A Evans, Man-Wah Tan

[International Worm Meeting, 2005]

The insulin/IGF-1 (DAF-2) signaling pathway regulates innate immunity in C. elegans. We and others have previously shown that mutations in daf-2 pathway genes and glp mutants enhance resistance to bacterial pathogens in a manner dependent on the FoxO transcription factor DAF-16 (1).Acetylation by CBP/p300, transcriptional cofactors with histone acetyltransferase activity, has been implicated as a regulator of FoxO activity in mammalian systems (2-5). These studies have suggested a model in which binding of CBP/p300 to FoxO factors is essential for FoxO-mediated transcription (6). Corroborating this model, we will present evidence that cbp-1, the worm CPB/p300 homolog, is essential for the immunity enhancements mediated by DAF-16. RNAi of cbp-1 at L4 suppresses resistance to Pseudomonas aeruginosa in the rrf-3(pk1426);glp-4(bn2) double mutant strain to the same extent as daf-16 RNAi does.We also observe that continuous exposure of daf-2(e1370) mutant worms to the cbp-1 RNAi feeding strain results in early death at a rate similar to daf-16 RNAi. Assigning significance to this suppression with regard to the distinction between aging and immunity is not straightforward because live bacteria are used to induce RNAi. Although E. coli is often regarded as a non-pathogenic food for worms, there is evidence that bacterial proliferation is a cause of death in aging worms (7). We believe the confounding of aging and immunity within these assays is a problem that warrants further investigation.We are investigating the relationship between aging and immunity in the context of the DAF-2 pathway. We will present evidence for a model of distinct regulation of aging and immunity.(1) Garsin (2003) Science 300. (2) Brunet (2004) Science 303. (3) Motta (2004) Cell 116. (4) Daitoku (2004) PNAS 101. (5) van der Horst (2004) JBC 279. (6) van der Heide (2005) Trends Biochem Sci. 30. (7) Garigan (2002) Genetics 161.