The Nematode as a Model Organism for Human Gene Function. Genome Canada and Genome B.C. funded project: Obtaining C. elegans Knockouts of 2000 Genes with Human Homologs.

Number of ORF’s, DNA content and genes with mutant alleles in the nematode Caenorhabditis elegans. The C. elegans 97 megabase genome encodes approximately 19,293 predicted proteins. Almost 7,000 of these proteins have human homologs. Only 7% of the total proteins (encoded by 1277 genes) have been studied at either the genetic or biochemical level by the C. elegans research community. The current problem facing the community is how best to characterize the remaining 93% of worm genes/proteins.

Visual screens for novel phenotypes Requires extensive mapping Reverse genetics (RNAi) Effect not heritable Reverse genetics (gene disruption) How can we obtain new mutations?

Constructed by Steven Jones and Sheldon Mckay and maintained by Sheldon Mckay.

Total requests in queue = 1059 Vancouver node queue = 369 Total complete by consortium = 380 Vancouver node complete = 80 (53 on Genome Canada Funds) Another 20 in various stages of finishing

Program designed by Steven Jones and Sheldon McKay and maintained by Sheldon McKay

- Mutagenize Po worms (several choices of mutagen) - Grow worms until F populations are started with 50 F1s (about 120,000 mutagenized genomes) - F2 generation sampled for DNA prep, each sample into individual wells of 96-well block (12 blocks total) - Samples from 12 blocks are collapsed into one 96 well block - Remaining F2 worms stored at 15°C for sib- selection Library Construction

- PCR amplify DNA (2 rounds) - Run gels - Stain gels - Collect and store images - Determine deletion bands - Confirm address With current capacity we can examine 128 genes per week. This is 12,288 PCR reactions. We are aiming to examine 200 genes per week. Primary Screen

Basic Amplification Strategy Two rounds of PCR used to amplify genomic fragments, using an external pair of primers in the first round, and a pair internal to the first set for the second round. Deletion mutations give a smaller product than does wild type

Modified amplification strategy The Poison Primer Scheme Wild-type Products: Deletion Product: Reverse primer er Forward primer el Poison primer [] Deletion [] External amplification round

Sample Gel Images Marker is a 100bp ladder indicates putative deletion

Sib Selection A population of worms corresponding to positive DNA well is recovered from library plate and subjected to sib selection: A number of sub-populations are established (384) with 5 worms each and their progeny are re-screened by PCR (sib 1 screen) A positive population is selected, distributed at one worm per plate and re-screened (sib 2 screen) This procedure continues until single worms carrying the deletion are identified (usually two rounds but possibly more)

All strains carrying ko’s are taken to homozygosity if they are viable as homozygotes. If ko’s are lethal as homozygotes we balance them at our center. All deletions are sequenced by us to confirm that the ORF is eliminated. All strains are shipped to the CGC for distribution. All data concerning these strains is provided to Wormbase The final product

Deletions recovered with poison primers Average deletion size: 486 bp Exon Intron Normal Deleted

Precision knockout of a gene within a gene Exon Intron Normal Deleted

C01G8.2 - CLN3 protein (Batten Disease) T21G5.3 -RNA helicase (Werner’s syndrome) F55H2.1 - sod-4 (superoxide dismutase) Y50E8A.16 - ABC transporter T19A5.2 - serine/threonine kinase F52B5.5 - p53 B MAP kinase T05A6.1 - cyclin-dependent kinase inhibitor F42E p-glycoprotein C37H5.1 - annexin C36E8.5 - beta tubulin T05C hedgehog like protein T10B cytochrome p450 K02B9.4 - elt-3, GATA binding transcription factor Y56A3A.20 - Pol II transcription factor C27B7.8 - ras related protein C54D2.5 - skeletal muscle calcium channel C05D human homolog function unknown T16G human homolog function unknown A sample of targeted genes for which we have obtained ko’s over the past several months.

Expression Profiles of Cells and Tissues in C. elegans Primary Goals  Determine RNA expression patterns for selected tissues and cells (muscle, nerve, skin, gut) at several points during development (SAGE analysis)  Determine protein expression patterns from genes that have human homologues  Develop protein expression baseline for specific tissues during development

Sequence resources available for gene prediction in C. elegans C. elegans genome sequence: 97 Mb C. elegans ESTs (Y. Kohara):70,000 EST clusters:7,400 Non-redundant protein database Proteins encoded:19,000

Serial Analysis of Gene Expression (SAGE) 1 … 1 Velculescu et al. Science 1995 Oct 20;270(5235):484-7 Has been used to identify genes in the yeast genome sequence. 2 2 Velculescu et al. Cell 1997 Jan 24;88(2): Has been used to identify differences in levels of gene expression in developmental and disease states. 3 3 Zhang et al. Science 1997 May 23;276(5316):

Description of SAGE From: Johns Hopkins University, Drs. K. Kinzler and B. Vogelstein CATG [11] Only one tag sequence per messenger RNA.

Do C. elegans SAGE tags provide a tool for improving gene prediction?

An error in gene prediction….

Or an alternative splicing event?

The most abundant mixed-stage tag identifies an unannotated gene ?

Expression profiles of cells and tissues in C. elegans Integration of data by bioinformatics teams in C and S SAGE analysis Muscle Intestine Neurons Blast cells Promoter fusions 4D analysis C and S Promoter fusions Protein 2D gels Canada Sweden Protein 2D gels Both

Expression Profiles of Cells and Tissues in C. elegans Timeline Year 1Year 2 Year 3 Functional fusions SAGE Major tissue classes Selected cell typesSingle cell GFP constructs Antibody production 4-D microscopy Promoter fusions Screening genes Screening for new genes based on results from years 1 and 2

Expression Profiles of Cells and Tissues in C. elegans Communication –Within group Lab heads meet with research team weekly –Between groups Dr. Baillie meets with lab heads once per month prior to videoconference with co-director and lab heads in Sweden Co-directors and Management Team meet at alternate sites twice per year Selected travel for individual researchers as required for completion of project Secure website for posting data among research groups –Scientific community Access to selected parts of website (e.g. bioinformatics analyses, spatial visualization of expression data from constructs in tissues and cells) for all interested users Deposition of SAGE data in public databanks (sagemap at NCBI) SAGE and GFP data in WormBase Protein expression data in BIND

Genome BC Canada David Baillie, Project Co-Director Karolinska Institute Sweden Claes Wahlestedt Project Co-Director Functional Genomics Don Moerman Sequencing Marco Marra Bio- informatics Steven Jones Expression Profiles of Cells and Tissues in C. elegans Management Structure BIND (protein interaction database) Francis Ouellette Functional Genomics Thomas Bürglin Bio- informatics Erik Sonnhammer Protein Expression Sabine Schrimpf Functional Genomics Ana Vaz Gomes