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Caenorhabditis elegans Functional Genomics

Caenorhabditis elegans Functional Genomics. Sheldon McKay Januar y 22, 2004. Introduction C. elegans as a model organism Functional genomics Gene knockout project Goals Methods Progress Gene expression project Goals GFP-promoter fusions SAGE. C. elegans as a model organism.

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Caenorhabditis elegans Functional Genomics

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  1. Caenorhabditis elegans Functional Genomics Sheldon McKay January 22, 2004

  2. Introduction • C. elegans as a model organism • Functional genomics Gene knockout project • Goals • Methods • Progress Gene expression project • Goals • GFP-promoter fusions • SAGE

  3. C. elegans as a model organism • A 1mm long nematode worm • Short generation time and large numbers of progeny. • A metazoan with differentiated tissues and comprehensively studied anatomy and developmental program • Sequenced genome

  4. C. elegans genome • ‘Essentially complete’ as of December 1998 • Contains ~100 million bp on 6 chromosomes • Predicted to contain ~20, 000 genes. • ~ 55% of these genes are similar to genes from other organisms. • ~ 20% associated with mutationally defined genetic loci

  5. Genetics with a sequenced genome • Knowing the sequence isn’t everything. • We need to identify functions for as many uncharacterized genes as possible • Identification of a mutant phenotype in a gene of previously unknown function can help in assigning a function

  6. Forward vs. Reverse Genetics • In classical genetic analysis, we start with a mutant phenotype, genetically map the gene(s) responsible to chromosomal locations then, hopefully, find the gene and study the DNA sequence • Reverse genetics: We start with a DNA sequence believed to encode a gene. We then attempt to learn about the gene’s function through expression analysis and perturbations of its normal function with tools such as RNAi and mutational analysis

  7. C. elegans Functional Genomics Projects: Examples • Large-scale EST sequencing • ORFeome • Microarrays • RNAi • Gene Knockout Project

  8. C. elegans Gene Knockout Project • Objectives: • Study gene function by • determining the null phenotype • Make knockouts for all genes, • with an emphasis on mammalian • orthologs • Approach: • Use PCR to detect, isolate and • sequence deletion alleles

  9. PCR Screening for deletions

  10. Problems • The approach is biased towards large deletions • Sensitivity is low in complex populations • Targeting is imprecise

  11. 1o PCR 2o PCR NO PRODUCT The Poison Primer Technique

  12. 1o PCR 2o PCR The Poison Primer Technique

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

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

  15. C. elegans Gene Expression Project

  16. C. elegans Gene Expression Project Objectives: Build on the knockout project and other large-scale functional genomics projects Study gene expression patterns, with an emphasis on human orthologs

  17. Approaches: • High resolution image analysis of gene expression with GFP/Promoter fusions • Serial Analysis of Gene Expression (SAGE) and microarray analysis of gene expression of life stages and cell types

  18. Goals: Understand patterns of gene expression through the course of development and in particular cell types and tissues Identify known and novel cis-regulatory elements and their role in transcriptional regulation at the gene and network levels Understand gene expression patterns and protein interaction networks in the context of space and time in a developing organism

  19. Green Fluorescence Protein Fusion Studies of Gene Expression

  20. Isolating Potential Promoter Regions PCR primers Gene model SOCKEYE -- BCCA Genome Sciences Centre

  21. PCR-based Promoter GFP fusion (Hobert, Biotechniques 32:728-30) Promoter GFP GFP Promoter

  22. Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO

  23. Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO

  24. Neurons C13F10.4 -- contains similarity to Listeria monocytogenes Probable DNA-directed RNA polymerase delta subunit (RNAP deltasfactor).; SW:RPOE_LISMO protein of unknown function -- expressed in neurons

  25. A case-study of tissue-specific upstream regulatory elements

  26. M03F4.3 7-pass G-coupled transmembrane recepter Expressed in head, gut, vulva, tail Use PCR-stitching technique to dissect the putative promoter region

  27. Tissue-specific Control Elements Vulva, Tail Head, Gut

  28. Serial Analysis of Gene Expression

  29. SAGE • Objectives: • Determine temporal and spatial • gene expression patterns • Approaches: • Construct life-stage and tissue • specific SAGE libraries • Use GFP markers to isolate • tissue specific cell populations • via FACS

  30. Isolate mRNA, RT to cDNA Digest with “Tagging enzyme” BsmFI Sequence Ligate tags SAGE: Procedure Digest with “Anchoring enzyme” NlaIII http://www.sagenet.org/home/Description.htm

  31. SAGE Data Analysis • Tag abundance  transcript abundance • Abundant tags = Abundant transcripts • Identify interesting tags, find out which genes they belong to

  32. Statistical analysis of tag frequencies DISCOVERYspace -- BCCA Genome Sciences Centre

  33. Mapping SAGE tags to genes NlaIII AAAAAAA

  34. Mapping SAGE tags to genes Many predicted genes are not confirmed by Expressed Sequence Tags (ESTs) and do not include 3’ Untraslated regions (UTRs) 3’-most CATG Predicted: 5’ UTR 3’ UTR Actual: 3’-most CATG

  35. The virtual transcriptome

  36. Predicted Gene Models Genomic DNA Sequence EST Data No EST Data UTR length distribution Transcripts with EST support Estimate UTR length For unconfirmed gene models Transcripts without EST support Conceptual mRNAs

  37. NlaIII 3’ 5’ AAAAAAAAAA 6 5 4 3 2 1 Real 3’ UTR Tags Estimated 3’ UTR Position of NlaIII site

  38. NlaIII 2

  39. Predicted Gene Models Genomic DNA Sequence Experimental SAGE tags EST Data No EST Data UTR length distribution Transcripts with EST support Map tags Transcripts without EST support Digest in silico Adjust 3’ UTRs Theoretical SAGE tags Adjust gene models Conceptual mRNAs

  40. Relating Gene Expression to Development Source: http://nema.cap.ed.ac.uk/Caenorhabditis/C_elegans_genome

  41. C. elegans Life Cycle Adult Embryo L4 L1 L3 L2 ~3.5 days

  42. SAGE data: embryo Sage_summary -- BCCA Genome Sciences Centre

  43. Developmental Series: Top 12 collagen genes Tags/100K egg larval adult old

  44. Stress Response and Ageing Adult Embryo L4 L1 dauer L3 L2 ~3.5 days

  45. daf-2 • Insulin-like growth factor receptor • Constitutive dauer formation at 25C; reversible by shift to 15C. • Increased lifespan at 20C; • Increased thermotolerance, UV resistance. • Most alleles hypersensitive to dauer pheromone.

  46. Transcribed Telomeric Sequence(tts-1) • Most abundant trasncript in a SAGE comparison of dauer larvae and a normal mixed population (Jones, et al, 2001) • corresponds to a previously unknown non-coding gene • implicated in dauer larvae induction or maintenance

  47. tts-1 expression patterns dauer longevity starvation 1-day adult 6-day adult 1-day adult 6-day adult 10-day adult Dauer larvae Mixed population Normal L1 larvae Starved L1 larvae daf-2

  48. Relating gene expression to anatomy

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