1. The evolution of the immune system in chicken and higher vertebrates @ Organon, Oss 2005-09-20 Tim Hulsen

2. Biorange Project SP3.2.2 Chicken immunosystem project is part of WP1, “Translational Medicine through Comparative Genomics and Integration” Partners: –Animal Breeding and Genetics Group, Wageningen UR (Prof. dr. Martien Groenen) –Avian Cytokines Group, Institute for Animal Health, Compton (UK) (Prof. dr. Pete Kaiser) Jack Leunissen (WUR) also part of WP1

3. M. Groenen: chicken sequencing

4. Kaiser: chicken immune system

5. Introduction Goal: developing an insight in the recent evolution of the immune system Usage of a more distant species: chicken (recently sequenced) Support by experimental data

6. Overview 1.Find IS-related proteins 2.Determine orthologies 3.Pfam annotation 4.Panther annotation 5.Zooming in

7. Step 1: Find IS-related proteins IRIS: “Immunogenetic Related Information Source” number of immune genes: 1562 (out of 21389 in LocusLink) percentage of genome related to immunity: 7.30% 1562 LocusLink proteins mapped to our Protein World set: 1381 proteins

8. Step 1: Find IS-related proteins

9. GO: Gene Ontology collaborative effort to address the need for consistent descriptions of gene products in different databases Checked human GO annotation for certain terms: “immunology”,”cytokine”,etc. 1515 proteins in human Potein World set

10. Step 1: Find IS-related proteins Result: –1381 proteins through IRIS –1515 proteins through GO –1929 proteins total

11. Step 2: Determine orthologies Study evolution from chicken (Gg) to rat (Rn), mouse (Mm) and human (Hs): –Hs Mm –Hs Rn –Hs Gg –Mm Rn –Mm Gg –Rn Gg Two methods: Best Bidirectional Hit (BBH) and PhyloGenetic Tree (PGT)

12. Best Bidirectional Hit (BBH) Very easy and quick Human protein (1)  SW  best hit in mouse/rat (2) Mouse/rat protein (2)  SW  best hit in human (3) If 3 equals 1, the human and mouse/rat protein are considered to be orthologs

13. Step 2: Determine orthologies BBHHsMmRnGg Hs192911451046704 Mm--863345 Rn---488 Gg----

14. PhyloGenetic Tree (PGT) Human Human, mouse, rat, chicken Hs, Mm, Rn, Gg Z>20 RH>0.5*QL ~25,000 groups PHYLOME SELECTION OF HOMOLOGS ALIGNMENTS AND TREES PROTEOME PROTEOMES TREE SCANNING LIST Hs-Mm pairs Hs-Rn pairs Hs-Gg pairs Mm-Rn pairs Mm-Gg pairs Rn-Gg pairs

15. Step 2: Determine orthologies PGTHsMmRnGg Hs1929230118191129 Mm--28732087 Rn---2142 Gg----

16. Step 3: Pfam annotation Pfam: “Protein families database of alignments and HMMs” collection of protein families and domains Pfam contains multiple protein alignments and profile-HMMs of these families 75% of protein sequences have at least one match to Pfam 1700 IS-related proteins mapped to 584 Pfam families (2814mappings)

17. Step 3: Pfam annotation BBHHsMmRnGgPGTHsMmRnGg Hs192911451046704Hs1929230118191129 Mm--863345Mm--28732087 Rn---488Rn---2142 Gg---- ---- BBH pfamHsMmRnGg PGT pfamHsMmRnGg Hs17761069974639Hs1776213517001040 Mm--795312Mm--28462070 Rn---442Rn---2125 Gg---- ----

18. Step 3: Pfam annotation

19. Step 4: Panther annotation PANTHER: “Protein ANalysis THrough Evolutionary Relationships” designed to classify proteins (and their genes) in order to facilitate high-throughput analysis proteins have been classified according to families and subfamilies, molecular functions, biological processes, pathways contains over 6683 protein families, divided into 31,705 functionally distinct protein subfamilies 1872 IS-related proteins mapped to 970 Panther families (4667 subfamilies, 14737 mappings)

20. Step 4: Panther annotation BBHHsMmRnGgPGTHsMmRnGg Hs192911451046704Hs1929230118191129 Mm--863345Mm--28732087 Rn---488Rn---2142 Gg---- ---- BBH pantherHsMmRnGg PGT pantherHsMmRnGg Hs187211251029688Hs1872226617931118 Mm--846339Mm--27291970 Rn---481Rn---2008 Gg---- ----

21. Step 4: Panther annotation

22. Step 5: Zooming in Which families are ‘new’ in human? Which orthologs have a different domain structure through evolution? Which human proteins don’t have orthologs in the other species? Any other interesting stuff

23. Future directions Include paralogs in our analysis (makes possible checking which families only exist in mouse/rat/chicken) Combine our findings with research at WUR: synteny between human and chicken Take a look at ratio of non-synonymous to synonymous substitutions (dN/dS)

24. Credits NV Organon: –Peter Groenen –Wilco Fleuren Wageningen UR: –Martien Groenen –Hindrik Kerstens Compton (UK): –Pete Kaiser