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Yeast 2-hybrid database Plasmid database ATCC plasmid distribution

Yeast 2-hybrid database Plasmid database ATCC plasmid distribution. Alliance for Cell Signaling Myriad Genetics Inc. American Type Culture Collection. Yeast 2-hybrid project has switched to a macrophage focus. Years 1 and 2 (to May ‘03) Baits screened against 3 libraries (all mouse):

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Yeast 2-hybrid database Plasmid database ATCC plasmid distribution

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  1. Yeast 2-hybrid databasePlasmid databaseATCC plasmid distribution Alliance for Cell Signaling Myriad Genetics Inc. American Type Culture Collection

  2. Yeast 2-hybrid project has switched to a macrophage focus • Years 1 and 2 (to May ‘03) • Baits screened against 3 libraries (all mouse): • Primary B cell + WEHI231 B cell line • Primary myocyte* • NK tag myocyte cell line • Year 3 (May ‘03-’04) • New macrophage library prepared • Resting RAW cells • LPS + IFNg stimulated RAW cells • Primary bone marrow-derived macrophages • Year 3 baits are currently being screened against 3 libraries • Macrophage • B cell • Mouse embryo (mixture of several embryonic stages)

  3. Current status of Y2H screens • New macrophage library ready for use late 2003 • 74 bait proteins selected for screening this year • 46 new proteins • 28 proteins previously screened against old library set selected for re-screening • See poster #07 for full bait list • Data pending on this years screens • Completion of screens from years 1 and 2 has provided a large quantity of new data • May 2003 473 interactions • May 2004 1081 interactions

  4. Y2H data files included in download

  5. 1081 total interactions

  6. Y2H data files included in download

  7. AfCS proteins in FXM pathway Other AfCS proteins Protein with no AfCS ID (GI# known) Protein with no AfCS ID (novel sequence) All 1081 interactions viewed in cytoscape

  8. Yeast 2-Hybrid summary • Website and data format improvements significantly enhance utility of Y2H data • We expect the current ‘year 3’ screens to provide valuable additional data, especially for proteins central to macrophage signaling pathways • Prevalence of FXM specific proteins in this years bait set should provide a wealth of protein-protein interaction data for this network

  9. AfCS Plasmid Database

  10. Parent vector template used for Parent vector template used for databasing databasing attB1 attB1 attB2 attB2 Gene Gene Gateway-based plasmid construction

  11. Automatic generation of plasmid sequences and feature information Promoter Amp resistance Parent template: attB1 attB1 attB2 attB2 Cloned gene: Gene Gene Promoter Amp resistance Construct: attB1 attB1 attB2 attB2 Gene Gene

  12. Automatic generation of plasmid sequences and feature information Parent: + Cloned gene: Construct:

  13. Construct barcode A05XK099A1NK ID of Parent vector Vector type (X=expression) Gene alias Gene version Gene form (N=no termination codon) Bacterial selection (K=kanamicin)

  14. Akt1 cloned genes Gene forms and versions describe characteristics of each clone

  15. What have we cloned for cDNA expression? • Parent vectors (200+, 50+ to ATCC) • Fluorescent protein tagging • All GFP variants • Epitope tagging • Flag, HA, Myc, TAP • Promoter options • CMV, EF1, viral LTRs • Expression platforms • Mammalian, retroviral, lentiviral • cDNAs • Full-length wild type (900+) • Kinases (550+) • Others (350+) • Mutants • Dominant –ve (180) • Constit. Active (6) • Domains • PH domains (136) • Subcellular markers (50+)

  16. Parent vectors Pol III cloning vectors (11) shRNA expression vectors Lentiviral (30+) Retroviral (2) Mammalian (10+) 17 to ATCC (website feature) shRNAs Genes targeted (70+) Total shRNAs (200+) Validated shRNA constructs to ATCC (114) mH1 or mU6 shRNA attL1 attL2 LTR FLAP attR1 attR2 Ubi-C CD4 Flag HA CD8 IRES or IRES2 Puro Hyg Zeo Neo WRE LTR What have we cloned for RNAi?

  17. 2,700+ Plasmids are now available through the ATCC Internal: 220 parents 5000+ constructs Public: 53 parents 2,709 constructs

  18. Molecular Biology Laboratory Genie Lee Qingli Mi Eileen Fung Joelle Zavzavadjian Jamie Liu Sam Couture Kavitha Dhandapani Estelle Wall Leah Santat Microscopy Laboratory Weisun Park Sarah Lim Grischa Chandy Bioinformatics Laboratory Stephen Lyon Andreia Maer Brian Saunders ATCC Judy Kantor Linda Holloway Jennifer Zinck Barry Westfall Yeast 2-hybrid AfCS Bob Sinkovits Gil Sambrano Myriad Genetics Terrece Pearman Brandi Williams Max Dufford Karen Heichman Acknowledgements

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