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Yeast 2-hybrid databasePlasmid database
ATCC plasmid distribution
Alliance for Cell SignalingMyriad 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):
• 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 + IFN 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)
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
All 1081 interactions viewed in cytoscape
Other AfCS proteins
AfCS proteins in FXM pathway
Protein with no AfCS ID (GI# known)
Protein with no AfCS ID (novel sequence)
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
Gateway-based plasmid construction
attB1
Gene
attB2
Parent vector template used for databasing
attB1
Gene
attB2
Parent vector template used for databasing
Automatic generation of plasmid sequences and feature information
attB1
Gene
attB2attB1
Gene
attB2
attB1
Gene
attB2attB1
Gene
attB2
Promoter Amp resistance
Parent template:
Cloned gene:
Construct:Promoter Amp resistance
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)
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+)
What have we cloned for RNAi?
• 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)
attL1mH1ormU6
attL2
attR1 attR2FLAPLTR Ubi-C WRECD4FlagHACD8
LTR
shRNA
IRESor
IRES2
PuroHygZeoNeo
2,700+ Plasmids are now available through the ATCC
Internal:220 parents5000+ constructs
Public:53 parents2,709 constructs
Acknowledgements
• 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