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

http://www.signaling-gateway.org/data/Y2H/cgi-bin/y2h.cgi

Y2H data files included in download

1081 total interactions

Y2H data files included in download

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

AfCS Plasmid Database

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

Automatic generation of plasmid sequences and feature information

Parent:

Cloned gene:

Construct:

+

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)

Akt1 cloned genes

Gene forms and versions describe characteristics of each clone

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