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The Yeast Resource Center

The Yeast Resource Center

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The Yeast Resource Center

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  1. The Yeast Resource Center An NCRR Biomedical Technology Resource Center http://depts.washington.edu/~yeastrc

  2. Focus • Technologies that extract information about macromolecular interactions from the genome sequence. • Regulation • Dynamics

  3. The people The technologies • The YRC is a consortium of five groups and five technologies • Aebersold mass spectrometry • Yates mass spectrometry • Fields two-hybrid • Baker protein struct. prediction • Muller/Davis microscopy and FRET • Davis/Baker informatics

  4. A P41 center has three missions • Technology development • Collaboration • Dissemination and Training

  5. By the numbers(for 2002) • 51 publications • Technology development: • 19 projects • Collaborations: • 132 collaborators, 80 projects • Dissemination and Training: • 130 individuals • 26 states, 12 countries

  6. Unknown ORFs • 105 unknown essential ORFs • Integrate all our technologies • Find a balance between quantity of data and quality of data

  7. Analysis of high throughput data von Mering et al., 2002

  8. Unknown essential ORFS • Tag with TAP tag and YFP tag • Purify each with TAP tag • Identify copurifying proteins by mass spec • Localize in living cells • Two-hybrid analysis • Advanced homology searches • Protein structure prediction

  9. YKR038C (47 kD) 212 212 Standards YGR046W (44kD) YJR013W (35kD) YOL034w (126 kD) YGR024C (28kD) YKR079C (97kD) YNL158W (22kD) YFR003C (18kD) YHR085W (38kD) YGR002C (55kD) Spc97p Standards Spc97p * 97 97 66 66 56 * 56 43 * * 43 * * 36 36 * * * * Optimization of purification with TAP tag New method Old method Graczyk, Sundin, Fox and Davis

  10. Summary of purification • 20 are part of a complex • 15 different complexes • 28 purify by themselves • 22 could not be tagged • 26 were inactivated by tag • 9 were not produced at detectable levels in logarithmic cultures

  11. Integration of technologies builds the story • New complex, ribosomal recycling • All four members are essential unknowns • One localizes to nucleolus, three others killed by tag • Two-hybrid confirms mass spec • Homology searching finds homology of one to ribosomal recycling factor

  12. Integration of technologies builds the story • An ER protein that purifies alone • Mass spec finds only that protein • Localizes to ER • Two-hybrid identifies a collection of membrane proteins • Homology searching identifies homology to mannosyltransferases

  13. Integration of technologies builds the story • YKR079c-TAP copurifies with 2 proteins. But localization reveals they are likely to form two complexes of two proteins. YKR079c NUC1 YMR099c

  14. Lessons • Mass spec is extremely powerful • Two-hybrid expands the story and provides critical information for proteins not in complexes • Localization is fast and easy • Great deal of information to be had merely by advanced homology searching • Protein structure prediction is underway

  15. Recommendations • Think in terms of optimal throughput which strikes a balance between quantity and quality. • More mass spec capacity is needed and the technology is not yet readily disseminated. • Promote the integration of technologies. • Increase capacity and speed of advanced homology searches.

  16. John Yates Scott Anderson Hayes McDonald Ruedi Aebersold Rick Newitt Eugene Yi Stan Fields Tony Hazbun Jennifer Aranda Eric Muller Brian Snydsman Trisha Davis Bethany Fox Beth Graczyk David Baker Lars Malmström Bill Schief Informatics Mike Riffle Yeast Resource Center