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Yeast Genetics Lab. Yeast two-hybrid system Yeast genetic analysis - tetrad dissection

Yeast Genetics Lab. Yeast two-hybrid system Yeast genetic analysis - tetrad dissection. Structural genomics. Functional genomics. Proteomics. DNA sequencing analysis . mRNA transcript analysis. Proteome analysis. Network analysis. DNA chips cDNA arrays

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Yeast Genetics Lab. Yeast two-hybrid system Yeast genetic analysis - tetrad dissection

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  1. Yeast Genetics Lab. Yeast two-hybrid system Yeast genetic analysis - tetrad dissection

  2. Structural genomics Functional genomics Proteomics DNA sequencing analysis mRNA transcript analysis Proteome analysis Network analysis • DNA chips • cDNA arrays • Differential display • DNA sequencing • S.N.P. • Gene mapping • Positional cloning • 2-D gel electrophoresis • Mass spcetroscopy • Protein chips • Yeast two-hybrid • Phage display • Affinity assay tech • Protein chips • Systematic gene knockouts • Transient gene inactivator

  3. Protein-Protein interactions • Biochemical approaches • 1. Protein affinity chromatography • 2. Affinity blotting • 3. Immunoprecipitation • 4. Cross-linking • Genetic approaches • 1. Extragenic suppressor • 2. Synthetic lethal effects • 3. Overproduction phenotypes • Library-based approaches • 1. Protein probing - gt11 library • 2. Phage display • 3. Two-hybrid system

  4. How the two-hybrid system works If the two proteins interact, the reporter gene (here: HIS3) is switched on and the diploids can grow on -His plates: If the two proteins don't interact, the reporter gene remains inactive and the cells can't grow on -His plates:

  5. New versions • Different characteristics • NLS • Transactivation capability VP16>Gal4AD > B42 • counterselection: CYH2 • Inducible expression: galactose, methionine • pB42AD pGilda (LexA) • e. With T7 promoter for expressing proteins in E.coli

  6. Problems and limitations • Post-translational modification: glycosylation, disulfide bond formation, phosphorylation • solution-ectopic expressed protein (such as kinase) • Bait fusion protein can activate expression of reporter in the absence o activation-domain fusion partner • solution - +3AT or to use different domains as baits • Some fusion proteins are harmful to yeast • solution – use inducible system (Gal induced protein expression • 4. False-positive clones • solution –use unrelted protein as a bait to reconfirm the specificity

  7. Advantages ad disadvantages of yeast 2-H • Advantage • direct identification of DNA sequence of interacting protein • No antibodies requries • Protein purification not necessary • In vivo-protein in native conformation? • Detect low affinity or transient interactions • Disadvantage • -failed to detect some know interactions • gene encoding target protein must be available • Bait and prey must be soluble for nuclear localization • Independent verification of interaction is recommended • False positives • Possible incorrect protein folding in yeast • Stable expression of fusion protein might be a prlblem • Not approapriate post-translational modifications

  8. False positives in general Found as false positives in other systems Real interactions (found in IT) Found as false positives in IT Proteins 16 5 - hsps Ribosomal proteins 1 14 3 Cytochrome oxidase 5 - 1 Mitochondrial proteins 3 1 2 Proteasome subunits 4 3 - - ferritin 2 4 tRNA synthase 1 3 - Collagen related proteins 3 - - 3 2 Zn finger proteins 4 vimentin 2 - - Inorganis pyrophosphate - - 2 PCNA 2 - - Others: 5 mitochondrial proteins (hsp, ribos, cyt.C oxidase, ATP-synth.) The most common trash reported: elongation factor, ferritin

  9. Hope for the best.. • Major traps for a hunter • Transactivation of bait protein itself • Failure to express the bail properly • Solution: abandon hunts without even starting a screen

  10. Total failure 16/115 library screening (no positives or only false positives • 4/16 – difficulties with protein expression • 3/16 – weak transactivation by the bait protein • 2/16 – primary transformants non-representative for the complexity of the genome • 1/16 – transformants were plated directly on selective medium • 2/16 – no obvious reasons • 4/16 – did not provide any clues

  11. Your chance of success • If your protein is properly expressed and is not activating the reporters odds are 6 to 1 that you will pull out womething which makes biological sense, if you screen an adequate number of clones (1-2 x 106 primary transformants for human cDNA library • If your protein is weakly transactiating, your chances drop, but not really dramatically. • If you cannot detect your bait protein in yeast, your chance drop substantially.

  12. The degree of your success can vary! The complexity of many genomes and the complexity of the web of protein interactions is beyond of the abilities of any human-made systems. So, you will find not necessarily what you want to find, it is better not to be ruled by preconceptions and to be aware of the limitation of the system. Two hybrid systems - > to uncover unanticipated interactions.

  13. PJ69-2A (bait) Ade+

  14. 提供之服務 Services Provided • 酵母菌雙雜交選殖系統 • 提供 pre-transformed libraries • 酵母菌四分孢子分析及單細胞分離 建立中之技術 Technology Development • 酵母菌雙雜交系統之自動化 其他酵母菌遺傳系統技術 Other yeast techniques • 酵母菌合成性致死基因選殖(如圖) L6

  15. Pre-transformed Libraries L6

  16. Reverse two-hybrid system

  17. Three hybrid system

  18. Other alternative two-hybrid system Cell membrane Sos Cell membrane Y Sos Ras activation Ubiquitin

  19. The Yeast Protein Linkage Mapis an attempt to identify as many protein-protein interactions among yeast proteins as possible by testing all possible protein pairs (I.e. ~6000 x6000 = 36 x 106 ) for interactions by individual two-hybrid tests. http://depts.washington.edu/sfields/yp_project/index.html

  20. How to make 6000 GAL4-AD clones First round PCR Using specific primers With common tails Re-PCR using common primers Recombination cloning

  21. Analysis of protein-protein interaction (PathCalling) e.g.AKR1http://portal.curagen.com/extpc/com.curagen.portal.servlet.PortalYeastList

  22. Yeast Resources for Funcional Genomic Studies Deletion strains I (from Research Genetics or from ATCC) http://www.resgen.com/products/YEASTD.php3 - ordering Search form http://www-deletion.stanford.edu/cgi-bin/deletion/search3.pl

  23. References for yeast genetics. Molecular Biology of the Gene 2nd ed. Chapter 18 An Introduction to the Genetics and Molecular Biology of the Yeast Saccharomyces cerevisiae Fred Sherman Department of Biochemistry and BiophysicsUniversity of Rochester Medical School, Rochester, NY 14642 1998 http://dbb.urmc.rochester.edu/labs/Sherman_f/yeast/Index.html Web sites: SGD: Saccharomyces Genome database http://genome-www.stanford.edu/Saccharomyces/ MIPS: Comprehensive Yeast Genome Database http://mips.gsf.de/proj/yeast/CYGD/db/index.html YPD: Yeast Protein Database http://www.proteome.com/databases/index.html

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