Post on 27-Dec-2015
Functional genomicsFunctional genomicsStructural genomicsStructural genomics
ProteomicsProteomics
DNA sequencing analysis DNA sequencing analysis
mRNA transcript analysismRNA transcript analysis Proteome analysisProteome analysis Network analysisNetwork analysis
•DNA sequencing•S.N.P.•Gene mapping•Positional cloning
•DNA chips•cDNA arrays•Differential display
•2-D gel electrophoresis•Mass spcetroscopy•Protein chips
•Yeast two-hybrid• Phage display• Affinity assay tech• Protein chips• Systematic gene knockouts• Transient gene inactivator
Protein-Protein interactions
I. Biochemical approaches 1. Protein affinity chromatography 2. Affinity blotting 3. Immunoprecipitation 4. Cross-linking
II. Genetic approaches 1. Extragenic suppressor 2. Synthetic lethal effects 3. Overproduction phenotypes
III. Library-based approaches 1. Protein probing - gt11 library 2. Phage display 3. Two-hybrid system
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:
New versions
Different characteristicsa. NLSb. Transactivation capability VP16>Gal4AD > B42c. counterselection: CYH2d. Inducible expression: galactose, methionine pB42AD pGilda (LexA)e. With T7 promoter for expressing proteins in E.coli
Problems and limitations
1. Post-translational modification: glycosylation, disulfide bond formation, phosphorylation
solution-ectopic expressed protein (such as kinase)2. Bait fusion protein can activate expression of reporter in th
e absence o activation-domain fusion partner solution - +3AT or to use different domains as baits3. Some fusion proteins are harmful to yeast solution – use inducible system (Gal induced protein expre
ssion4. False-positive clones solution –use unrelted protein as a bait to reconfirm the s
pecificity
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
False positives in general
Proteins Found as false positives in IT
Real interactions (found in IT)
Found as false positives in other systems
hspsRibosomal proteins
Cytochrome oxidase
Mitochondrial proteins
Proteasome subunits
ferritin
tRNA synthase
Collagen related proteins
Zn finger proteins
vimentin
Inorganis pyrophosphate
PCNA
16 5
14 1 3
5 - 1
3
4
4
3
3
3
2
2
2
1
3-
-
-
4
-
-
-
-
2
-2
1
-
2
-
-
-
Others: 5 mitochondrial proteins (hsp, ribos, cyt.C oxidase, ATP-synth.)
The most common trash reported: elongation factor, ferritin
Hope for the best..
Major traps for a hunter
1. Transactivation of bait protein itself2. Failure to express the bail properly Solution: abandon hunts without even starting a screen
Total failure 16/115 library screening (no positives or only false positives
1. 4/16 – difficulties with protein expression2. 3/16 – weak transactivation by the bait protein3. 2/16 – primary transformants non-representative for the complexity
of the genome4. 1/16 – transformants were plated directly on selective medium5. 2/16 – no obvious reasons6. 4/16 – did not provide any clues
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.
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.
L6
• 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
提供之服務 Services Provided
• 酵母菌雙雜交系統之自動化
建立中之技術 Technology Development
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 )
LIBRARY VECTOR YEAST STAIN
Human liver cDNA library (4024)
pACT2 Y187
Human fetal brain cDNA library (4028)
pACT2 Y187
Human fetal liver cDNA library (4029)
pACT2 Y187
Drosophila 0~3 hr cDNA library
pGAD10 Y187
Yeast genomic two hybrid library
pGAD-C(X) Y187
Pre-transformed Libraries
L6
The Yeast Protein Linkage Map is 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
How to make 6000 GAL4-AD clones
Recombination cloningFirst round PCRUsing specific primersWith common tails
Re-PCR using commonprimers
Analysis of protein-protein interaction (PathCalling) e.g.AKR1 http://portal.curagen.com/extpc/com.curagen.portal.servlet.PortalYeastList
Yeast Resources for Funcional Genomic Studies
Deletion strains I (from Research Genetics or from ATCC)http://www.resgen.com/products/YEASTD.php3 - ordering
Search formhttp://www-deletion.stanford.edu/cgi-bin/deletion/search3.pl
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 cerevisiaeFred ShermanDepartment of Biochemistry and BiophysicsUniversity of Rochester Medical School, Rochester, NY 14642 1998http://dbb.urmc.rochester.edu/labs/Sherman_f/yeast/Index.html
Web sites:SGD: Saccharomyces Genome databasehttp://genome-www.stanford.edu/Saccharomyces/MIPS: Comprehensive Yeast Genome Databasehttp://mips.gsf.de/proj/yeast/CYGD/db/index.htmlYPD: Yeast Protein Databasehttp://www.proteome.com/databases/index.html