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									Yeast Genetics Lab.

Yeast two-hybrid system
Yeast genetic analysis
  - tetrad dissection
   Structural genomics                Functional    genomics
                                                          Proteomics


DNA sequencing        mRNA transcript
                                           Proteome analysis    Network analysis
analysis              analysis

•DNA sequencing       •DNA chips         •2-D gel              •Yeast two-hybrid
•S.N.P.               •cDNA arrays       electrophoresis       • Phage display
•Gene mapping         •Differential      •Mass spcetroscopy    • Affinity assay tech
•Positional cloning   display            •Protein chips        • 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 gro
on -His plates:
               New versions




Different characteristics
a. NLS
b. Transactivation capability VP16>Gal4AD > B42
c.   counterselection: CYH2
d. 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
    the absence o activation-domain fusion partner
     solution - +3AT or to use different domains as baits
3. 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
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
                                                                        Found as false
               Proteins       Found as false      Real interactions     positives in other
                              positives in IT     (found in IT)         systems
                  hsps             16                     5                   -
     Ribosomal proteins            14                     1                   3
    Cytochrome oxidase              5                     -                   1
  Mitochondrial proteins            3                     1                   2
    Proteasome subunits             4                     3                   -
                 ferritin           4                     -                   2
          tRNA synthase             3                     -                   1
Collagen related proteins           3                     -                       -
       Zn finger proteins           3                     4                   2
               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
 Hope for the best..

Major traps for a hunter

1. Transactivation of bait protein itself
2. 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 expression
2.   3/16 – weak transactivation by the bait protein
3.   2/16 – primary transformants non-representative for the complexity
     of the genome
4.   1/16 – transformants were plated directly on selective medium
5.   2/16 – no obvious reasons
6.   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.
PJ69-2A
(bait)




          Ade+
     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
             Pre-transformed Libraries
     LIBRARY                    VECTOR    YEAST STAIN


     Human liver cDNA library pACT2       Y187
     (4024)
     Human fetal brain cDNA     pACT2     Y187
     library (4028)
     Human fetal liver cDNA     pACT2     Y187
     library (4029)
     Drosophila 0~3 hr cDNA     pGAD10    Y187
     library
     Yeast genomic two hybrid   pGAD-C(X) Y187
     library


L6
Reverse two-hybrid system
Three hybrid system
                     Other alternative two-hybrid system

                 Cell membrane



Sos




                 Cell membrane

Y

      Sos




            Ras activation                    Ubiquitin
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




First round PCR
Using specific primers   Re-PCR using common   Recombination cloning
With common tails        primers
Analysis of protein-protein interaction
(PathCalling)

e.g.AKR1
http://portal.curagen.com/extpc/com.cu
ragen.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 form
http://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 cerevisiae
Fred Sherman
Department of Biochemistry and Biophysics
University 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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