Yeast Genetics Lab.
Yeast two-hybrid system
Yeast genetic analysis
- tetrad dissection
Structural genomics
Functional
genomics
Proteomics
DNA sequencing
analysis
•DNA sequencing
•S.N.P.
•Gene mapping
•Positional cloning
mRNA transcript
analysis
•DNA chips
•cDNA arrays
•Differential
display
Proteome analysis
Network analysis
•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 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
Proteins
Found as false
positives in IT
Real interactions
(found in IT)
Found as false
positives in other
systems
hsps
Ribosomal proteins
Cytochrome oxidase
16
5
-
14
5
1
3
1
Mitochondrial proteins
ferritin
4
1
3
-
2
Proteasome subunits
3
4
tRNA synthase
3
-
1
Collagen related proteins
3
-
-
Zn finger proteins
3
4
2
2
-
-
Inorganis pyrophosphate
2
-
-
PCNA
2
vimentin
-
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.
2.
3.
4.
5.
6.
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
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
L6
VECTOR
YEAST STAIN
Human liver cDNA library pACT2
(4024)
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
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
With common tails
Re-PCR using common
primers
Recombination cloning
Analysis of protein-protein interaction
(PathCalling)
e.g.AKR1
http://portal.curagen.com/extpc/com.curag
en.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/cgibin/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