Yeast Genetics Lab.

Yeast two-hybrid systemYeast genetic analysis - tetrad dissection

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.

PJ69-2A(bait)

Ade+

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

Reverse two-hybrid system

Three hybrid system

Cell membrane

Cell membrane

Other alternative two-hybrid system

Ras activation Ubiquitin

Sos

Sos

Y

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