Progress of Researchgenome

Genomic sequence ・ Polymorphism（ SNP etc ）

Transcriptome

Gene transcription profile

Proteome Expression profile of proteins

Functional proteomeMetabolome

Genetic functionPost translational modificationProtein interation etc.

Time consuming and enormous costElucidation of functional network of cellular

molecules　　　　　

DNA Chip

mRNA extractionLabeled withCy5 and Cy3

Hybridization

Genes in which transcription levels are affected with the disease can be determined.

Normal ratDisease rat

Importance of RNA

Species and genomic size ・ number of genes　　　　　　　　　　　　　 Genomic size （ Mbp) 　　　　 The number of genesHuman 　　　　　　　　　　　　　　２３５１　　　　　　　　２２２８７

C-elegance 　　　　　 　　　　　　　１０３　　　　　　　　１９８９３

Drosophila 　 　　　　　　　　１８０　　　　　　　　１３６７６

Arabidopsis thaliana 　　　　 　　　　１２５　　　　　　　　２５４９８

Cellular slime 　　　　　　　　　　　　　３４　　　　　　　　１２５００

Yeast 　　　　　　　　　　　　　　１３　　　　　　　　　５５３８

In E. Coli., 80% of genomic DNA encodes proteins.On the other hand, human genomic DNA contains only 3% for genes.However, 70-80% of human genomic DNA is transcripted! → non-coding RNA

Human Accelerating Regions (HAR)The genomic region that has the most different transcription activity between human and chimpanzee.The transcript from this region was small RNA.

Lung cancer ：Causing by a transcriptional suppression of a microRNA which suppresses ras gene

Limphoma ：Causing by a overtranscription of a microRNA which suppress E2F (apoptosis inducible protein)

MicroRNA controls gene expression level.(miRNA can find out the specific mRNA by using the sequence of its own. ）

MicroRNA also sometimes regulate protein activity with binding to it.Ex ： MEI2 protein that progress meosis of fission yeastlocalize in nucleus with meiRNA. The protein localizes in cytosol without the meiRNA.Ex ： 7SK small nuclear RNA suppresses PolII transcription

Mechanism of RNA interference by siRNA

2. Formation of RLC(RISC loading complex)

3. Formation of RISC

1. Dicer binds to dsRNA

DicerTRBP

dsRNA

AGO2Dicer

3’-pp-3’

4. Activated RISC (RISC*)

mRNA

cleavage

Nucleus5. Activated RISC bind to mRNA

6. Regeneration of RISC*

cleavage

siRNAp-5’

3’-p

p-3’

5’-p

19 nt duplex2nt

Sense

Antisense

Dicer

TRBP

genome

m-RNA

Protein

Non-coding RNA

What is post-genomic research?

Genomic researchWhole sequence of human genome was determined ！Individual difference （ SNP) of genes are elucidated.

We can access how effective of drugs or how strong of the adverse effect to individuals. （ Tailor-made medicine ）

What is the cause of particular disease?How we can treat the medicine?

How can we discover the effective drug?

Transcription

Gene （ DNA)

Copy（ massenger RNA)

Protein

Translation

Expression

Progress of Researchgenome

Genomic sequence ・ Polymorphism（ SNP etc ）

Transcriptome

Gene transcription profile

Proteome Expression profile of proteins

Functional proteomeMetabolome

Genetic functionPost translational modificationProtein interation etc.

Time consuming and enormous costElucidation of functional network of cellular

molecules　　　　　

Proteome

The situation of whole proteins that are expressed in particular condition

In living cell.

To know the function of gene or life phenomena,we have to know how many proteins are expressed

and what is the way of relationship of such proteins on given conditions.

Difficulties with comprehensive analysis of proteinsDiversity of proteins characteristics 　　　　Difficulty with development of universal techniques ⇒ case by case handlingOccurrence of post-translational modificationThey often form complex with other proteins and moleculesExpression profile is various depending on tissues or temporallyDynamic range of their expressions are very wide (1,000,000 folds difference)

Proteomics techinologies

Identification

２ D electrphresis + MS

SDS PAGE

Isoelectric focusing electrophoresis

Restricted hydrolysis

MALDI-TOF MS

Sypro-Ruby stainingProtein quantification

Autoradiography imaging（ 35S-methionine pulse-

labeling ）Quantification of newly synthesized proteins

＋

Proteins with already stopping synthesis

Newly synthesized proteins with the stimulation

Continuously synthesizing proteins

Dual-Channel Imaging(Detection & quantification of protein

synthesis dependent on particular stimulations)

 

Various techniques for proteome analysis

For protein indentification and differential display

For investigating protein-protein (ligand) interaction

Peptide sequence using charged tag (SMA or SPA reagent)Isotope labelICAT assayICAT assay with 15N-enrich medium2-dimensional PAGECapillary LCIdentification of phosphorylation siteProtein array

Base on two-hybrid system Yeast two hybrid system (Y2H) Large scale Y2H Y2H in mammalian cell Three hybrid system One hybrid system

Based on protein complementation assay Using Dihydroforate reductase(DHFR) Split Ubiquitin Using protein splicing Using b-galactosidase Using rasGEF+V-src myristoylation signal Using adenylyl cyclase

Other Using isotope-labeled crosslinker Protein array

Assay of protein-protein interaction

Yeast Two Hybrid (Y2H) system

GAL4 DNABinding domain

baitPray GAL4 transcription

activating domain

DNA

Reporter gene expression

Highly sensitive, but easy to get false-positiveNot available to proteins difficult to express in yeastOnly available for 1:1 interactionCostly & time consuming

Apply to HSP format

Reporter gene expression

Bait Prey

Proteins of interest: GAL4 yeast transcription factor

Transcription activating domain

DNA binding domain

GAL4 promoter Reporter gene

Conceptual scheme of Y2H

BaitPray

Binding site Reporter geneDBD

Bait Pray AD

a) b)

GAL4BE TATAGAL4DBD

Bait Pray

AD

OriP

Pray AD

c)

Cellular mambrane

Membrane localization factor

Ras or hSos

d)

cAMP/CAP dependentpromoter

T25 　 T18

ATP 　　ｃ AMPCAP

Adenylate cyclasee)

Schematic outlines of Two hybrid systemsa) Yeast Two Hybrid System, b) effect of homodimer-forming in bait or prey in Y2H, c) two hybrid system in mammalian cell, d) protein recruitment system using Ras(RRS) or hSos(SRS), e) two hybrid system in bacterial cell

＋

Protein A Protein B

Ｓｐｌｉｔ　Ｌｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ

Fragment of a protein(DHFR or β-Gal)

a)

Reconstitution of the protein

FKBP FRB

rapamycin b)

Reconstitution of ubiquitin

X Y

Cleavage with UBP

Reconstitution of intein

c)

Luciferase or EGFP

splicing

X Y

Schematic outline of protein-fragment complementation assaya ： Original protein-fragment complementation assayb ： Split ubiquitin assayc ： Split enzyme reconstitution based on protein splicing

Gene A Gene AGene B

Application of Intein-Extein system for reconstitution assay

Protein BProtein A

Intein

extein

Protein A

Luciferase’ Luciferase”

Gene X Gene Y

Protein X Protein Y

Native Luciferase

Binding site Reporter gene

DBD

AD

a)

DBD

A B AD

Receptor forligand A

Receptor for ligand B

Binding site Reporter gene

b)

DBD

A B AD

RNA

Binding site Reporter gene

c)

Schematic outlines of one- and three-hybrid assaya ： One hybrid assay for detecting DNA-protein

interactionb ： Three hybrid assay for detecting protein-ligand

interactionc ： Three hybrid assay for detecting protein-RNA

interaction

DNA ・ RNA-linkage

DNA

Biotin

StreptavidinPhage Coat protein

Target proteinDNA

Phage display STABLE assay

RNA

ribosome

Ribosome display

In vitro virus

RNA

Puromycin

Phage library

Target-coating plate

Washing out of unbound phages

Collect of bound phagesProliferation

Panning in Phage display

Reverse micelle

gene biotin

Encapsulated gene encodes fusion protein between target and streptavidin

DNA

biotin

expression binding

STABLE assay

Ribosome display

In vitro virus

Expression vector without terminal codon is used for each target protein expression. Ribosome can’t detach from mRNA so that RNA-Protein fusion is obtained.

Puromycin binds to ribosome pocket when the transcription completes.Then pyromycin connects between mRNA and translated protein in covalent bonding.

RNA

ribosome

RNA

Puromycin