Progress of Research
genome
Genomic sequence・Polymorphism（SNP etc）
Transcriptome
Proteome
Gene transcription profile
Expression profile of proteins
Functional proteome
Metabolome
Genetic function
Post translational modification
Protein interation etc.
Time consuming and enormous cost
Elucidation of functional network of cellular molecules
DNA Chip
Disease rat
Normal rat
mRNA extraction
Labeled with
Cy5 and Cy3
Hybridization
Genes in which transcription levels are affected
with the disease can be determined.
Importance of RNA
Species and genomic size・number of genes
Genomic size（Mbp)
Human
C-elegance
２３５１
The number of genes
２２２８７
１０３
１９８９３
Drosophila
１８０
１３６７６
Arabidopsis thaliana
１２５
２５４９８
Cellular slime
３４
Yeast
１３
１２５００
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
p-3’ Sense
p-5’
siRNA
3’-p
5’-p
2nt
Antisense
19 nt duplex
dsRNA
TRBP
6. Regeneration of RISC*
Dicer
cleavage
1. Dicer binds to dsRNA
mRNA
Nucleus
TRBP
AGO2
Dicer
3’-p
5. Activated RISC bind to mRNA
p-3’
Dicer
2. Formation of RLC
(RISC loading complex)
4. Activated RISC (RISC*)
3. Formation of RISC
cleavage
genome
m-RNA
Protein
Non-coding
RNA
What is post-genomic research?
Genomic research
Whole 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?
Gene（DNA)
Expression
Transcription
Copy
（massenger RNA)
Translation
Protein
Progress of Research
genome
Genomic sequence・Polymorphism（SNP etc）
Transcriptome
Proteome
Gene transcription profile
Expression profile of proteins
Functional proteome
Metabolome
Genetic function
Post translational modification
Protein interation etc.
Time consuming and enormous cost
Elucidation 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.
Proteomics techinologies
Difficulties with comprehensive analysis of proteins
Diversity of proteins characteristics
Difficulty with development of universal techniques ⇒ case by case handling
Occurrence of post-translational modification
They often form complex with other proteins and molecules
Expression profile is various depending on tissues or temporally
Dynamic range of their expressions are very wide (1,000,000 folds difference)
Identification
２D electrphresis + MS
SDS PAGE
Isoelectric focusing electrophoresis
Restricted
hydrolysis
MALDI-TOF MS
Dual-Channel Imaging
(Detection & quantification of protein synthesis
dependent on particular stimulations)
＋
Sypro-Ruby staining
Protein 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
Various techniques for proteome analysis
For protein indentification
and differential display
Peptide sequence using charged tag
(SMA or SPA reagent)
Isotope label
ICAT assay
ICAT assay with 15N-enrich medium
2-dimensional PAGE
Capillary LC
Identification of phosphorylation site
Protein array
For investigating protein-protein
(ligand) interaction
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
Pray
bait
GAL4 DNA
Binding domain
GAL4 transcription
activating domain
Reporter gene expression
DNA
Highly sensitive, but easy to get false-positive
Not available to proteins difficult to express in yeast
Only available for 1:1 interaction
Costly & time consuming
Apply to HSP format
Conceptual scheme of Y2H
Transcription
activating domain
: GAL4
yeast transcription factor
Proteins of interest
DNA binding domain
Bait
Prey
Reporter gene expression
GAL4 promoter
Reporter gene
a)
b)
Bait
Pray
AD
c)
Bait
Bait
DBD
Binding site
d)
GAL4DBD
Reporter gene
Membrane localization
factor
Cellular mambrane
OriP
Pray
Pray
Pray AD
AD
GAL4BE TATA
e)
T25 T18
ATP
Adenylate cyclase
CAP
ｃAMP
Ras or hSos
cAMP/CAP dependent
promoter
Schematic outlines of Two hybrid systems
a) 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
Ｓｐｌｉｔ Ｌｕｃｉｆｅｒａｓｅ Ａｓｓａｙ
＋
Protein A
Protein B
b)
rapamycin
a)
FKBP
Y
X
FRB
Reconstitution of
ubiquitin
Cleavage
with UBP
Fragment of a proteinReconstitution of
the protein
(DHFR or β-Gal)
c)
X
Y
Luciferase or EGFP
splicing
Reconstitution of intein
Schematic outline of protein-fragment complementation assay
a：Original protein-fragment complementation assay
b：Split ubiquitin assay
c：Split enzyme reconstitution based on protein splicing
Application of Intein-Extein system for reconstitution assay
Gene A
Gene B
Gene A
Intein
extein
Protein B
Protein A
Protein A
Gene X
Gene Y
Luciferase’
Luciferase”
Protein X
Protein Y
Native
Luciferase
a)
b)
AD
Receptor for
ligand A
Receptor for ligand B
A
DBD
Binding site
DBD
Binding site
Reporter gene
c)
B
Reporter gene
RNA
A
B
AD
DBD
Binding site
AD
Reporter gene
Schematic outlines of one- and three-hybrid assay
a：One hybrid assay for detecting DNA-protein interaction
b：Three hybrid assay for detecting protein-ligand interaction
c：Three hybrid assay for detecting protein-RNA interaction
DNA・RNA-linkage
Phage display
STABLE assay
DNA
Target protein
Biotin
DNA
Phage
Streptavidin
Coat protein
Ribosome display
In vitro virus
RNA
RNA
ribosome
Puromycin
Panning in Phage display
Washing out of unbound
phages
Phage library
Target-coating plate
Proliferation
Collect of bound
phages
STABLE assay
expression
gene
biotin
Reverse micelle
Encapsulated gene encodes fusion protein
between target and streptavidin
binding
DNA
biotin
Ribosome display
In vitro virus
RNA
RNA
ribosome
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
Puromycin binds to ribosome pocket
when the transcription completes.
Then pyromycin connects between
mRNA and translated protein in covalent
bonding.