Upload
feng
View
37
Download
0
Embed Size (px)
DESCRIPTION
Progress of Research. genome. Genomic sequence ・ Polymorphism ( SNP etc ). Transcriptome. Gene transcription profile. Proteome. Expression profile of proteins. Functional proteome Metabolome. Genetic function Post translational modification Protein interation etc. - PowerPoint PPT Presentation
Citation preview
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
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?
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
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
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
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