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Progress of Research genome Genomic sequence ・ Polymorphism ( SNP etc ) Transcriptome Gene transcription profile Proteome Expression profile of proteins.

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Presentation on theme: "Progress of Research genome Genomic sequence ・ Polymorphism ( SNP etc ) Transcriptome Gene transcription profile Proteome Expression profile of proteins."— Presentation transcript:

1 Progress of Research genome Genomic sequence ・ Polymorphism ( SNP etc ) Transcriptome Gene transcription profile Proteome Expression profile of proteins Functional proteome Metabolome Functional proteome Metabolome Genetic function Post translational modification Protein interation etc. Time consuming and enormous cost Elucidation of functional network of cellular molecules

2 DNA Chip mRNA extraction Labeled with Cy5 and Cy3 Hybridization Genes in which transcription levels are affected with the disease can be determined. Normal rat Disease rat

3 Importance of RNA Species and genomic size ・ number of genes Genomic size ( Mbp) The number of genes Human 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

4 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)

5 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

6 Mechanism of RNA interference by siRNA 2. Formation of RLC (RISC loading complex) 3. Formation of RISC 1. Dicer binds to dsRNA Dicer TRBP dsRNA AGO2 Dicer 3’-p p-3’ 4. Activated RISC (RISC*) mRNA cleavage Nucleus 5. Activated RISC bind to mRNA 6. Regeneration of RISC* cleavage siRNA p-5’ 3’-p p-3’ 5’-p 19 nt duplex2nt Sense Antisense Dicer TRBP

7 genome m-RNA Protein Non-coding RNA

8 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?

9 Transcription Gene ( DNA) Copy ( massenger RNA) Protein Translation Expression

10 Progress of Research genome Genomic sequence ・ Polymorphism ( SNP etc ) Transcriptome Gene transcription profile Proteome Expression profile of proteins Functional proteome Metabolome Functional proteome Metabolome Genetic function Post translational modification Protein interation etc. Time consuming and enormous cost Elucidation of functional network of cellular molecules

11 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.

12 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) Proteomics techinologies Identification 2 D electrphresis + MS SDS PAGE Isoelectric focusing electrophoresis Restricted hydrolysis MALDI-TOF MS

13 Sypro-Ruby staining Protein quantification Autoradiography imaging ( 35 S-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) Dual-Channel Imaging (Detection & quantification of protein synthesis dependent on particular stimulations)

14 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 label ICAT assay ICAT assay with 15 N-enrich medium 2-dimensional PAGE Capillary LC Identification of phosphorylation site Protein 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  -galactosidase Using rasGEF+V-src myristoylation signal Using adenylyl cyclase Other Using isotope-labeled crosslinker Protein array

15 Assay of protein-protein interaction

16 Yeast Two Hybrid (Y2H) system GAL4 DNA Binding domain bait Pray GAL4 transcription activating domain DNA Reporter gene expression 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

17 Reporter gene expression BaitPrey Proteins of interest : GAL4 yeast transcription factor Transcription activating domain DNA binding domain GAL4 promoter Reporter gene Conceptual scheme of Y2H

18 Bait Pray Binding siteReporter gene DBD Bait Pray AD a)b) GAL4BETATA GAL4DBD Bait Pray AD OriP Pray AD c) Cellular mambrane Membrane localization factor Ras or hSos d) cAMP/CAP dependent promoter T25 T18 ATP c AMP CAP Adenylate cyclase e) 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

19 + Protein A Protein B Split Luciferase Assay

20 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 assay a : Original protein-fragment complementation assay b : Split ubiquitin assay c : Split enzyme reconstitution based on protein splicing

21 Gene A Gene B Application of Intein-Extein system for reconstitution assay Protein B Protein A Intein extein Protein A

22 Luciferase’Luciferase” Gene X Gene Y Protein X Protein Y Native Luciferase

23 Binding siteReporter gene DBD AD a) DBD A BAD Receptor for ligand A Receptor for ligand B Binding siteReporter gene b) DBD A B AD RNA Binding siteReporter gene c) 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

24 DNA ・ RNA-linkage DNA Biotin Streptavidin Phage Coat protein Target protein DNA Phage display STABLE assay RNA ribosome Ribosome display In vitro virus RNA Puromycin

25 Phage library Target-coating plate Washing out of unbound phages Collect of bound phages Proliferation Panning in Phage display

26 Reverse micelle gene biotin Encapsulated gene encodes fusion protein between target and streptavidin DNA biotin expression binding STABLE assay

27 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.


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