Progress of Research

1. 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. Time consuming and enormous cost Elucidation of functional network of cellular molecules

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

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 RNAsuppresses PolIItranscription

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

7. genome m-RNA Non-coding RNA Protein

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

10. 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. 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. Proteomics techinologies Difficulties with comprehensive analysis of proteins Diversity of proteins characteristics Difficulty with development of universal techniques ⇒ case by casehandling 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 Restricted hydrolysis MALDI-TOF MS Isoelectric focusing electrophoresis

13. Dual-Channel Imaging (Detection & quantification of protein synthesis dependent on particular stimulations) ＋ Autoradiography imaging （35S-methionine pulse-labeling） Quantification of newly synthesized proteins Sypro-Rubystaining Protein quantification Proteins with already stopping synthesis Newly synthesized proteins with the stimulation Continuously synthesizing proteins

14. 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 15N-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 b-galactosidase Using rasGEF+V-src myristoylation signal Using adenylyl cyclase Other Using isotope-labeled crosslinker Protein array Various techniques for proteome analysis

15. Assay of protein-protein interaction

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

17. Conceptual scheme of Y2H Transcription activating domain : GAL4 yeast transcription factor Proteins of interest DNA binding domain Prey Bait Reporter gene expression GAL4promoter Reporter gene

18. a) b) c) OriP Pray AD Bait Pray Bait Pray Bait Pray AD DBD AD GAL4DBD Binding site Reporter gene GAL4BE TATA e) d) Membrane localization factor Adenylate cyclase T25T18 Cellular mambrane CAP ATP　　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

19. Ｓｐｌｉｔ　Ｌｕｃｉｆｅｒａｓｅ　ＡｓｓａｙＳｐｌｉｔ　Ｌｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ ＋ Protein B Protein A

20. a) rapamycin FRB FKBP Reconstitution of the protein b) c) Y X Luciferase or EGFP Y X splicing Reconstitution of ubiquitin Cleavage with UBP Fragment of a protein (DHFR or β-Gal) Reconstitution of intein • Schematic outline of protein-fragment complementation assay • ：Original protein-fragment complementation assay • ：Split ubiquitin assay • ：Split enzyme reconstitution based on protein splicing

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

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

23. a) b) Receptor for ligand A Receptor for ligand B AD B AD A DBD DBD Binding site Reporter gene Binding site Reporter gene c) RNA B AD A DBD Binding site Reporter gene • Schematic outlines of one- and three-hybrid assay • ：One hybrid assay for detecting DNA-protein interaction • ：Three hybrid assay for detecting protein-ligand interaction • ：Three hybrid assay for detecting protein-RNA interaction

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

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

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

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.