(gene)RNA DNA--(exon)--(intron)5'-3'-(UTR)()(split-gene)(genome)()DNA3X1 09(30bp)10DNA C(C-value Paradox)human genome project, HGPgenomics,structural genomics functional genomicsproteome proteomicsDNA(2--3)DNA•()(>lO5)DNA(Satellite DNA)()1DNA()2(long interspersed repeated segments) LINES(Short interspersed repeated segments) SINESSINES<500bp>105AluLINEs>1000bp(7Kb),104-105LINEl()(Unique Sequence)(gene family)(ancestral gene)(duplication)(gene cluster)(tandemly repeatedgenes)rRNA tRNA(Pseudogene) a1a1(processed pseudogene)1tRNA1300tRNA tRNA2rRNA>l00copy rRNA(28S18S 5.8s-rRNA)330-40copy7q32-q36(H1H2A H2B H3H4)intron Poly(A)- RNA416p13(24Kb)5'------1--2--1--3'11p15(60Kb)5'----Gr--Ar--------3'(Supergene family Superfamily)1A1u50-1003-6Kb AluA1u300bp2X130bp+31bp()7-21bp(direct repeats)?Alu90%AluAlu2• •Variable number tamdem repeat VNTRDNA(minisatellite DNA)(6-40bp)(6-100)VNTR----DNA fingerprint.DNA H-Ras3short tandem repeat,STRDNA microstallite DNA2-6(10-60),l0kbDNA STR{CA)n50000-100000116569bp(H)(L )mtDNA1324RNA(22rRNA 2tRNA)2DNA3mtDNA4mtDNA5DNA DNA1--23DNA1DNA RNA23X1033X106bp34(RNA DNA)RNA56--(chromatin)--(chromosome)--()(nucleosome)DNA(H2A H2B H3H4) 1.8(146bp),Linker DNA(0-80bp)+Linker=(180-210bp)DNA Ladder()(histone)<Lys Arg)DNA1H2A H2B H3H4(102-135aa)DNA2H1(220aa)Linker DNA•130nm2(looped domain)3=Chromatid)46(Karyotype)()centromere)()(telomere)DNADNA1G(kb)DNA TTAGGC212-163'DNADNADNA(DNA Replication)DNA1. (Semi-Conservative)Meselson-Stahl2. ()(origin)+=(, Replicon)3.(Semi-discontinuous)(leading strand)-(Lagging Strand)-,(okazaki fragment).DNA()1.DNA.2RNA(RNA Primer)8-14nt.3'-OH.DNA(DNA Helicase),DNA.(Proliferating cell nuclear antigen.PCNA).(Telomerase)(),RNA.RNA.():.PCR"TRAP"Telomerase repeat amplificationprotocol Kim,N et al.,science,266,2011-2014(1994)5%.DNA DNA repairDNADNA1DNA C U A IReactiveoxygen species ROS2X(Base excision repair BER)(glycosylase)()DNA(AP)DNADNABER(1)DNA(UDG)DNA(2)3--(3MeA)DNA(3)DNA Fpg MutM DNABER DNA()(Nucleotide excision repair NER)DNADNA DNA20(CPD)(6--4)((6--4)PPs)NERNER Xeroderm pigmentosum,XPNER(1)(Global Genomicrepair GGR)DNA(2)(Trancsription--coupledrepair TCR)()DNARNA II TFIIH(Mismatch repair)DNASTR-(loop)-expansion microsatellite instabilitycoli MMR mutS,mutH,mutL uvrD DNA DNA mutS,mutHHNPCC(Recomhinant repair)DNAFurther readings:1 Wood RD DNA repair in eukaryotes Ann RevBiochem199665135--1672 Krokan HE,et a1., DNA glycosylase in the base excisionrepair of DNA Biochem J19973251--163 Vrieling H et a1., Transcription coupled repair and its impacton mutagenesis Mutation Res1998400135--142(recombination).2DNA DNA, ,DNA..(Homologous recombination)DNA,DNA..(transposition)DNA(mobile DNA elements)-(Transposable element).DNADNA DNA.-:1.,DNA.2.(transposase).(M2+)3.3-12bp(),DNA..(transposon):DNA.(retroposon),RNA(DNA RNA DNA):Alu.LINE1.(retro-pseudogene)-DNA mRNA""-(heterochromatin)()(euchromatin)10%()()DNaseI()DNase IDNase I(DNase I HyperSensitive Sites DHSS)()H3CyS110)(Phased positioning)1(rotational positioning)DNA DNA`2(translational positioning)DNA DNA()1H12Lys)HMGHMG high mobility group)--, HMG14/HMG17.DNA()DNA(Methylation)5%C(m C)mC CpGCpG CpG(CpG islands)10Kb CpGCpG()()DNA1(X23()(housekeeping gene)(Constitutivegene)(genomic imprinting)(imprinted)--DNA()(+1---)RNA Pol(GTFs)()(promoter)DNA RNApoII100-200bp1(core promoter)TF DTATA(TATA box) -25-30bp(initiator Inr) InrTATA Inr2(Upstream Promoter element UPE)(-30-110bp)CAAT GC GC SPl()RNA(mRNA)7-10(CTD)7(Tyr-Ser-Pro-Thr-Ser-Pro-Set)CTD()(basal transcription factor)RNA--RNA(generaltranscription factor)1RNA II(TF II)TFIID,TF B,TFIIF TFIIE TFIIH TFIIATFIID TATA(TATA binding protein TBP)8TBP(TBP associated factor TAF)TFIID(TATA Inr)TF B C TFIID DNA N-TF F RNA II TFIIE,TFIIHTFIIH RNA Pol CTDTFIIA TFIID TATA2TAFTFIID8TBP,30--250kD,TAF-30250 TAF150(1nr)TAF(cis-regulatingelement)DNA DNAconsensus sequence)()(promoter)()(enhancer)8-12bp)1()235'-3'45'-3'-5(30Kb)6()--(silencer)()1(responsive elements)--()2DNA(trans-acting factor)()()()(domain)(motif)1DNA(DNA binding domain)//Helix-turn-helix,HTHDNA(Homeodomain)HTH Homeodomain (Zinc finger)(His2Cys2)2(cys2cys2)DNAGC SPl WT1 (Leucine-Zipper)6 a aLeu Leu----LeuC-Jun/C-fos(APl)--(Helix-loop-Helix HLHt)2(loop)C-myc MaxHLH Leu---aa DNAbzip bHLHb=basic)2(Transactivation domain) `DNA30-100aa(acidic a-Helix domain)Jun(glutamine--rich domain)SPl(proline-rich domain)()1POUDNA-POIJPOUN POU(POUs)+C POU(POUHPOU Pit1octl oct2unc86POU2(GR(ER AR)(TR) (RAR)VitD3(VD3R)DNA2Cys2Cys2(2)DNA(HRE Hormoneresponsive elements)3NF-BRelA(P65)P60RelB C-Rel P50P50RelAP50RelA•I B()I B(mRNA)(openreading frame ORF)mRNA-mRNA--RNA(heterogeneous nuclear RNA hnRNA)hnRNA(hnRNP), mRNA hnRNP()5'-(capping)--mRNA5'-(m7pppN)5'm7G1mRNA23mRNA)3'-(poly(A))--mRNA3'200--250(A)poly(A)1mRNA2mRNA3)mRNA(sp!icing)--1Gu AG5'-()3'-()branchsite)mRNA2mRNA-lariat intermediate)23(spliceosome)RNA(300)--SnRNA small nuclear RNA)U1--U6 snRNA-SnRNP mRNA snRNP--mRNAU2U6U4U6 SnRNP()RNA(RNA editing)mRNA mRNADNARNA apo--B C U(alternativesplicing)mRNAmRNApoly(A)mRNA mRNA(isoform)>5%(IF)1eIF-4F2eIF-2mRNA)5'-UTR1mRNA5'm7G2"AUG"(AUG AUG)3AUG Kozak GCCAUGG()3'-UTR1poly(A)2UAmRNAmRNA3'--UTR1poly(A)mRNA23'-UTR UA mRNA1N-O-2Proprotein protein.1N-202 N-3SV40 T pro-lys-lys-lys-Arg-lys(127-132)Viral oncogene V-onc).(cellular oncogene c-onc)(protoncogene)--v-onc(tumor suppressor gene)1(PCD)23(germ 1ine)l22•3()C--ras121361C-ras(21kD,P21)RAS GTP GTP()(translocation)1t(922)c-abl bcr P2102t(814)c-myc()(gene amplification)HL-60C-myc8-22c-erb B c-net()LTRLTR(long terminal repeat)1sis(PDGF-)fgf(int-2csf-1)2()neu ht met erbB trk fms ros-13()src src syn fyn abl lck ros yes fes ret4()raf raf-1mos pim-15G()GTP GTP ras H-ras K-ras N-ras mel ral6DNA()myc fos Jun ets rel erb A()()(Lossof heterozygosity LOH)LOS DNA(RFLP VNTR STR SSCP) LOHp53P5317P1311393p535-8, 2493G T,B1P53P53DNA N C1P5310bp N-P53DNA2P53DNA3P53C-4P53cki p21, G1.5p53 GADD456P537p53DNA P538Mdm2p53Mdm2)(signal transduction)12()3(intracellular messenger)(secondmessengers)cAMP{)ATP cAMP;cAMP cAMP5'-AMPAcGMP()cGMPGinositol triphosphate,IP3diacyglycerol, DAGG-C IP3DAG1IP3Ca2+2DAD Ca2+C(SR)[Ca2+]>>[Ca2+]()()()()IP3()Ca2+(calmodulin, CaM)Ca2+•CaMCa2+•CaM(NO)NO L-NONO(NOS)(nNOS)(ecNOS)iNOS)cGMPNO(Proteinkinase PK)()ATP (Protein phosphatase PPase){)(Ser Thr PK)1A PKA)-cAMP.PKA C RPKA cAMPPKA()2C(PKC)-Ca2+Ca2+DAG TPA()PKC N-C--PKC(>12)PKCEGFRMap TauNa+-H+Ca2+-ATPRaf-13Ca2+•(Cam-PK)Cam-PKII4cGMP(PKG)5DNA(DNA-PK)DNADNA SPl Fos Jun Myc P53DNA,TF;(Checkpoint).6(Mitogen-activated protein kinase MAPK)MAPK-MAPKK MEKMyc Jun Ets()Tyrosine protein kinase TPK)--1srcc-src Src C-N--SH1SH2SH3(SH=srchomolog)SHlSH2SHSrc src yes lyn fyn lck blk fgr bcd yrk TPKSH2SH22JAKJAK(Janus kinase)Jakl Jak2Jak3Tyk2Jak TPK Src TPkJaK SH2SH3Jak()PPl PP2A PP2B PP2CPP2A()(PTPase)PTPase21()SH2 domain PTPlC PTPlB2(PTPR)2CD45PTPlC()N2SH2{Tyr•P)CJak PTP1CPTPaseG(IGF EGF PDGF NGF SCF HGF )()TPKJak(cytokine)(IFN)(IL)LIF M (IL-8R G)()(ACH)5-HT GGG(effector)GG-(G-protein)GG Gs,Go,Gi,Gq CT PT G-G-G-pr C A2RAS-MAPK1Ras G-)RasSOSRas•GDP Ras•GTP() GTP GAPGrb2SH2TyrSH3pro SOS SOS Ras2Ras-MAPK()SH2(Grb2) SOS Ras-GTP Rafl MAPKK MEK MAPK3Ras-MAPKRas-MAPK(Ras)cAMP-PKA Raf-1PKC Raf~lJak-statSTAT signal transducer and activators oftranscription684-113KD SH2()SH3DNA StatJak-Stat()Jak Stat Stat()•(cell proliferation)(cell cycle)G1S G2M(G1S G2)G1(Gap1)DNA()DNA;S DNA;G2DNA;M(Mitosis)MS+G2+M G1G11)S()2)Co()3)S DNA M21G1DNA G1RNA(restrictionpoint R)G1DNADNA(checkpoint)DNA2S DNA()S2 •S G2DNA3G2RNA4M(cyclin-dependent kinases CDKs)(cyclin)-(CDK)cyc-CDK cyc-CDK1G1cyc-CDKG1C D D1D2D3)ED CDK4(CDK2CDK5CDK6)R(G0G1E CDK2cycE-CDK2G1S2S M cyc-CDKscycA-CDK2ScycB-cdc2G2M(cdc2p34cdc2CDKl)(cyclin-dependent kinase inhibitor CKIs)CKls CDKsCKIsCKls1Kip Cip P21P27P57cyc-CDKP21(WAFI CIPl)P53P21cycDl-CDK4cycE-CDK2 (DNA)P53P27TGF-2INK4P16P15P18P19INK4cycD-CDK4cycD-CDK6CDKP15TGF-G1'1cyclin CDKs2CKI CDKp16(MTSl)(myc)pRB •106KDG0G1pRB E2F-1()GF cycD-CDK4cycE-CDK2pRb()E2F DNAp53()-DNA(P21 G1)1GF GFR Ras-MAPK fos/jun cycD-CDK4pRB E2F2TGF-G1(Ubiquitin Ub)(P27)apoptosis, programmed cell death,PCDapoptosis vs. necrosis1homeostasis,2341shrinkage2blebbing34DNA DNA ladder56()Fas TNFBcl-2B bcl-2ced-91814bcl-22Bcl-2Bcl-Xl, Bcl-w, Mcl-1, and A13Bcl-2Bcl-xlBax-Bcl-21Bcl-2Bax Bak Bik Bid Bim HRK2Bcl-23Bcl-2Bax4Bax P53DNA P535Caspase -1cysteine aspartic acid2Caspase procaspasecaspase prodomain3caspase IL-1(ICE=caspase-1) pro-IL-1ICE CrmA CrmA caspase412caspasecaspase(e.g. DEV D)a. DFF -DNA fragmentation factor- cleavage of an inhibitorysubunit activates this nuclease which cuts the DNA into a distinctladder-likepattern and for chromatin to condense.b. PARP -Poly-ADP ribose polymerase, a DNA repair enzymeinactivated by caspasesc. nuclear lamin - causes nucleus to lose its structural integrityafter cleavaged. cytoskeletal proteins - cleavage causes membrane blebbing5T-granzymes granzymescaspase6caspasesCaspases1TNF TRADD FADD Fas FADD2FADD caspase-8 (FLICE),3caspase-8.4.Fas FasL Fas Fas,5FasL FasLFasApaf1- caspase1Caspase-8Bid C-2Bid Bcl2C.3Bcl2cytochrome c4Cytochrome c Apaf-1Apaf-1/cytochrome c(Apoptosome)procaspase-95procaspase-96Caspase-9caspase-3caspases effector phase ,IAPs, caspaseA. IAPs are inhibitors of apoptosis protein. There are 7identified IAPs in human.B. IAPs inhibit apoptosis by inhibiting caspase activities.C. IAPs provide a safeguard mechanism against minimal activationof the apoptosis program.D. Several IAPs are overexpressed in tumors.A. CancerB. Autoimmune diseaseC. Neurodegenerative disease1. ALS - Amyotrophoic lateral sclerosis or Lou Gehrig's disease2. Spinal muscular atrophygenecloning,DNADNAclone DNA DNA(cloning)DNADNADNADNA DNADNA1DNA2DNA5'-3'-35'-3'-DNA4MboI Sau3A ""Sambrook,J et al . molecular cloning(vector)DNA DNA,DNADNA DNADNADNADNADNADNA(plasmid)DNAkbDNADNA<15kbDNA50kb+DNA12COS20kb,12-24kb,10kb,DNA DNA5-20kb DNA5-7kb DNA,cDNA gtDNA13136407bp DNADNA DNA DNA100-200DNA,M13507DNA M13M13M13Mp18M13<1000bp,M13DNADNA DNADNA COS(20-bp)4-6kb,40-50kb DNADNA(cen4)(ARS1)(Tel)DNA200-1000kb DNADNADNA DNAcDNADNADNA()DNADNA20-24kbDNAcDNAcDNA mRNA DNA.mRNA cDNA cDNA cDNAcDNAmRNAcDNAcDNAcDNA;cDNADNADNADNA cDNADNADNA DNA DNA DNADNADNA DNA5'-3'-DNA DNA T4 DNA ATPT4 DNADNADNADNA DNA DNA 5'-DNAT4DNA DNA3'-5'-5'-5'-DNA klenow3'-3'- T4 DNA3'5'T4DNADNAT4DNA DNADNA DNADNADNADNADNA DNADNADNA DNADNA DNAampr tetrrDNAlacZ N-146lacZ15C-X-galX-galDNA,DNADNA PCRDNADNA5x105-5x10612PCR3Nucleic acidhybridizationDNADNADNA Denaturatioin DNATm DNA DNA DNADNA Tm GC GC TmDNA Renaturation DNA TmTmTm15Tm0.4M Tm1%DNA/DNADNA/RNA Tm0.7250%1DNA cDNA2RNA RiboprobeRNA()Northen34mRNAP14.335S87.133P(25.4)1Random primingDNA klenow2(Nick translation0DNase DNA3RNARNA RNANorthern4DNATaq DNA dNTP,sk c DNA 55'-T4DNA5'-3'-32P-dNTP ddNTP65'-123blot456Dot/slotblot DNA RNASouthern DNASouthern1DNA;2DNAa3DNADNASouthern DNANorthern RNAmRNANothern1RNA2RNA3RNA45RNA DNA SouthernRNA1RFLPpolymorphism DNA1%RFLP DNAsouthernblotting PCRRFLPVNTR RFLP DNA fingerprinting2ASO3Northern blottingPCRPCRPCRPCR DNA1Denaturation DNA2Annealing3Extension DNAPCR5'DNAPCR1Taq DNADNA5'-3'DNA5'-3 3'-5'PCRPCR Taq 0.5-2.5U2PCRPCR15-30G/C A/T G/C45%-55%3'3'-3'G C T5'-ATGPCR0.1-0.5uM3MgCl2 Mg2+Taq DNA Tm PCRPCR MgCl2 1.5-2.5mM(Mg2+Mg2+ dNTP EDTA4dNTP dNTP 4 dNTP200umol/L,dNTP Mg2+5DNA RNA cDNAPCR Taq/PCR()PCR1Initial denaturationDNA PCR953-52Primer annealingPCR372Taq49530DNA5PCR25-356725-15PCRPCR12PCR carry-overPCRPCR Reverse transcription PCR,RT-PCRRNA PCR.PCR RT PCRPCRRT PCR cDNA gDNAPCR Multiple PCRPCRPCR DNA() PCR nested PCRPCR PCR,()PCR asymmetric PCRPCR50100:1PCR,DNAPCRPCRPCR1Restriction fragment length polymorphism-RFLP;2(Sequence polymorphism);3(Length polymorphism)PCR-RFLPPCRPCRPCR ASOASO Allele specificoligonucleotide1PCR AS0ASO ASO2ASO PCR-reversedot blotrasHLA typingA11eles specificamplification ASA-(Amplification refractory mutationsystem ARMS)PCR3'DNAPCR3'PCRPCR-SSCPDNADNA DNA --Singlestrand conformation polymorphism SSCPPCR SSCP PCR PAGEPCR SSCP200bp175-345ntAmp-FLPVNTR STRPCRPAGEPCRDNAPCR PCR DNAPCR cyclesequencing PCR ddNTPPCRPCRPCR PCR.PCR-RFLP Amp-FLPPCR1HBV HCV HIV HPV21ras,p5323CML Minimal residual disease MDR4multi-drug resistance,MDR5Gene chip/DNA chip DNA DNAMicroarray DNA1Genechip,Affymatrix,Inc2DNA DNAMicroarray DNADNADNA1mRNA,cDNA2microarray3element mRNA123/( single nucleotide polymorphism, SNP)45mRNA cDNANorthern Blot12345Further Readings1. Ekins R. and Chu F.W. Microarrays: their origins andapplications. Trends in Biotechnolology, 1999, 17, 217-218.2. Sinclair, B. Everything's Great When It Sits on a Chip - Abright future for DNA arrays, The Scientist, 1999 May 24, 13(11),18-20.3. Nature Genetics published a special issue (January 1999Supplement), The Chipping Forecast. It's a collection of morethan 10 reviews (60 pages) on different aspects of microarrayanalysis. All the reviews are freely available online.4. Schena, M., Heller, R.A., Theriault, T.P., Konrad, K.,Lachenmeier, E., and Davis, R.W. Microarrays: biotechnology'sdiscovery platform for functional genomics. Trends inBiotechnology 1998, 16, 301-306.5. Service, R.F. Microchip arrays put DNA on the spot. Science1998, 282(5388), 396-399.6. Ramsay, G. DNA chips - states-of-the-art. Nature Biotechnology1998, 16(1), 40-44.7. Marshall, A.; Hodgson, J. DNA chips - an array ofpossibilities. Nature Biotechnology 1998, 16(1), 27-31.。