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by: Cassie Koepp


Cassie Koepp
GPA 3.61

P. Dimario

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P. Dimario
Class Notes
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This 336 page Class Notes was uploaded by Cassie Koepp on Tuesday October 13, 2015. The Class Notes belongs to BIOL 3090 at Louisiana State University taught by P. Dimario in Fall. Since its upload, it has received 16 views. For similar materials see /class/222830/biol-3090-louisiana-state-university in Biological Sciences at Louisiana State University.

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Date Created: 10/13/15
Nonhistone Chromosomal Proteins Relatively low in abundance compared to the histones They include 1 proteins for transcription SSB DNA poly etc RNA polymerases activators and repressors later disc Transcription Factors 2 DNA polymerase for replication 3 High Mobility Group HMG proteins moderately abundant average 1 HMGnucleosome role in transcription Structural Functional later disc 4 Other structural proteins Topoisomerase etc a Hinge domain Structural Maintenance qg emquot Chromosome proteins domain SMC proteins Smcz Head Can hold two circular Mai DNA molecules together cl Chromatin loop containing a transcription unit Most interesting SMC proteins may reside in the chromosome scaffold and help establish the radial loops Figure 638 Molecular Cell Biology Sixth Edition L 2008 W H Freeman and Company 30 nm 100130 nm chromonema fiber 200250 lllll middle prophase chromatid 500150 nm mataphase hromatid Chromosomes are 39 39 Telurnere at their most m WWWWMW condensed state W quot X39gm E t 3333 wmmmm during h je apS39gggg canquot39quot9 9 r0quot e W t WWW Micrograph of a 21m c nlei m53 Mitotic Chromosome Certain d es e Giemsa stain mitotic chromosomes in a banded attern 1 Banding pattern is unique for each chromosome ocate genes 2Allows clinicians to identify chromosomes and their aberrations 3 p petite the smaller arm g 641 q the larger arm just the next letter in the alphabet ransmcat39o Philadelphia chromosome gt ON der 22 22 Chronic myelogenous Leukemiaa break occurs in a proto oncogene Oncogene now out of matrBMZa der 9 to detgot dorm and dgquz Normal chromosome 9 Phi adelphia chromosomequot der22 Normal chromosome 22 O der 9 Fix mw 77 gleW Q FAOJ OJKQ o gv 1 r A if my a3hj1gtli 7 j n22 TL a mw LVan hIVN W quota Kg mmmgm Wmn HYi J v fe h U H r aN Elm fa tg 9mg Td 131 r is mmsgwedl m shrgw 16 and human Wq lrgfemgd ta a5 Primate ancestor Homo sapiens 11 12345 x 9 12 456 X 23 X 78 3 1 4 5 6 15 I I I U m i107H12H81314I15I6 21 910111213 7391011121314 m I D I I I I Is w w w 23 2 14 I D 2 22 H21 15 16 17 1s 19 20 21 22 23 15 16 17 18 19 20 21 22 MW111w m mamHtmmmnmumpw The various chromosome paints allowed us to propose a karyotype for the ancestral primate Euchromatin versus HetercHatetlaim rem ati nondensed after mitosis ie chromocenter of polytene Many stranded chromosbmes Clh romoce nter E39Yi paw 339 EN W n A E 3 iF 3955 K1 l N F 3Wiu I 7 quot 3 i 39 397 i E ZR 4 T 39 1f3 f Picture by V 39 J 39 4 Joe Gall 39 39 y f m f thechrom v m osome guy Fig 644a Polvtene Chromosomes of Insect Larval Salivary Glands Both bands and interbands are considered euchromatin Banding patterns establish an alphanumeric mapping Sy itlemBands are considered to be condensed eucl fgr39zcjatr irahItall Centromerez t 1 telomere Telomere t I a F 3 Maternal 5 pm e 39 tzrszw39 telllomere Polytenization Maternal and Paternal each have 1024 l o6a gs bl Centromere Telomere TeIomere In situ hybridization of a singlestranded probe to a specific gene 4 Alphanumeric cytological mapping 4 system established by 97 TH Morgan s Lab 98 f l 99 100 Fig 644b to localize a particular gene within the cytological map What else are Polytene Chromosomes good for Anitbodies can locate various chromatin proteins But recall nascent RNA is a part of chromatin So proteins that bind nascent RNA can be localized mm Gunningnmmm Yeast Artificial Chromosomes YACSL Necessary requirements for a functional eukaryotic chroma 1 Autonomous origins of DNA replication ARS Shuttle Vectors 2 Centromeres for kinetochore formation during mitosis and faithful separation to daughter cell 3 Proper ends telomeres for chromosome stability Protect the ends of the chromosome 4 At least 50 kbps aka 50000 bp 5 YACs now used as vectors to clone genomic frang up to 1000 kbps 1 Autonomousl Plasmid with Sequence fro nonnal yeast Re licatin Se uence ARS 100 bp element at which DNA replication initiates nganv ai translated cell Growth without leucina Mitotic seglagztian plasmid Conclusion ARS required for lasmid replinalion In presence of ARS plasmid replicaiion accuvs bui mimic segregation is faulty Fig 646a 2 Centromere allows replicated plasmids to separate into daughter cells at mitosis Plasmid with Transfemd Pragenv of man cell canclusion sequanm from Lsu call normal yeast Gmwm Mitotic without segregation b leunine Yes 1 Good enumic 5 Eu gt won iragmsm i A of cells have required for Yes plasmidl good segregation I u III i A A r VBISICEN GYCACGTGli75 asbpv H39GYTYCTGNTYTCCGAAA DmMphl39lnGICACATAGlizelhpglYGATTA39IYTGATGACCGAAA lDomSSBl Centromeric sequences are highly repetitive in higher eukaryotes Centromeric DNA in all h eukaryotes is associated with nucleosomes that contain a a a i i i i Domains that associate Domains that assomate wiihamicrotubule withiheCEFacomplex CEN PA Ndc80 complex lcl Centromeric chromatin In budding yeast it s only can one Specialized nucleosome du ommex to the very short centomeric DNALW altachm CBF3 complex of proteins 533 I binds the special nucleosome 323 l Ndc80 links CBF3 to mitotic spindle microtubules first on the side m2 quot w then with Dam1 for w 39 endon attachment Fig 545 In fission yeast 8 pombe centomeric DNA is longer 40100 kb recruits multiple CBF3 complexes attaches several microtubules to the centromere during mitosis Plants and animals have highly repetitive elements 171bp element in humans make up tandem arrays 24 mega base pairs in length called alpha alhpoid DNA In higher eukaryotes eg mammals Kinetochore forms at the centromere during mitosis associates with multiple mitotic spindle microtubules some proteins are similar to those in yeast other proteins are different Kinetochores in mammalian mitotic chromosomes Sisaer duomasids Cohesins i i i i i U i i Itinerer Fig 1839 3 Telomeres stabilize linear chromosomes Pinsmid with Transkmd Leu cell sequence from manual vean in enzyme produces ilnear Diasmid Progeny ormnmaed uii Conclusion Growth Mimlic wuhom segregation cannm replicate 7 Jam iasmid Unstabl quot e Linear plasmlds conuining ARS and CEN behave 5 like normal chromosomes if genomic fragment V55 TE L is added to man ends 50 linear chromosomes need at least ARS CEN and Telon for stability and proper inheritance Fig 646c TEL OMERESII The End Replication problem known for some time prior to work on telomeres u Lagging strand Rightend of remains incomplete unle some mechanism 39 fill in the last gap 5 Leading strand Lagging strand 5 3 SS 5 3 Leading strand Lagging strand iShortened i 5 3 Now what ngm 54u Moiezulav CeiiaioingylsinhEimnn nmnzw H human and amnany Two maior considerations dealing with the ends of chromosomes 1 Inherent problem in replicating the ends of the lagging strandl5 Template strand 3 j 3 5 Newly replicated lagging strand V Last Okazaki fragment s RNA primer leaves a gap 2 Special structures must exist at the ends of chromosomes to prevent the ends from fusing McClintock 1940 s This is bad McClintock s bridgebreakagefusion cycle Causes Aneuploidy She was the one who did McClintock s bridqebreakaqefusion a b c c cle I Telomeres protec w FI Loss of teleomeres 4 B mil 25asasaasasssssssssssssea 4 Fusion of I Broken ends Cycle b repeats Mitotic a Spindle quot Breakage occurs at G b ltanaphase Breakage to daughter E cells Translocation but could also cause aneuploidy Aneuploidv abnormal loss or qain of normal chromosomes or chromosome seqments qenes Telomeres solve both problems simple sequence repeated many times at chromosome ends a few hundred bps in yeast and protozoans eg Tetrahymena a few thousand in vertebrates forms heterochromatin GGGGTTGGGGTTGGGGTTGGGGTTGGGG3 CCCCAACCCCAAE J V Elizabeth Blackburn first to determine 1246 F 5mg39e Strand OVerhang a telomere sequence while a postdoc in 39 Tetrahymena Joe Gall s lab Classic paper in 1978 Used Sanger s ddNTP method of J 3 Telomerase Greider and Blackburn an enzyme that extends the telomere simple sequence Telomer a protein and RNA complex RNA serves as the template to extend the 3 overhang a special reverse transcriptase reads the RNA template to synthesize the repeating single stranded DNA sequence Classic Mechanism of Telomere Extension parental strand 3 m 393939GGGGT39GGGGTTGGGGTI39G AAC 39 CC in i3mpltele newly synthesized lagging strand TELOMERASE BINDS 3 l TTGGGGTTGGGGTTGGGGTI39G direction of l AACCCQ CCCCAA n elomere TELOMERASE 5 SVMheS39S EXTENDS 3 END telomerase with bound RNA template lHNA lempaled DNA synthesis 3 I393939GGGGTI39GGGG39I39I39GGGGTI39GGGG39ITGGGG39I39I39G IgAACCCQ ACCCCAA COMPLETION OF LAGGING STRAND BY DNA POLYMERASE Actually thousands of bps In leng h DNAitemplated DNA synthesis o 3 I 393939GGGG39I39I39GGGG39I39I39GGGG39I39I39GGGG39I39I39GGGG39I39I39G IgAACCCC CCCCAACCCCAACCCE DNA polymerase Figure 543 Molecular Biology ofthe Cell 41h Edition From Al berts et a A Structural Model of the Telomere 2TLoo w p o Telomere I as a if Loo 394 39 p HIE quot 11m quot5 DLoop w E553 Displaceme v u nt Loop A m E G 539 o 39 DIoop This is the 3 end Incredibly not In LOdlSh et al What the Ends of Human Chromosomes Really Look Like Fig 544 from Alberts et al Telomeres are Heterochromotin AD 1 um I loop McClintock predicted i1 the 1940 s the ends mLs have a special structure to prevent fusions 9 307mm chromatin ber w 1 heterochromatin euchromatin E Figure HA Molecular Biology onhe Cell 4th Edition Telomeres Loss of telomerase leads to a loss of telomere sequence Mouse knockout for telomerase gene OK for 3 generations But thereafter the telomeres are so short that 1 ends of chromosomes fuse 2 leads to loss of chromosomes during mitosis Aneuploidy BridgeBreakageFusion Cycle Sterility b 6th generation Phenomena discover d by McClintock 1940 Normally telomerase is active in qerm cells and adult stem c but not in most differentiated somatic cells Aging They get shorter and shorter over time Teomerase expression is reactivated in cancer cells Design anticancer drugs that block telomerase activity Telomeres Elizabeth Blackburn Professor Dept of Biochemistry and Biophysics University of California San Francisco March 2005 Franklin Medal ptember 2006 LaskerAward hiqhest scientific prize awarded in the USL October 2009 Nobel Prize in Physioloqy and Medicine This was for the telomeres Elizabeth Blackburn is a great scientist the only stem cell biologist who served on the President s GW Bush s Council on Bioethics under Leon Kass and those who watched as she was dismissed from the Council were appalled Chapter 7 Transcriptional Control of Eukaryotic Gene Expression Read 276320 Single cell yeast vs metazoan mumcellular organisms Like Whas genes that respond to the environmer sugars nutrition temperature oxygen tension Not so much in metazons mumcellular organisms but metazoans display developmental programs in their gene expression Most genes in higher eukaryotes are controlled by transcription rates rate of transcription Initiation starts per min Is most important aspect in regulating gene expression how often do you actually start transcrip on on a particular ga Classic runon to measure 39 quot rates GeneA emergenas r 6 6 8 RNA FNA m pulymerass H mm a Nascent RNA chain Pulse Ia Jl Laazbiai wuh radioactive nuciermdes i P 32 P 1 e o o a a a a a radioactive nucleotides Label thndize m excess gene A DNA separate hybrids and mum radinamvitv in mm RNA and A RNA Relalive lranscriplian rate of gene A Application of runon experiments W L 000 39 M eeo ti 390 m 0 Here liver kidney and brain cells were pulselabeled with 32Pribo lgidRBv nucleotides and RNA hybridized 7 several different liver cDNAs and other cDNAs spotted onto Nitrocellulose in equal amounts Brain Gene 1 is the most active of RNA those assayed for the liver but 0 gene 1 is not expressed in kidney and brain Forerunner t0 CHIP Technology 3 Bacterial RNA polymerase bl Yeast RNA polymerase II V r27 5 Bacterial RNA polymerase b Yeas RNA polymerase II cl Vsasl RNA polymerase I Figure no Molecular Cell Biology Sevenlh Edilion ma WH Freeman and Company RNA Pol I resides within the RNA Pol II and III are in the TABLE 72 Classes of RNA Transcribed by the Three Eukaryotic Nuclear RMA Polymerases and Their Functions POLYMERASE RNA TRANSCRIBED RNA FUNCTlON RNA polymerase I N uCIeOlus Pre rRNA 285 185 585 rRNAsl Ribosome components protein synthesis RNA polymerase ll mRNA Encodes protein snRNAs RNA Splicing miRNAs Posttranscriptional gene control N ucleoplasm RNA polymerase III tRNAs Protein synthesis 55 rRNA Ribosome component protein synthesis snRNA U6 RNA Splicing N UCIeOpIaSm 7S RNA Signalrecognition particle for insertion of polypeptides into the endoplasmic reticulum Other stable short RNAs Various functions unknown for many Table 72 Molecular Cell Biology Sixth Edition 2008 W H Freeman and Company Differential sensitivity allows you to determine Pol 1 low which polymerase transcribesPOl 2 high a certain gene Sensitivity of alpha aminitin Pol 3 intermediate E coli core RNA polymerase laZBB39m Qnmo Eukaryotic RNA polymerases Ill I iilike subunits CTD unlike subunits nlike Subunit Common 0 O 0 subunits l39 D D I I I Additional enzymespecific 5 3 7 subunits 160220 kDa RPB1 128140 kDa RPBZ Note CTD carboxy termin domain on RPB1 of Pol II Note oclike subunits for Pol II are different Fig 710 Carboxy terminal domain CTD of mi ll RPB1 subun Seven amino acid stretch repeated 26 times in yeast 52 times in mammals Tyr Ser Pro Thr Ser Pro 002642 39 Tyr and Ser residues will be phosphorylated during transcription elongation But the unphosphorylated form is needed to assemble transcription initiation complex Phosphorylating the tail of RPB1 may be the trigger to begin transcription once the complex is assembled Drosophila polytene ch ro mo so mes Puffed out regions are genes su peractive in trans cription Fig 7 11 Eukarvotic Transcription Control Reqions 1 TATA box at approximately 25 to35 bp upstream positions the initial start site of transcription at 1 if sequences are deleted between TATA and true 1 initiation will still be 1826 bases down stream of TATA 3 to 32 z 31to 2IB 2 to 4 28 to 32 BFIE inlrl mpg TFIIB I initiator Dawnstream core Recognition Drasaphila 7 1391 G T promoter element element 39TCA TTC r G EGG V T A GAC A CEACGCC Mammals WANAW G TT c 712 Eukaryotic Transcription Control Regions INot all Pol II genes have TATA elements Instead they could I f23Vl itiator elements 5 YYA1 N TAYYY3 where Y C or T pyrimidines N any one of the 4 deoxynucleotides 3 Promoters for genes encoding enzymes for intermediary metabolism No TATA element No initiator element Initiate transcription within a 20 200 bp extended region Leads to multiple start sites multiple 5 ends Typical of house keeping genes CpG rich promoters where C s can be methylated 5 methyl C f nf MA I IIAIIAn UHIn NARA Ilappinq transcription start sites using restriction fragmen1 and in vitro transcription assay An old fashioned Runoff expt f i ii quot51quot T i i i y Initiation site 32pabeed ribonucleotides 205 nt Hm l I Incorporate into RNA 430 nt xmam RNA pol merase y M runs Off the end Smal l l of the DNA K fragment DNASequence quot39GTCCTCACTCTETTCCG39quot S endo39meNA cap AC UCUCUUC C GIquot and in vitro RNA transcripts Electrophoresis of labeled RNA followed by autoradiography Smal Hindlll Xmalll Smalocamanitin 39 I RNA Pol II 560 430 quot 39 o uamanl In A m A 0quot a i I Treat living ceils or tissues with a membrane permeable crosslinker such as formaldehyde Chromatin ImmUno Scnicatelo shearceliuiar Precipitations ChIPs AWN H l 2i i fquot Nascent m RNA i1 is d g y A powerful technique Ammowain to e Paused polymerase Paused polymerase Elongation inhibitor immunoprecipitate to isolate Pol II crosslinked to DNA Elongation inhibitor sequences associated in 539 DWNAWMMH with defined chromatin l 19552Faiizn n zii sqi izd we proteins in this case 9 RNA Pol II ib Bidirectional initiation Unidirectional initiation 20 93955000 Chrorn Position 93962000 121457000 Chram Position 121463000 RNA Pol i CountsMlillun gt D T533 RNA Hsd17b12 Ra Rplb LH l Gear Change General Pol merase ll Transcri tion Factors Formation of the RNA 7 limerase ll Transcriyt 39 quot39 quot Complex TATA ox lll quotklIll TB P TF stands for m I Iranscription Eactor for RNA Pol TBP TATA Binding Protein and 11 other proteins TFIID 50 actually TFll I binds first TBP is a monomer but its two halves have 2fold symmetry TBP binds the minor groove to bend DNA similar to HMG1 Formation of the RNA Iquot limerase Transcriyt 39 quot39 quot Complex After TFIID binds TFIIB joins TFIIB is a monomer Fig 7 31 TBP and TFIIB Bound to the TATA Sequence For what it s worth Formation of the RNA Polymerase ll TranscriptionInitiation Complex Pol II TFIIF K CTD Then a preformed complex of RNA Pol II and TFllF joins TFllF is a tetramer az z Fig 731 Formation of the RNA Iquot Ivmerase Transcriyt 39 39 39 Complex Then TFIIE joins TFIIE is also a tetramer 1292 TFE creates a docking site for TFIIH f TFIIE I ll Fig 7 31 I u 4 Formation of the RNA lquot 39 ll Transcriyt Complex TFllH consists of 9 proteir subunits One subunit has helicase activity to create an open TFHH O DNA complex using ATP hydrolysis Preinitiation complex Fig 7 31 a Closed complex b Open comptex Point of DNA opening Downstream DNA Formation of the RNA Polymerase ll TranscriptionInitiation Complex As transcription begins RNA Pol II moves away from the initiation complex Cause and Effect are still under investigation TFllH phosphorylates tyr and ser of RPB1 Most of the general factors then dissociate except for TBP Formation of the RNA lquot 39 ll Transcript Complex Preinitiation complex 1quot Yeast genetics sugge ts NTPS ATP 6070 proteins involv cl a 3 Mda complex ADP g fent Elongating r Pol II with Release of phosphorylated general factors f gt CTD except TBP pr V It What about th J Nucleosomes quot 0 Fig 7 31 5 RNA Pol HI Fig 720 Linkerscanning technique to determine what promoter regions are important for efficientFter g ntscripetion tk mR rge fineFiner e n seuence NA 1 l Mutant Short overlapping 10 bp deletions or 10 bp inserted linkers 139 m 2 3 4 5 6 7 9 Control Fig 7 14 Back to introducing DNA into eukaryotic cells DNA introduced into eukaryotic cells by transfection could f DV 1 randomly integrate Into chromosog3tgilzrsggct gt 2 undergo homologous recombination with normal endogenous gene 3 remain as an extrachromosomal episome but soon lost if no integration in genome Change gears here for next three slides to explain 1 promoter fusion ln transgenic versus animals 2 protein fusions Transcription a Promoter starts here Fusion Promoter DNA encoding GFP for the gene that encodes odorant receptor Transcr39pt39on This promoterworksiT M mRNA only two neurons l Translation Green Fluorescent Protein GFP distributes randomly within those cells that actually use the specific promoter Transcription starts here Promoter for the gene that encodes odorant receptor 1 Transcr39pt39on mRNA W l Translation b Fusion Protein DNA encoding GFP DNA encoding receptor protein GFP now fused to the receptor protein which localizes to the very anterior of two neurons Transgenic C elegans u Promoterfusion ODRIO promoter fused to the DNA a The prom oter for a gene that gicsdlng encodes an odorant receptor is ligated to the DNAthat encodes the Green Fluorescent Protein GFP b When the coding sequence of the same gene is ligated to the D encoding GFP the cell produces a fusion protein the receptor prot fused to GFP Il 10pm f9 Mdlezulnr Cell Binlugy Sixth Edition o 2008 w H Freeman and Company DNA Chip MicroarrayTechnology 532113 wC J7u39I39 se mRNAs are prepared from two sample groups I serum 1 ssoImmnnunl giversefaenzcriml cDNAs are made from the mRNAs 1 3331534533 using deoxynucleotides tagged with quot dyes Mlx Hybridize to DNA miuoarray cDNAshybridized to l DNA lam single gene Two groups of cDNAs are meed 1 together and annealed to the 8600 1 genes on the microarray a competiti w between pools of RNAfor the signal gene in that spot Wash Measure green and red uorescence aver each spot 1 If a gene spot shows red then it grim was turned up upon serum addition 39 If a gene shows green then A Irsponsgeexpssion ofthatgeneideueases In cells after serum addition there s a lack of red cDNA ass 39 lt I herserum addition that this one gene was turned off dbgg g in the cells treated with the serum TZ li39 ilfj fff l fi quot FiQ 529 Know these inherited human diseases Chang 2 TABLE 5 2 Common Inhe ted Hu eases genes msmz Moucuun AMDCELLUAR nzrm mrmzucz mom mum 5ddeltel MIMIIII Md hlno mlhwhkh In became I059 In upillarles Ilsownkrs ESISBMA lo NIII I mum nemawmb ammcnmasn morzupg Ind mm m lungs Pmnyxlwmnm u mm mummy WWW mm IIIEMDDMEurnpunangln Nhlecu39 Iym n hydmxyhu 5qu 1 um phenyl a ar In My la mnul Kurd munll sx remluzd by diet genEtIC YIySaxhsdlseue Dekulve hexonmlnldase Anrymeleads tollcllmulilloll IUJDOQISkm Euwnsn Jews technl q of en sphlngo plds 1 m Wm uI neurons ues lmpalvlng neural dmlopmenx AutosoMAL nommm mungm 4 mm m p mummy my 5 in mm mumquot 0 wk mm 4 a mm NYPeI dIoieslelnkmh anulve LDI mmorluds ll exusslvedsolnlem n 1712271200 Canalas blond Ind ulIY hurt unk ermKEn masswz nd umqummumuuinaymmn4 13501 qu dynmphy 0M0 Impind muxxlllunninn HlmDDhIIIiA le l a bluad Iolllng 1am VIII In sw I ZIIUNDO males uncnmmlled mam ml 52 Mnkzulm llilu39ugyyslxm 54 umw H mm Cammny and how they are inherited a Huntington39s disease Autosomal Dominant MF and only need one Affected Not affected b Cystic d AWEN gtlt AW VN Q Autosomal fibrosis Carrier Carrier recess ive Males and females MF need to ACFWA AVACF A vA homologous Affected Carrier Carrier Noncarrier c Duchenne d XY gtlt XDMDX 9 Carrier XlInked dystrophy recess we Males Females M bad X from mom X VY XDMDXquot xx 5 5 Affected Unaffected Carrier Noncarrier d ANDA gtlt AVA Q Affected Males and females AA estriction Fragment Length Eolymorphisms RFLPs are used as Physical Markers on Chromosomes a Ehmmmnmal mangemm Hybridiutinn banding Pl um Eruva Enzyme A a M 39 t i gggm Southern blots e s demg i ResxnctinnendonucmzsaA quot differe t Hesmcuonendonuc euea Polymorphlsms have been mapped in the human ger i e5393 a mum singtecnpv legion bl RFLP analyses in human genetics and police for nsics Grandparents Parents 536b Children Alleles Fragment lengths GP GP P P GP GP 10 kb Agood example of Southern 2 quot V quot 39F O l 77 kb 039 I39 a 65 kl blotting 3 in articuIar with different Linkage Disequilibrium haglotype m haplotype Genera of New mutation I Chromosome to map mutations l Meioti recombination Generation 2 1 RFLPs 1 Several generations with meiotic l recombinatio 2 Singlenucleotide polymorphisms SNPs 3 Simple Sequence Repeats SSRs that lie within 011 centimorgan are good indicators to locate the mutation n Close set of polymorphisms referred to as l w llllllllllllll ngm 537 Maluulnr Cell l39ol gn Sim Edlllwl oman Freeman and anpany Generation Folymorphk markers Plasmid or BAC I Innes 5 Polymorphism are DNA markers EA Track those markers that um 2 remain 75m g associated with ll g the disease after 5 many l g generations of I recombination i LEVELOF RESOLUTION tyioganolh Linkage thsiml Sequm map map may Inup METHOD OF Chromosome Linkage 0 restriction Hybridiution Sanger DEYECTION banding pallem lragmem lenggh poly Io plasmid dideoxyl Fluoms 39n lump isms lanes 5 39 d miun single quotnaming poly h39 V d 39 g3939ZE 39quot Recall the partlal Sau3A n m muss restriction digestion when MglqumKeHBiolngnSinhEduinn making a genomic library monaw H mm and WW Gene Disruption in Yeast knockout 20 nt flanking sequence Designed PCR DNA synthesis primers 4 3 ZOnt flanking sequence kanMX for G418 resist Primer 2 kanMX 3 5f Primer 1 DNA synthesis EH gt 1 PCR Fig 5398 I kanMX Disruption construct 1 Diploid cell PCR m Transform diploid cells with disruption construct prody PCR product from previous slide Homologous recombination lf the disrupted gene is essential these spores will be nonviable Fig 539b Gene knockout is yeast is easy compared to that in mouse The Holliday intermediate in the recombination between two homologous DNA sequences Figure 561 Molecular Biology of the Cell Sle 9 Garland Science 2008 Gene Knockout in Mice To study cell function in development physiology and behavi Embryonic Stem ES cells taken from blastocyst embryos mouse totipotent easily cultured easily transfected by electroporation homologous recombination will disrupt or replace endogenous genes normal gene in the chromos can select for homologous recombination yields a transgenic knockout animal Gene Disruption to Generate Transgenic Mice a Foimzlion nfES ceiis carrying a kneekem mulalion Disrupt gene X rkHSV by molecular techniques in the lab Fig 540a Gene x replacement veeinr Disrupted gene x Homologous Nonhomoiogous iecombinalinn recombinzlinn What you Wm aka random insertion I I I CW3 Wn i you DON T Wan v ES neiis i i antitan 4mquot DNA Gene X othy genes Generlargeted Random insertion insanion tk gene lost upon homo ogous I recombinatw Mmsunn in gene X No mutationm geneX tk gene 51m presen nenmycin and neemyozn bul sensitive t gmcydw39r39 ganciciovir to gznciclovii quott WC agent But ES cells are diploid What about the other endogenous copy Selection usin neom cin and Hancl ElW i39rfbegative selection of recombinant ES cells 0 O O Q O Nonrecombinant cells Recombinants O with random 0 O O O Recombinants with insertion 0 O genetargeted insertion positive selection 0 O O o AllEScellsw hth O 0 O neo gene SUrVIVe 0 0 Treat with ganciclovir negative selection 1 Treat with G418 Kills cells that still have the tk o t 0 Use to make ES cells lacking t O O O A tk gene survwe O ransgenlc mous 0 Fig 53940b ES cells with targeted disruption in gene X How to Make a Trans enic Mouse Inject ES cells into blastocoel cavity of early embryos Brown mouse Want these injected cells to AA X X Black mouse eventually give rise to sperm aa XHX or eggs germ line transformatio 45 day blastocyst A a fur COIOF Surgically transfer embryos A brown dominant into pseudopregnant female a black recessive X normal gene X39 knockout gene Foster mother Fig 541 Foster mother Injected ES cells AA XX mixed with endogenous ES cells aa XIX gr gt Black Chimeric Possible progeny Injected ES cell AA XX39 didn t survive Fig 5 41 Chimeric Black Select chimeric mice for crosses to wildtype black mice begb Possible germ cells All germ cells AX AX aX aX I l Li laploid germ cells sperm or eggs apl d germ cells sperm or egg F 5 derived from injected ES cells eriv d from endogenous ES cell 399 39 some or giving rise to germ cells WI gtlt Possible germ cells All germ cells AX AX aiX aX I ES cellderived progeny will be brown Aa X ZX Aa xX 83 XV r J Progeny from ES cell derived germ cells But how do you distinguish xx or xx F39g39 53941 Aa XX Aa X7X aa X VX a J Progeny from ES cellderived germ cells Genomic Southern Screen brown progeny DNA Blot 039 PCR to identify X X heterozygotes ll Mate X X heterozygotes Genomic Southern I Screen progeny DNA to identify Blot or PCR X X homozygotes Knockout mouse 3quot What fgenex A germ line Knockoutig 541 is necessary for viability Genomic PCRs to distinguish X39X versus X39X39 Disrupted gene X referred to as X39 Fgt lt R lt R1 neo R2 Use genomic PCR with Forward primer F and both Reverse primers R1 and R2 to test if the mouse is X39X or X39X39 45o bp gt lt X39 when neo is present PCR product 600 bp gt lt X when neo is not present Heterozygous X39X animal would have both PCR products Homozygous X39X39 animal would have just the shorter product Germ line gene knockout could be lethal as homozyglt So a more recent Somatic Cell gene knockout strateg loxP Cre Prepare by mouse mouse Promoter homologous 3 fusion All cells carry endogenous gene Heterozygous for gene anock recomblnatlcn XWIth loxP Sltes flanklng exon 2 out all cells carry cre gene n n n n exp exp Cell typespeclflc promoter Cells not expressing Cre Cells expressing Cre Gene function is normal You can 33 9 Exon 2 is deleted genetically I manipulate All cells carry one copy of loxP modified gene X one copy of l n transQenes gene X knockout and cre genes G f d d in various 39 ene unctlon IS Isrupte ways39 Flggjj iell iologysixthEdition in a o 2008 WH Freeman and Company RNA Differs from DNA 1 ribose instead of deoxyribose RNA has a 2 OH group which makes RNA very stable 2 uracil instead of thymine Can be linear or circular single or doublestranded Doublestranded RNA provides secondary structure tRNA AND rRNA Secondary structures can fold into tertiary structures RNA is exceedingly sensitive to ribonuclease activity And basecatalyzed hydrolysis pH should be at 8 Cleave by doing this So it is subject to base catalyst hyc i i i oilz o o io 0To o u i a 3 7 I I o 2 1 mm cuZ quot10 cu ml in monuphuphnu u u gt H u or N u 1 dulmm n n n H u n n H o n on n a H n F I I n o P o o p o 0quot 0 so I I quot 2 muunphnsphnla on 339 monuphosphulo o H Mu A m ul tlm H H H H H H N H 0 DH 0 UN 390 l o 390 l 0 l E Figure 5 Mammary Biology sixm mm 4 1008 w H human and ompany RNA Stemloop and hairpin or secondary si39amsetunes structu re Double helicai stem region Stemloop Hairpin Fig 49 RNA a lot of GCGCGCGCGC Tertiary structure G c A C G U A Loop 6 c 1 A U C 6 II A U A U A Stem G C 1 quotA C G G c 539 2 5 Pseudo mol C A Figure 49I1 Marsala Cell Biology Sixth Edition 2008 W H Freeman 1 nd Lorn pany tRNAs and rRNAs have lots of secondary and tertiary structu 3 Structurepseudoknot Fig 49b RNA Some RNAs fold to achieve enzymatic functions Called Ribozymes Some ribozymes display LARGE RIBOSOMAL RNA PEPTIDYL ACTIVITY nucleotidyl transferase activity site specific nuclease activity This is 1 RNA working on another RNA Some RNAs selfsplice Some RNAs catalyze premRNA splicing called small nuclear RNAs snRNAs rRNA displays catalytic activity in protein synthesis specifically peptidyltransferase activity LSU RNA is amazing Transcription 7quot g 5 The synthesis of RNA mi 3 071CD D Incoming rNTP base W pairs with the base in an the DNA coding strand 3 OH attacks a phosphate This results in the PP Hyrdolyis a N truth Synthesis is 5 to 3 Incoming mm anquot v I vinw p2 RNA polymerase reads the DNA template 3 to 5 Blue arrow DNA strand 5 to n Fig 43910 Promoter Transmptmn Cnding sequente Upstream Downstream F DNA emprere wanna J a Primary RNA transcript numeral Murnururk aiofvgyliixlhEduinn r znnzw e new and mm 5 Untranslated Region UTR the AUG is not the first three codons on the mRNA RNA polymerase Start site Stop site INITIATION on template on template u r strand Polymerase binds to promoter sequence in duplex DNA quotClosed complexquot Polymerase melts duplex DNA near transcription start site forming a transcription bubble quotOpen complexquot irllumi 39 Polymerase catalyzes phosphodiester linkage of two initial rNTPs ELONGATION Polymerase advances 5 3 gt 5 down template 3 strand melting duplex DNA and adding rNTPs Nascent I to growing RNA R 5 DNARNA hybrid region Hey What kind of helix does this DNARNA hybrid have A type DNA with in RNA polymerase This is where we find it in a human Are the two strands making up the helix antipEigll l 1 Ln TERMINATION At transcription stop site 3 olymerase releases completed RNA and dissociates from DNA Completed RNA strand Fig 4 11 3 subunit r T 3932 A tit lal Model for bacterial RNA polymerase as it synthesizes RNA Notice how the DNA is bent NA Several subunits make up the holoenzyme Note how the DNA is bent at the transcription bubble Bacterial RNA polymerase h Four subunits held together Quaternary structure Sgl uiezieuaiaiogy ixmEdition 412 2003 W H freeman and cumpany a Prokaryotes Gene Organization inE Wigenome trp operon Bacterial Cells an Operons trp and IacLactose Start sue for Irp mFiNA Polycrstronlc mRNAs synthesis T 39 t39 Repressor corepressor l ranscrlp Ion trmeNA 5 I I 39t 3 Start sites for protein synthesis Trp has five genes linked on the promoter Lac has three genes linked on the promote E Each has a separate Pm ins L c ShineDelgrano sequence For protein A syr be i sa IlTranslation The lac Operon Negative control oREPRESSOR Positive control CAP BINDING PROTEN C This is an enzyme a lot of the enzymes 1 transcription start she Promoter v ri i CAP site Operator E coli iactranscripiionconlrol genes CAF ai 7 lactose glucose iavZ 0W CAMP N0 mRNA transcription lacloie a a3 lactose glucose IDW cAMF i LOW transcription cl CAMP a 7 Euka ryotes Yeast Chromosomes 1550 IV 91o VII 4 mp3 680 XI RNA processing trp lTranscription and 1 3 mRNAs F F 45 tt lTranslation 1 2 3 4 5 Proteins Figure413b Molecular Cell Bialngy Sixth Edition 3 2008 W H Freeman and Company Gene Organization in Eukaryotes Separate genes for the 5 enzymes necessary for Trp bibtqtatmasis poly cistronic mRNAs All 5 TRP genes are coordinately rengagad41 3b 56mm 31 32 105 106 1 genomic DNA Start site for PoyA PremRNA Spllcmg RNA synthesis quot site in Eukaryotes Primary 539 339 RNA 339 cleavage and transcr39m addition of Notice the 5 Cap VpoyA tail Referred to as m7prp Mn I5 PenA Notice the PoyA tall 1 tall V n UTH PonAbinding protein 0 A called PABP1 quot176mm ntronexc15ion Notice 5 and 3 UTRS n exon ligation A Bee 1 Fig 415 F O quot1 a 5 7mthylgunylnte a 9 o 0 ow oTo Figure 414 Molecular Cell Slalogyp Sixth Edition 6 2008 W H Freeman and Company The 5 cap on mRNAs and some small nuclear RNAs snRNAs in eukaryotes 5 Cap found on ANY RNA transcribed RNA polymerase II Note 7Methylguanylate 5 5 triphosphate linkage This is a posttranscription modificati Methyl groups on the first two nucleotides encoded by the gene Eukaryotic PremRNA can be spliced in alternative ways This is how we can essential proteins in different or ans EmB EmA Fibronectm gen Fibroblast 5 fibronectin mRNA Hepatocyte 5y fibronectin mRNA Fig 416 Three RNAs in Protein SvntheSIs a aj39tRNA7 arriving Growing H I polypeptide 2NCF 7 chain 0 Ribosome tRNA4 leaving 0 0C mRNA 5Ill illill 3 631 aaz 383 Flg 43917 Movement of ri bo some gt The Genetic Code Four nucleotides in DNA and RNA Nucleotides read as triplets 43 64 possible triplets Each triplet is called a codon 6164 codons encode AA s 61 FUNCTIONAL CODONS Remaining 364 codons are stop codons Most AA s are encoded by two or more codons called redundancy or degenerate TABLE 4 1 The Genetic Code Codons to Amino Acids SECOND POSITION U C A G Phe Ser Tyr Cys U U Phe Ser Tvr Cvs c Len Ser Stop Stop A Leu Ser Stop Trp G a 5 Leu Pro His Arg U a g c Leu Pro His Arg C E E Lou Pro Gln Arg A 5 Pro Gln Arg G a 39 n E lle Thr Asn Ser U 3 A lle Thr Asn Ser C lle Thr Lys Arg A Thr Lys Arg G lal Ala Asp Gly U Val Ala Asp Gly C G Val Ala Glu Gly A Glu Ely G un A A nuu a hunting hut usualquot I 4 Table 41 Molecular Cell Biology Sixth Edition quot 2008 W H Freeman and Company I Val lMetl l Ala usually codes or valine and CUG for w initiate a protein chain Only Met and Trp are encoded by single codons AUG encoding Met is used as the translation start codon and wherever Met is encoded in the mRNA Notice that upon rare occasion CUG and GUG can encode the initial Met The blue boxes are EXTREMELY rare There are three translation stop codons Table 41 0 Notice that when several Three possible reading frames in a given mRNA Similar to Fig 418 The Genetic Code Initial AUG start codon sets the reading frame DOWNSTREAM OF THE FIRST NEUCLEOTIDE AUG is also used to encode internal downstream Met s Never ambiguous THERE ARE A FEW RARE EXCEPTIONS Example CCC always encodes Pro in all cells and in all organisms You can but EColi genein EColi and it will make the same thing Generally true for all the other codons Allows us to clone and express human genes in E coli Never say never There are some rare exception where stop codons encode for amino acids FYI TABLE 42 Known Deviations from the Universal Genetic Code OUON UNWERSAL ODE UNUSUAL GDP OCCURRENCE UGA Stop Trp Mycopiasma Spiroplasma mito chandria of many species ICUG Leu Thr Mitochondria in yeasts UM UAG Stop Gln Acetabularia Tetrahymena Paramecium etc UGA Stop Cys Euplotes Found in nuclear genes of the listed organisms and in mitochondrial genes as indicated sounce S Osawa et al 1992 Microbiol Rev 56229 Table 42 Molecular Cell Biology Sixth Edition 5 2008 W H Freeman and Company How the Genetic Code was deciphered Using synthetic mRNAs Bacterial extract Synthetic mHNA Polypeptide This was a sweet experiment Synthetic transcripts now have two different nucleotides with three distinct reading frames More experiments like this cracked the genetic code Transfer RNAs tRNAs dihydrouridine inosine ribothymidine 1 pseudouridine m methyl group attaches to 3 CCA Note Codon and Anticodon interaction is anti Qarallel Fig 4Zoa s I Figure 410 Molecular Ce Biology Sixth Edition A 39 2008 W H Freeman and Company K Amino acid covalent A tRNA has 3D structure 1 T ch 1009 Acceptur St l f l ml D ID p Variable loop Anticgdon loop Fig 420b tRNAs in Bacteria 3040 different tRNAs in bacteria Problem here Remember 61 functional codons In these orqanisms a sinqle tRNA mav recoqnize more than one codon within the mRNA How Answer Wobble base position base position the first base in the tRNA anticodon and the third base in the correspondinq mRNA codon Let me explain Recall codon and anticodon are antipaella Wobble Positio I tRNA AAG u n m 539 UUC 339 7 mRNA or So this particular UUUquot tRNA can bind two codons UUC or UU I UUC and UUU botr Wobble Position Encode Phe Nonstandard basepairing between G and U can occur 3 IRNA Base Pairing Rules for the Wobble Position 5 321 1 23 539 mRNA 339 539 mRNA 339 1 23 321 INOSINE IS OFTEN I FOUND IN THE 5 WOBBLE 3 POSITION CAU BUT NOT tHNA G Mostly FY Inosine If these bases are in first or wobble position of anticodon C d ICIAIGIUI I iLto G U C A C then the thN y U G A recognize co in U mFiNA havinm ser bases in third position T If these bases are in third or wobble position of codon of an mRNA I C l A G U G U C A l U G I then the action may be recognized by a tRNA having these bases in first position of anticodon Fig 423 Nonstandard basepairing that occurs in the wobble position n 5 W i Hc cvo c UIidine i u C CC NH LcMH Aduloime i u c 0 NvN a c H L N i n N c H C N H CH In 39 N C um i 1 1H Gunning sNCw C C Hm N C C E Hc o quotname I N cw c N Cy dine c D k 0M g cN N cNE g CH lnosin mi bMH Innslne s N s r m c The point is one tRNA can base pair with two or three codon For MCAT ers Note that inosine is the deaminated version of adenine TAB LE 4 1 The Genetic Code Codons to Amino Acids SECOND POSITION U C A G Phe Set Ty Cys U U Phe Set Tvr vs C Leu Ser Stop Stop A Leu Ser Stop Try 6 g L m is Mg U 3 One tRNA for these three 3 c Len codons encoding Leu wl a P 539quot 399 A E lnosine in the tRNA s wobl 2 Pro Gln Arg G 3 5 g pOSItlon E Ile Thr Asn Set U S 39 n Ile Thr Ash Ser C Ile Thr Lys Arg A M Thr Lys Arg 6 Val Ala Asp Gly U Val Ala Asp Gly C 6 Va Ala Glu Gly A I VaHMetP Ala Glu Gly G quotr39 A A quot 39 and Lm uiorvallne andCUGfor Innau Ma8393 J 39 J quot 39 nuinhiateapmtelnchain Tabletli Molecular Cl Biology Sixth Edition b J 2008 W H Fleeman and Company 41 tRNAs in Eukaggotes 50 100 different tRNAs in eukaryotes Remember only 20 AA s but 61 functional codons So in eukaryotes there are more tRNAs than codons Problem here Answer more than one tRNA can attach a certain AA to one codon One specific AA is said to have a few cognate tRN This will become clearll Let39s summarize Cognate tRNAs in eukaryotes tRNA Single tRNA2 gt Single Codon RNAa Now let s take a slight diversion Xray Crystallography Determined Precise Nucleosome Structure 07 T LO 939 LL 0 H E Remember eukaryotic DNA is supercoiled No gyrases in eukaryotes So how Answer One nucleosome introduces one negative supercoil as it wraps 146 bp of DNA nearly twice Eukaryotic Chromatin Organization In low salt buffers mM At physiological salt mM 30 nm solenoid fiber 11 nm beadsona string fiber Fig 628 The 11 nm fiber winds into the 30 nm fiber Linker DNA 1555 hp connects one nucleosome core to the nex H1 binds linker DNA often called the linker histone H1 lines the inside of the solenoid Structure even the existence of the 30 nm fiber recently called into question Chainof nucleosome 39 Twoustart 7 he llx la b Figure 630 Molecular Ee Biology Sixth Edltian gt5I 2003 W H Freeman and Company terminal tails of the core histone proteins project from lt These tails contain residues that interact with 1 DNA within the same nucleosome 2 linker DNA that connects neighboring nucleosomes E 3 DNA or grotein Histone Deacetylases These Lys residues undergo reversible aoetylation HDACs 1 aoetylation unwinds 30 nm fiber to 11 nm fibe c Eransferases 2 deaoetylation rewinds 11 nm fiber to 30 nm fi r HATS Many different HATS g n 1 The amino terminal tails of the core histoneI 39 q39 proteins project out 39 to make contact with the neighboring nucleosome Amino terminal tails can be reversibly acetylated mum mm umiwnsmsmm H g 6 3 1 zonaw H quotmm a n m mum A Ac AK u n 1 The 5 9 u U pm 119 Histone Code M M k m I I I I PEPAKSAPAPKKGSKKAVTKA AVSEGTKAVTKVTSSK 5 2 I415 20 120 differential ME osttranslationa f A REIAonrllgTDLnFQ df t f quot2 Nlle Melt Rf Mz AI Milne quot9 We ARTIEQTARKSTGGEAPSKQLATgAARKSAPATGGV KPH the histone tails 5 3 If 27 ocell type specific w Al AI 1quot SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT I Z 5 8 II X518 1 Phos ho Iation mark chromosome Mj p 39y I M Methylation mark SpelelC VKKKARKSAGAAK w 39 26 A Acetylauon mark developmental 26 I Uh Uhiqui nation mark speCIfICIty r Fla5164 Murmtmcm mmquot mm Edition 20st Freanmnanuimhpmy Dave Allis in green sweater celebrating his election to The National Academy of Sciences NAS Repiy rmm Dave Aiiis in my eengramiamiy Emaii Fume me NAS news came as a umpiae su rise and it has mare man a bit humming and WEMhEiming i feeifununate m a m recite in trum me best pan Dfitfurme was gemngme chance in re ect eri aii erme penpie andme great life it is to be an experimenzsi scientist itwas nice that me Turiy Let s Review and then Build a Chromosome Linker quotBeads onastring form of chromatin Spg gegocilgle 2E1m helix Gene is silent 30nm chromatin fiber of packed nucleosomes quotBeads onastring form of chromatin Gene is active Classic Experiment in 1976 with DNase Decondensed chromatin Condensed chromatin i W5 T BamHl DNase BamHl Bam HI DNase Bam HI 14day erythroblast MSB ggif faummmimmgquot Expresses globin gene cell type that does 2008 w H Freeman and Company M express globin gene DNA from 14day DNA erythroblasts from A MSB DNasepgml 0 0105 1 5 11515 39L u it r Mi globin v A V a oquot gene I in quot3 t39 quot v a Loading control 6 Southern blot showing globin gene Susceptible to Dnase in erythroblasts Fi mes32b MilecularCellBialagMSixthEdition o In o 2008 WHFreeman and Company nucleosomes packed chromatin fiber of 30nm i 300 nm Chromosome scaffold T Still making a chromosome Su mmary 235335 m Rule ofthumb 3911 m flbef 395 n a z a T transcrIptIonally actIve gggg d ma gg mm Wm 30 nm fiber is silent due to solenoid winding Ems summed 7mm Chromusama mum 30 nm fiber is cast into loops th are anchored by a matrix or am scaffold called the radial loop model of chromatin structure Lifs 39m l m 2minquot i Classic gure taken 39om the align Kama m competing text book Alberts et al EN AdoAblr Mitotic Chromosomes W H 39 histone proteins depleted One of the first indication of a radial loop model es oompoeeo w momahie tome pmteimeu FYI E iii id Picture by Ulrich Laemmli the SDSPAGE guy Fig 635 Scaffoldassociated regions SARS also called matrix attachment regions MARS are DNA elements that anchor the loops to the scaffold matrix F SARSIMARS are AT rich Loop of 307nm chromatin fiber Chromosome scaffold Fig 636 Two DNA segments genes on the scaffold could be millions of base pairs apart on the linear DNA sequence but closely spaced within the scaffold Fluorescent probes for these segments genes would labe defined regions of the interphase nucleus SARS are at the base of the loops perhaps to regulate insulate transcription in one loop from transcription in neighboring loops topoisomerases bind SARS to relieve torsional stress within the looped domain generated by transcription Varietv of Fluorescent Probes Label Chromosomes 7 and 8 in a HumanCeH Nuclear domains occupied by individual chromosomes 1 I i F iii 11 1 11 i A Ui iiiiwivf2 riilt however Figure 637 Molecular Celt Hiology Sixth Edition P TJURW H Freeman and Company Euchromatin versus Heterochromatin chromatin Figure 633a Molecular Cell Bialngy Sixrll Edition r9 zoos w H Freeman and Company Heierothromu n inmlivetondensed M 30 nm 3993 More Methylated H3 ARTKQTARISTGGKAPRKQLATKAARKSAPAT Notice Lys 9 is Trimethylated by HMT M93 3 ARTKQTARKSTGGKAPRKQLATKAARKSAPAT 27 Euchromulin dive open 11 n m A AI More acetylated H3 ARTKQTAREEFGGSAPRKQLATKAARKSAPAT ME A I H3 ARTKQTARKSTGGKAPRKQLATKAARKSAPAT 4 14 ngm 53 Mmmnrc new ma w H ny Forming Heteroch romatin I e H3K9 methyl transierase Aspecific histone methyl transferasg Mg m Me Me Me HMT trimethylates Lys 9 in H3 Heterochromatin Protein 1 Eggg39ggggi ntmmm HP1 then reads the Histone Code and binds to Me3H3 to H ms Me in m begin chromatin condensation Bound HP1S bind to lHPlnligomerizatiun themselves and to more HMT m M 3 ME 3 3 V3 Thus heterochromatin wumiv mv can spread down the a J J I chromosome is Lima pm I i w W miniruin din viii r Heterochromatin ngm 534 Mamimzenaiorogy mm Edltv39nn n2sz H Free and mum What serves as bounda elements between condensed Heterochromatin and decondensed euchromatin And how do they work LSU s David Donze in yeast And Craig Hart in Drosophil Me3 Ac Ac Ac Ac me3 Me3 me3 A 39 h t39 we c roma m Spreading of silenced and HP1 coated heterochromatin 11 nm mw H F nnnnnnn and mm n m Xchromosome Inactivation One of the two X chromosom in female mammals is mostly 37 174Ty393Pwpxaxa Luna2V 19 T HEW Lquot Li KERN ELquot ngl ab ULquot 39Jx w39i 3 l 1 L LCv g Called SIZE for inactive also Called the Random decision between Xp and Xm very early in embryonic development in Note the location of Xi just unlt1 RNA fluorescence the nuclear envelope in situ hybridization FISH shows Xist RNA as it binds to Xi Blocking iactor7 Way stations LiNEs7i Figure 5 A model for X inactivation The model indicates speculative roles of some of the proposed players in the initiation of X inactivation a Before inactivation Xisr RNA is expressed in an unstable form dotted red lines and the I 39 39 lupieguiaiiuii andor its association with the chromosome in as b i Xist RNA becomes upreguiated through stabilization transcriptional upreguiation or reiease oithe blocking factor LiNEs might participate in the spreading process in some way either W iii a Xist RNA Coating Xist or by a mechanism such as REPEATlNDLiCEDGENE 5 siLENlJNi RIGSi c i Stabilized Xisr RNA coats the m m mm M m m X chromosome before its inactivation d i Transcriptional Hg hg g i i Silencing of genes on the X chromosome occurs as a result of AVquot Xisr RNA coating LISing an unknown mechanism and is rapidly Egtabiighmem oi me followed by a shirt to asyncnlonous replication timing of the X New chromosome e i Chromatin modi cations such as the histone leacetylatlon and metiiylation of promoters of leinketl genes as well as the recruitment ottne histone variant macroHZA pi i i ii i WtheXi39stDMquot 39 a stany inactive and condensed chromatin state state asynchronous replication Histone H3 and H4 Wp ce y a Nuclear Elements retro transposons loMacroi lEArecruitinent Nonhistone Chromosomal Proteins Relatively low in abundance compared to the histones They include 1 proteins for transcription SSB DNA poly etc RNA polymerases activators and repressors later disc Transcription Factors 2 DNA polymerase for replication 3 High Mobility Group HMG proteins moderately abundant average 1 HMGnucleosome role in transcription Structural Functional later disc 4 Other structural proteins Topoisomerase etc a Hinge domain Structural Maintenance gnaw j Chromosome proteins domain SMC proteins Can hold two circular dnmain DNA molecules together Klein Chromatin loop containing Most interesting a transcription unit SMC proteins may reside in the chromosome scaffold and help establish the radial loops 30 nm 100130 nm chromonema fiber 200250 lllll middle prophase chromatid 500150 nm mataphase hromatid Chromosomes are 39 39 Telurnere at their most m WWWWMW condensed state W quot X39gm E t 3333 wmmmm during h je apS39gggg canquot39quot9 9 r0quot e W t WWW Micrograph of a 21m c nlei m53 Mitotic Chromosome Certain d es e Giemsa stain mitotic chromosomes in a banded attern 1 Banding pattern is unique for each chromosome ocate genes 2Allows clinicians to identify chromosomes and their aberrations 3 p petite the smaller arm g 641 q the larger arm just the next letter in the alphabet W Philadelphia chromosome 0 O a E der 22 22 Chronic Reciprocal W TranSIOCBtion Leukemiaa break occurs in a proto oncogene W now out of ega slzaa der 9 Chromosome Paintin Multicolored FISH Normal chromosome 9 quotPhiladelphia chromosome der 22 Normal chromosome 22 Paint probes specific The same paint probes now for chromosome 16 used to anneal to human of the tree shrew a chromosomes Long q primate distantly related arm of chromosome 10 to humans labeled In fact gene order is conserved on shrew 16 and human 10q referred to as 39 39 Primate ancestor Homo sapiens 11 12345 x 9 12 456 X 23 X 78 3 1 4 5 6 15 I I I U m i107H12H81314I15I6 21 910111213 7391011121314 m I D I I I I Is w w w 23 2 14 I D 2 22 H21 15 16 17 1s 19 20 21 22 23 15 16 17 18 19 20 21 22 MW111w m mamHtmmmnmumpw The various chromosome paints allowed us to propose a karyotype for the ancestral primate Aminoacyl tRNA synthetases link a specific AA to its cognate tRNAs Recall CCA3 end of tRNA it has a h ydroxyl group Amino acid Highenergy EPhBI ester bond H O H 0 I I II I ll HZN cc UH HEN CC O HzN C C O 39 CH CH CH 2 0H n 2 I 2 I Ph Net result 5 Linkage of tRNA 3 binds Phe is selected Phe to tRNAPhB to the UUU codon by its codaquot ATP AMP e gt PF Aminoacyi AAA AAA 39 AAA tRNA synthetase tRNA specific for AminoacyletRNA 5 RNA specific for Phe Phe tRNAPhei m 20 different aminoacyl tRNA synthetases Each enzyme is specific for only of the 20 AA s Fig 419 Aminoacyl tRNA Synthetases Once coupled to the appropriate M the tRNA is said to be activated or charged Synthetases have a proofreading function Stop and think about this for a second how important is um functionquot One synthetase can recognize the cognate tRNAs for a given AA Means that the enzyme recognizes some Identity site on the cognate tRNAs Identity site anticodon in part Other parts of the tRNA also contribute to recognition Ribosomes 39 Most abundant RNAProtein complexes in the cell 95 of all the rRNA v Machines nanoorganelles that 1 read the mRNA 2 bind activated tRNAs and growin peptide chains 3 catalyze peptide bond formation 3 5 amino acids incorporated per sec 100 200 per min 39 Two ribosomal subunits to make one functional riboson Large subunit LSU Small subunit SSU One intact functional ribosome contains 0 four ribosomal RNAs EUKARYOTES 0 83 ribosomal proteins in eukaryotes Ribosome Assembly LSU and SSU Subunit Aunmhlld Pmlawnlic as 1500 bum g 2xs 2 5115 a 135 r 5 35 as guano ha 1w bases me ba quot as won bum Total of 83 Similar to Fig 422 LSU assembly in the nucleolus in eukaryotes rRNAs 288 585 and 58 Proteins 50 large subunit proteins called L1 L2 L3 SSU assembly in the nucleolus in eukaryotesj rRNA 18S Proteins 33 small subunit proteins called S1 82 3 Ribosome LSUSSU E coi s16SrRNA PROKARYOTES 1 SU 20 Ribosomal RNAs ha conserved Peptidyltransferase secondary 7 Activity and tertiam Formation of the structures 1 eptide bond is a fxn c This Enzymatic function now attributed to the LSU 23S rRNA w nuclamides The bacterial ribosome at work 9 Polypeptide A site Aminoacyl LOW P site Peptidyl RESOIUtlon E site Ejection CryoEM 3 Fig 426 Crystallization of the Bacterial Ribosome for Xray Diffraction Studies c 305 p m ATruIy Amazing Feat in Structural Biology Fig 4 23 RNARNA helix Protein alpha Hm mmauwwnsmm quot he I IX WW H and WW Protein Synthesis 1 Initiation 2 Elongation 3 Termination Usually the first AUG codon is the start codon This first AUG sets the reading frame Two different tRNAs for Met COGNATE TRNAS 1 tRNAiMet for Initiation 2 tRNAMet for Elongation Only one aminoacyl tRNA synthetase for Met Cognate tRNAs Eukawoles ml Amhaeans gtrgt Bacteria CHO 1 AH cells gt initiation Protein Synthesis Initiation in Bacteria Only fMettRNAiMet can bind the P site at initiation MettRNAMet cannot at initiation only during elongation Note CHO formyl group Figure not in the book Protein Synthesis Initiation in Bacteria U 1 303 SSU plus Initiation Factors 1 Z and 3 form a pre initiation complex 2 Complex binds fMettRNAMet and the mRNA 3 ShineDalgarno I helps position 303 complex 4 503 LSU joins F2GDP pops off 5 fMettRNAi39Vlet in the P site 5 Ready for Elongation as mm imnunon Vaclav ursi Q row Freimuuion wmpiex KMRNAQMeiilRNAMH W v Mquot Shinzrbaiwamn I mum Q7 mm 305 mmauou compiex sus subuml bimlnrenmm As in prokaryotes eukaryotic Translation Initiation now in LSU and ssu are kept separae Eukanlotes prior to translation by gukaryotc initiation Eactors eIFs a vquot w 1 ma 1 I K E eiFE ternary 339 mpml ME 7 i SWEDEN m ft n i l 7 I iGTP AESmmnlex nrmaLiJn 3 39 39 1 u GW Q W3quot quotI saw 5 v u an MAL attiwaiinri I Alzat mmenzlo m thl 5 sequence CCAGCCAUGG Once SSU with its bound tRNAiMet 438 complex is in place it is released from eF4E 4E le all alone SSU begins to scan looking for s sequence AUG w elF4A is an RNA that l Lt11323333323a and P release unwinds any stemloop structures M Once SSU finds initiation AUG elF2 5 t P hydrolyses its bound GTP elF5 is the 315 lacturdlsplacement 4 Game causing complex to change shape now 7 Hydrolysis oi DlF Bbound GTP and LSU and 5BGTP join as most early elFs release 55 5B hydrolyses GTP to GDP 1A pops off oReadv for elonaation note sites and now Translation Initiation in Eukaryotes elF6 LA 1 ll LSU 6 and SSU 3 ar kept separate prior to translation by gukaryot c initiation factors 6 and Fig 424 eF1A ternary complex k eIF2GTP MettRNAi39VW Jt Preinitiation complex Fig 424 Recall the 7methylG cap AUG AAA 39IAA ISeveral subunits in eF4 AAA Initiation cplex Figure 421 part 3 Mofeculur Cequot Eiaiugy Sixth Edition many 2008 W H Freeman and Con Met GTP He g AUG AAA n Q i Initiation cmplex 2 structure ATP unwinding I scanning and ADP pi Start 3 elF1A elF3 elF4 complex recognition eF2GDP Pi Met AAAns39 AAAn 3 608 subunitelF6 elFEOGTP elFB elF50GDP Pi AAAn 80 riosome Fig 424 Eongation Elongation Factors EFs facilitate ME How does the ribosome know to bind aminoacyltRNA 2 instead of 3 or 4 in the A site 803 ribosome G e GTP Entry of next n y39GTP e A site aatRNA at GTP Ans The codon in mRNA is ii the bottom of the small SUbL This determines the next pro tRNA39 Fig 425 top Elongation Step 1 PeptidyltRNA already in P site change GTP hydrolysis ribosome 2 7 conformational 39GDP Pi Step 2 Aminoacyl tRNA arrives in the A site complexed with EF1aGTP Fig 425 upper middle Elongation Step 3 Peptidyl transfer Peptidyl transferase activity is a function of the 288 rRN A Recall ribozyme Peptide bond formation E Fig 425 lower middle Elongation Amino group of incoming AA attacks bond of the PeptidyItRNA in the Psite Ester linkage H O R 39 H N I C N IZZCILI I C H2N Cl C 2 l I ll H 0 R1 H H 0 R3 aminoacvl tRNA peptidyl tRNA attached tFlNA molecule to Cterminus of the freed from growing polypeptide its peptidyl chain linkage LJ i Asite P site Figure 6 61 part 1 of 2 Molecular Biology of the Cell 4th Edition Fig taken from rival text book I like this figure Elongatio n Notice the peptide chain has been temporarily transferred to the A site EFZIGTP Ribosome translocation EF2GDP pi Step 4 Ribosome shifts to the right By one codon while the tRNAs V remain bound to their respective codon Free tRNA in E site is now ejected A site again open ready for next round Fig 425 bottom Termination like tRNAs mimicry Enter A sit In Bacteria RF1 or UAG UAA UGA In Eukaryotes RF1 Once RF1 or RF2 binds A site RF3GTP binds and provides enerqv to cut the last peptidvl tRNA bond by GTP hydrolysis PeptidyItFiNA cleavage I eRF l eFiF339GDP P Fig 427 Polyribosomes and PoyAbinding protein PABP1 interacts with eF4G v quotquotquotquotquotquot at for efficient use of x ribosomes so cooll Fig 428b Atomic Force Microscopy Maktularfell ialngnslnhldilfau and Cnmpany rum tan n 2063 w H Freeman RNA Interference RNAi a In vitro production of doublestranded RNA Sense transcript Antisense transcript 8 E N S E E S N E S Selectively block expression of a particular target mRNA Special enzymes recognize and cleave the dsRNA Short RNA products from this cleavage anneal with the target mRNAs that are then cleaved Fig 545a RNA Interference RNAi Targeted RNA degradation A defense mechanism foreign dsRNAs virsuses s dsRNA transposable element 39quotte39mediates Found in fungi plants nematode worms ies mice probably human Think of the eukaryotic Model Organisms an ancient mechanism RNAi Mechanism 1 Free dsRNA attracts a protein complex a nuclease activity b helicase activity make dsRNA to ss 2 Protein complex DICER cleaves dsRNA into small 23 nt fragments small interfering RNAs siRNAs 3 siRNAs then associatedwith the RISC enzyme complex acts as a catalyst to degrade more dsRNA with complementary sequences secondary RNAs could be dsRNAs RNAi Mechanism 4 Small 23 nt fragments can be ampli ed and transmitted to neighboring cells or daughter cells RNAi first used to study gene function in nematode worms C elegans Andy Fire at the Carnegie Inst of Wash Dept of Embryology Baltimore M Now at Stanford Medical School Craig Mello working in Arabidopsis Share 2006 Nobel Prize in Physiology or Medicine Fire Introduced dsRNA into C elegans by 1 Injection into intestines or simply 2 Feed worms E coli expressing dsRNA rA B C D lgtCDNA D C B A Transcribe inverted A B C D CDNA segments IIIIIIIIIIIIIIIIIIIIIII A B C D This dsRNA is not endogenousintroduce it in some manner In situ hybridization showing RNAi eliminated the presence of a particular particular mRNA no mRNA In situ hybridization signal Noninjected Injected r RNAi is known to work well in all eukaryotes test Regardless of which Model Organism usedig 545b DICER nuclease that specifically cleaves double stranded RNAs into siRNAs of 2123 nt Delivers them to RISC RISC EMAinduced silencing complex contains an Argonaute protein Argonaute protein with functional peptide domains RNAi now an extremely powerful tool to knock down expression of a particular gene product Chapter 6 Genes Genomics amp Chromosomes Read pp 215266 Up to 229 for Test 2 The Gene The Physical and Functional Unit of Heredity Cell Biologist s De nition of a Gene the entire DNA sequence necessary for the production of a functional protein or RNA mRNAs that encode proteins ribosomal transfer and other functional RNAs as well v includes noncoding transcriptional control regions promoters includes introns and exons includes enhancer seguences that could lie some distance awaf y ro th t m 9 promo er39fh ei er ups39 39t re d am or owns39ram Fundamental Differences between Prokaryotic and Eukaryotic Gent Recall the operon and polycistronic mRNAs in bacteria In Eukaryotes monocistronic mRNAs Simple transcription unit 50 kb k gt b Cap me C d PolyA Slte a Gene Control regions a mRNA 539 war Es39 logy sum Edm39on a umvany Fig 632 Introns Extremely rare in prokaryotes Yeast have introns but not as prevalent as in higher eukaryotes Higher animals and plants have introns Alternative splicing of a single premRNA yields different protein products Alternative Splicinq of Complex Eukaryotic Genes b Complex transcription units Cap site a b l C d PolytAi i xiv G r i7 ene Exon i Exun 2 Exon 3 Exam 4 internal mFiNA splicing 0 mHNAg 539i 1139 Cap site PolyiA PoyA a b c d e 439 Jr W i i f Gene I7 w11mm 1mm Exon 1 Exon 2 Exam 3 51rquot n m 3 3 ends or N side sa 3 mRNAg 511 Emmy v Cap site Cap site f d g e PolyA1 r Gene EXOH 1A quotEian 1B Exon 2 N Exon 3 5 ends mRNA1 52222222139 quotquotquotquotquotquotquotquot 13 I 1 A I C Slde sa 6 mRNAz 5mmquot 113 F39g 6393b Different Eukaryotic Genome Organizations 80 kbp region of human genome lots of nonfunctional DNA T32 and I B1 are noncoding members of the family pseudogenc Pseudogenes do not have a promoter you cannot initiate these ge Alu sequences are short 300 bp repeat elements 3 Human Bglobin gene cluster chromosome 11 ll Exon I Pseudogene TAIL site I we e GT A t a 3 TT bl S cerevisiae chromosome Ill rR gene mmmmi mm ii iii ill lillll lll H ill 80 kbp region of S cerevisiae genome very little nonfunctional DNAnoncoding Yeast genes are tigth linked Human av a lot of seperation Fh39g39 96 4 The Cvalue Paradox Sizes of various genomes yeast 0015 pghaploid genom fruit fly 015 pghaploid genome chicken 13 pghaploid gen m human 32 pghaploid genome Phylogen Qvalues But many amphibians and plants have more DNA per haploid genome than does human North American newt 50 pghaploid lily plant 100 pghaploid This is the paradox why the C values do not correlat with phylogeny Why is there so much nonfunctional DNA Most of it is repetitive DNA It is spacer DNA TABLE 61 Major Classes of Nuclear Eukaryotic DNA and Their Representation in the Human Genome DP MEMBER IN HUMAN GENOME FRACTION OF HUMAN GENOME 96 USS ENGTH Proteinacoding genes 052200 kb 2 5000 Tandemly repeated genes U2 snRNA 61 kb 20 rRNAs 43 kh HMilli Repetitinus DNA Simplesequence DNA 1500 hp Variable Interspersed repeats imalbiie DNA elements DNA transposons 23 kb 300000 LTn retrotlransposons 6 1 1 lib 440000 NonUR retrutransposons LIMEs L1 element 68 kb 850000 SINEs AIU element 100 400 bp 1500000 Processed pseudogenes Variable 1 and 00 Unclassified spacer DNA5 Variable na quot55 13quot lt0001 04 6 w 1 3 04 15 Complete transcription units including introns i Transcription units not including introns Proteincoding regions exonsi total 1 1 of the genome Length of tandemly repeated sequence 5 Sequences between transcription units that are not repeated in the genome me not applicable saunce International Human Genome Sequencing Consortium 2001 Nature 409860 and 2004 Nature 431931 Table 61 Maiecuiar Ce Eulogy Sixth Edition 390 2008 W H Freeman and Company 3 interesting facts only 11 of the human genome encodes protein A lot of the DNA is made of the repeative DNA Alu element 13 Alu sequences are short 300 bp repeat elements L1 line elements 21 Just one example of a solitary gene The chicken lysozyme gene Solitary genes comprise 2550 of haploid genes not DNA 50 60 Rb Spacer DNA A Spacer DNA Lysozyme No mRNA r l 1 i Miwmi i gt Blue exons Tan introns Red arrows Alu repeat sequences Gene Families Genes that encode functionally related homologous proteins or RNAs Gene duplication Bglobin gene L1 5srsz39somesf x Recombination unequal crossing over Elglobin gene Bglobin gene Recombinant quot quot 39 chromosomes Figure 62 Molecular Cell Biology Sixth Edition quot 3008 WVHVFreran and Company L1 Line 1 repetitive element Unequa crossing over resulted in the initial duplication of an ancestral globin gene Duplicated genes then diverged initiating a f rgil qb Other gene families include those that encode 1 protein kinases 2 immunoglobulins lGGlGAect 3 tubulin 4 intermediate filament proteins 5 actin Remember that we are talking about genes not DNA Subtle differences in function between proteins within a family a globin versus B globin Celltype speci c expression of particular family member genes Tandemly Repeated Genes o encode identical nearly identical proteins or functional RNA provide abundant gene product for growingdividing cells Examples Tandem repeats Preribosomal RNA genes in the nucleolus 5S ribosomal RNA genes outside the nucleolus Genes that encode histone proteins wrap up genomic Dl Genes that encode tRNAs and small nuclear RNAs Tandemly repeated rRNA genes Nascent prerRNPquot PrerRNP nascent Ribosomal RNA complexed With processing proteins Transcription unit l l Notice 5 Nucleolar J 39 393 chromatin It inquot 1 Nontranscribed spacersQ 3 quotNo lfa39nacribed p g V 9539 I sspacer 2 Many RNA Pol I complexes transcribe one gene at the same time Called a V Transcription Unit TU quot32a Transcriptionquot uniftg 39 Direction of tr Renaturation Kinetics chop genomic DNA into small pieces heat to denature then allow to reanneal over time A function of initial DNA C0 X time ssDNA Highly repetitive DNA 1015 of genome AszSO nm Referre d to as Middle repetitive Cot 2540 of genom curve 0 dsDNA Single copy 5060 o of genoine C0 multiplied by time Cot Highly Repetitive DNA 1015 of the mammalian genome o simplesequence DNA Centromeric and Telomeric DNA very short sequences repeated over and over in tandem Middle Repetitive DNA 39 2540 of the mammalian genome large number of copies tandem repeated genes prerRNA and histone genes mobile genetic elements Line elements and Alu elements Single copy DNA bulk of our genes that encode proteins 5060 of the mammalian genome contains single copy genes but only 5 of the entire genome actually I Satellite DNAs r Cesium Chloride CsCl isgu yy density gradient Heavy sali Repetitve That dissolves In water f 3 39ilite DNA AT rich Light band Main Band DNA 1 Satellite DNA GC rich Heavy band Equilibrium densitygradient centrifugation Satellite DNAs in a Chromosome Telomeric DN Centromeric DNA at Telomeric DNA the primery protects the end constriction of Just one element Chromosomes m Over again to give on long stretch Telomeric ON Here the chmmosames b Manse K w 539 te acemtriicg I Figure 6 6 Molewl nr Ce Bio09y mm moan 1111 W N fEL39II EJH and fIquotIJ n39 E H if ff 1 3 v c 59quotquot 4129 9 quot 9 3 1 f fg i rLgr 7 77 FHMQM gsxxgemu I31 Hm gmqu U137OJI JQMJQSJth 11 KO n lt9 439 Minisatellite seguences 31 mime Example of Single nucleotide polymorphism M A 335 minisalellite Red residues represent sequence differences between people Now used to fingerprint an individual bc minisatelites are different with different people Distinguishable PCRs to Fingerprint People with Different MiniSatellite la Paternity determination MCF1F2 i 4 4 Figune 64 Molecular Edl inlagy ZUOG W Hinge I I Sluh Edr39rmn mar a 1d Company Is Criminal identifimlion Victim Specimen I 0 P l M Suspects LU Each lane represents a single PCR reaction that used many DNA primer sets Forward and Reverse primers Each band in a lane represents a separate PCR product Primer sets amplified previously characterized minisatellites that are known to differ in length among different people Minisatellites simple sequence repeats can be Polymorphisms Each lane is a Fingerprint m w ncE 8E 29 Qwh E3 55 x33 Llon n o E U Simplesequence tandem array f Parental chromosomes X equal tandem m arrays lMeiotic recombination Germcell m chromosomes unequal tandem m Unequal crossing overcauses changes in the number of minisatellite sequences arrays Xeiotic division Germ 8 unitarray cens 4unitarray Fig 106 a Normal replication Another way of creating SSR polymorphisms t El SSR Simple Seguence Repea mm It Inlkwnrd slippage E3 3 m During DNA 339 momma s39 replication new daughter strand slips backwards 15mm replication t Semnd replitnlinn Daughter DNA with one extra repeal Normal daugmer DNA 539 mammal 239 239 mmmm 539 Figure 65 Muluulnr ellaiolvgyrsinh Edition rizmw H reman and omvnny 262011 73400 PM Chapter 1 Life Begins with Cells 1 Four aspects of the cell theory 0 All living things are composed of cells and their products Schleiden and Schwann 0 Cells are essentially alike in chemical constitution 0 All cells come from cells Omnis cellula e cellula Virchow 0 The activity of the organism as a whole is the sum of the activities and interactions of essentially independent cell units 2 Archaea are in between prokaryotes and eukaryotes in terms of evolution Fig 13 0 Eukaryotes and Archaea diverged from bacteria before they diverged from each other leading to similarities between archaea and eukaryotes 3 Halophiles vs Thermophiles vs Methanophiles 0 Halophile 7 High salt environment 0 Thermophile 7 High heat environment 0 Methanophiles 7 very low 02 environment reduce C02 carbon dioxide to make CH4 methane 4 What is the nucleoid in a prokaryotic cell 0 Nucleoid 7 irregularly shaped region within the cell of prokaryotes where nuclear material is localized no membrane nucleus 5 What important internal features distinguish eukaryotic cells from prokaryotic cells 0 Extensive internal membrane systems bilipid layer such as isolated nucleus golgi apparatus lysosomes etc 6List the names and functions of the various eukaryotic organelles Would you consider a ribosome an organelle What about the nucleolus inside the nucleus 0 Nucleus 7 contains most of cell s DNA enclosed by envelope consisting of two membranes 0 Mitochondria 7 inner and outer membrane produces most of the cell s energy ATP matrix contains DNA RNA and ribosomes 0 Rough ER 7 synthesis of membrane proteins and secretory proteins Rough because of ribosomes on membrane 0 Smooth ER 7 No ribosomes on membrane synthesis of lipids 0 Golgi apparatus 7 Transport and modification of membrane and secretory proteins 0 Peroxisomes 7 degradation of fatty acids and amino acids 0 Lysosomes 7 animal cells only Degradation of old organelles and foreign materials 0 Chloroplasts 7 plants only photosynthesis light and dark reactions contains DNA and RNA 0 Vacuoles 7 most plant cells large uid lled organelles store nutrients and waste materials 0 Cytoskeleton 7 cell shape and strength organelle movement 7 What are the three components of the cytoskeleton and what proteins make up these components 0 Microtubules 7 made of tubulin 0 Intermediate Filaments 7 made of IF proteins 0 Microfilaments 7 made of actin 8 Review the model organisms and what features each is good for 0 Budding yeast 7 SaccIaromyces DETEVIiS39iaE O O O O O 0 control of cell cycle and division protein secretion and membrane biogenesis function of cytoskeleton cell differentiation aging gene regulation and chromosome structure 0 Roundworm 7 CaenorIabdittls elegans O O O O O O 0 Development of body plan Cell lineage Formation and function of nervous system Control of programmed cell death Cell proliferation and cancer genes AgingBehavior Gene regulation and chromosome structure 0 Fruit Fly 7 Drosoplz a melanogaster O O O O O O O 0 Development of body plan Generation of differentiated cell lineages Formation of nervous system heart and musculature Programmed cell death Genetic control of behavior Cancer genes and control of cell proliferation Control of cell polarization Effects of drugs alcohol pesticides 0 Zebrafish 7 O 0 Development of vertebrate body tissues Formation and function of brain and nervous system 0 Birth defects Cancer 0 Mice 0 Development of body tissues 0 Function of mammalian immune system 0 Formation and function of brain and nervous system 0 Models of cancers and other human diseases 0 Gene regulation and inheritance 0 Infectious diseases 0 Arabidopst39s Italiana 0 Development and patteming of tissues 0 Genetics of cell biology 0 Agricultural applications 0 Physiology 0 Gene Regulation 0 Immunity 0 Infectious disease plants 0 Viruses 0 Proteins involved in DNA RNA protein synthesis 0 Gene regulation 0 Cancer and control of cell proliferation 0 Transport of proteins and organelles inside cells 0 Infection and immunity 0 Possible gene therapy approaches 9 What s an ES cell what features does it possess in terms of mammalian development and why is it in the news How do you obtain ES cells 0 ES Embryonic Stem cells ES cells are totipotent can differentiate into any type of cell ES cells are obtained through extraction of the inner cell mass from the blastocyst in human embryos destroying the embryo which raises ethical debates Can be used for nuclear transplant technology cloning sheep cows etc 10Ofthe principal macromolecules which has the most diversity in terms ofstructure and function 0 Proteins are the most dynamic of the macromolecules serve as enzymes directors of motion hormones cell shape antibodies carriers etc 11 What macromolecule indirectly regulates the synthesis of all macromolecules 0 DNA through transcription and translation of genes in the cell 12Who were Watson and Crick Franklin and Wilkins Meselson and Stahl 0 Watson and Crick were the first to propose the double heliX structure of DNA using the Xray crystallography of Rosalind Franklin Maurice Wilkins was awarded the Nobel Prize for Physiology or Medicine along with Watson and Crick Meselson and Stahl proved the semiconservative model for DNA replication one original strand acts as a template for a newly synthesized strand 13 What s a karyotype 0 A karyotype is the number and appearance of all the chromosomes in a specific organism 14 Review the eukaryotic cell cycle What s Go Roughly what percentage of the cell cycle is mitosis 0 Cell cycle starts in G1 proceeds through S phase and into G2 before continuing to mitosis G0 is the cell state without division such as brain cells Roughly 10 of the cell cycle is mitosis Chapter 2 Chemical Foundations 1We said that water ions and small molecules make up 77 ofa cell s mass What makes up the remaining 23 0 The remaining 23 is made of macromolecules proteins mitochondria etc 2 Electronegativity to explain polar versus nonpolar covalent bonds 0 Polar covalent bonds 7 atoms making up the bond are Slgmficantlydifferent in their electronegativity electrons orbit one atom more often than the other leading to partial charges Nonpolar bonds 7 electronegativity of atoms in nonpolar bond are very similar electrons orbit each atom evenly roteinDNARNA interactions ionic bonds 0 Electronegative atom is attracted to nonelectronegative atom Cl 9 H hydrogm bonds 0 A H atom is covalently bonded to more electronegative atom 0 such that the bond is polar The partial positive charge on the H allows for a partial negative charge on another molecule to attract it by electrostatic interactions 3van der Waals and hydrophobic interactions Figs 210 and 21 are good 0 Van Der Waals 7 occurs both polar and nonpolar molecules electron distribution in shell makes molecule have slight dipole forming interaction between molecules over a very brief period Hyrdophobic interactions 7 deals with energy required for inserting a nonpolar molecule into water no hydrogen bonding with nonpolar molecule Surrounding water is forced to resume a rigid structure around nonpolar molecule energetically unfavorable Thus the molecules aggregate together by van der Waals forces leading to higher entropy of water energetically more favorable 4 Fatty acids Fig 221 versus Triacylglycerols page 48 versus phospholipids Fig 220 0 Phospholipids 7 necessary building blocks for biological membranes amphipathis hydrophobic and 7philic39 hydrophobic fatty acid tail 9 hydrophilic head glycerol 9 phosphate 9 alcohol 5 Know the 20 standard amino acids TF Righthanded D amino acids make up proteins 0 False All proteins used for protein synthesis is Lamino acids 0 Test Q All proteins soluble in water Yes C00 and NH3 are hydrophilic 6 Which amino acid forms disul de bonds 0 Cysteine forms disul de bond through oxidation only amino acid that has sulfur group 7 What happens when you acetylate the side chain of Lysine Lys 0 When lysine is acetylated you lose the charge on the R chain of the amino acid 8 Know the nitrogenous bases A T G C and U 0 Purines Adenine and Guanine double ring structure 0 Pyrimidines 7 Thymine Cytosine Uracil Single ring T found in DNA U found in RNA 9 What s the di 39erence between a nucleoside and a nucleotide 0 Nucleoside 7 Nitrogenous base sugar no P04 0 Nucleotide 7 Nitrogenous base sugar phosphates l 2 or 3 Ps 10Glucose and ring closure What s the difference between alpha and beta glucose 0 Glucose 9 glucopyranose much more stable than glucofuranose 0 Alphaglucose 7 OH on C1 down Opposite of CHZOH Betaglucose 7 OH on C1 up 11Reaction rates and chemical equilibrium Why is the K3 useful 0 Catalyst speeds up both forward and reverse reactions by speci cally binding to transition intermediates but does not change Keq or AG 0 Kd is the dissociation constant 7the point where half DNA is bound to protein and the other half is free The Lower the Kd the higher the af nity and vice versa 0 Steady state vs equilibrium Steady state is the state where the product formed is rapidly consumed to form new products Steady state is where the concentrations remain constant 12De nition of pH both the mathematical formula and what the formula means 0 pH log H Example H of water is lxlO7 so 7loglx107 7 pH of water 7 13 HendersonHasselbalch equation and what it means regarding biological buffers pKa What is the middle pKa of phosphoric acid and why is that one so important 0 pH pKa log AHA 0 When half of the acid is dissociated the pH pKa o pKa is de ning that particular organic acid Descibes where the equilibrium is 0 HendersonHasselbach explains how buffers work 0 Example Acetic acid 7 pH dos not change much around the pKa 90 of molecules dissociate pH goes up 1 unit 90 of molecules undissociated pH lowers 1 unit 0 Cell pH is 72 Phosphoric acid is the intracellular buffer because it s middle pKa 72 14 Positive and negative AG39s ATP hydrolysis to drive coupled reactions forward 0 AG AH 7 TAS 0 Cell constantly couples endothermic to exothermic reactions Hydrolysis of ATP to ADPP04 is usually the exothermic rxn a cell couples to most endothermic rxns Energy mostly comes from P04 bonds in ATP Catalyst simply stabilizes intermediate products in rxn thereby lowering the Activation Energy and speeding up the rxn Chapter 3 Protein Structure and Function 1Know what atoms make up the peptide bond and how these atoms contribute to secondary structures Fig 33a 0 Carbonyl oxygen and amide nitrogen that make up the peptide bond are electronegative Amide N binds to carboxyl C kicking off H2O in the process condensation o NaC bond is the phi bond 0 aCCO bond is the psi bond 0 Alpha helix is a series of H bonds through the Carbonyl O and the amide N located 4 peptide bonds apart AHelix has a dipole top being amide N and bottom being carbonyl O 2 Primary secondaryFigs 34 35 and 36 tertiaryFig 38 and quaternary structures of proteins 0 Primary 7 speci c AA sequence contains al the information on how a protein folds into 3D conformation AlaLeuProetc 0 Secondary 7 alpha helix and beta sheet makes up 60 of 3D structure Alpha helix series of H bonds stabilize structure Carbonyl O bonded to amide o N every four peptides R groups point outward don t contribute directly alpha helix has a dipole with carbonyl O pointing down amide N pointing up 0 Beta sheetstrand 7 58 AAs per strand H bonds between bacbone amide and carbonyl groups hold one strand to another R groups project above and below the sheet and aren t involved in stability could be parallel or antiparallel 0 Reverse turn 7 Glycine and proline often found in Reverse turns 0 Random coil 7 connects alpha helices and beta sheets no defined structure 0 Tertiary 7 overall folding of chain into a specific 3D structure hydrophobic interactions are main driving force 0 Quaternary 7 one protein interacts with another protein by same set of interactions used for tertiary structure 3 Protein motifs and domains Fig 39 0 Motif 7 region of the protein that contains various secondary structures Also has its own defined function o Leucine Zipper 7 dimerization motif for several DNA binding proteins 0 HelixLoopHeliX Loop domain binds calcium used to bind DNA 0 Zinc Finger 7 Binds DNA Zn ion coordinates structure of motif 0 Domains are usually larger than motifs could contain several motifs 0 Domains are duplicated for the same reactions in different proteins 4 What interactions help fold proteins Section 32 ofChapter 3 but Fig 212 is good 0 Important interactions include o Hydrophobic interactions 1 Hydrogen bonds Ionic interactions Disulfide interactions cysteine O O O 0 Van Der Waals interactions 0 Proteins fold incrementally and each step is thermodynamically more favored than the previous 5 What other proteins help fold proteins and how do they work Fig 316 and 317 0 ChaperonesChaperonins 7 family of proteins that facilitate the folding of most cellular proteins by binding and stabilizing unfoldedpartially folded proteins preventing degradation Chaperone is an individual protein where a chaperonin is a large group of proteins 6 What mechanisms are used by cells to degrade proteins Section 34 cf Chapter 3 Fig 329 0 Extracellular proteases o Intestines contain trypsin and chemotrypsin o Endoproteases cleave within the protein basic or hydrophobic AAs o Exopeptidases cleave from ends of protein carboxy starts on C terminus amino starts on N terminus 0 Intracellular o Lysosomal pathway I Hydrolytic enzymes operate at pH 5 if lysosome lyses enzymes won t function at cell pH 72 o Cytosolic pathway I Ubiquitin pathway 7 Three enzymes I E3 7 most important recognizes target protein to degrade covalently linking target to ubiquitin El 7 binds ubiquitin to E2 E2 7 interacts with E3 transferring Ub to target protein a n n Ubiquitin is never destroyed reused over and over Target proteins contain target sequence to direct ubiquitin I PEST sequences 7 Proline P Glutamic acid E Serine S Threonine 7 Enzymes kinetics basics Activation energy what enzymes do and don tdo Fig 320 Basic definitions of Vmax Kcat and Km pages 8081 You should be able to recognize the MichaelisMenton equation and have an idea of what it39s describing 0 Enzymes speed up both forward and reverse reactions by lowering the Activation energy necessary for the rxn Enzymes do NOT alter the Keq or the AG of the rxn 0 VmaX 7 measures the maX velocity of rxn when substrate concentration S is saturating with respect to what the enzyme can handle Kcat7 rate constant the number of substrate molecules processed per enzyme molecule per second turnova number 0 K1117 Michaelis Constant measures the affinity that an enzyme has for its substrate analogous to Kd 0 MichaelisMenton eqtn 7 Velocity v VmaXSSKm 8 Methods for protein puri cation Section 36 of Chapter 3 o Ionic and nonionic detergents o o What does SDS do to proteins Does SDS break covalent bonds 0 SDS interacts with hydrophobic side chains to unravel protein giving the protein an overall negative charge Does not break 0 Centrifugation Differential vasus ratezonal Electrophoresis Differential centrifugation 7 separates particles according to mass heavy particles 0 clump at bottom while lighter particles stay suspended in supernatant Ratezonal centrifugation 7 uses gradient to separate particles based on layers of 0 gradient sucrose percoll o Isoelectric focusing and SDSPAGE What does isoelectric mean SDSPAGE separates proteins primarily based on their size SDS denatures and 0 places negative charge on proteins Proteins migrate toward the pole The smaller the protein the farther through the gel matrix it will move 0 Isoelectric point is the pH where the protein has a net neutral charge 9 Methods for protein puri cation continued 0 Column chromatography 0 a gel ltration separation based on size of protein small particles trap on beads large proteins ow through 0 b ionexchange separation based on charge of protein Basic proteins ow through but acidic proteins bind to gel bead dissociated by adding NaCl 0 c affinity chromatography separation based on a nity to the antibody antibody binds specifically to acidic protein then dissociated by adding low pH 0 Western blotting What is it goodfor 0 Used to determine expression of proteins in different types of cells Hydrophobic interactions 0 Radioactive isotopes Which isotope would you use to label a piece of DNA A protein during its synthesis in the cytoplasm Radioisotopes can be introduced into a molecule and tracked by its radioactivity O emitted Use P32 to label DNA Phosphorus in nucleotide Use Sulfur35 to label protein 10What is a PulseChase experiment Describe how Meselson and Stahl s classic experith O was actually a pulsechase experiment 0 PulseChase experiment 7 0 Step 1 Pulse Incubate cells for NS mins with 35SMet radioactive Step 2 Wash cells to get rid of excess 35SMet Step 3 Chase Add back Met NOT radiolabeled All proteins labeled in the 5 min period can now be observed osttranslational O O O modi cation secreted localized to organelle 0 MeselsonStahl experiment used the same general technique They radioactively labeled DNA in a short time frame then tracked each labeled strand of DNA in its replication 1 1Physical biochemistry techniques 0 Timeof ight mass spec and its relationship to proteomics o TOFMS Laser ionizes particles off target As detector is hit by particles mass and charge of the particle is determined lightest ions arrive at detector rst allowing one to identify the composition of the target particle 0 Cryoelectron microscopy Lowlevel resolution of proteinparticle structure Protein frozen in liquid helium and examined computers help determine 3D structure 0 NMR spectroscopy Strong magnetic elds used to change spin on atoms effect is detected in neighboring atoms Distance between atoms is determined then structures determined 0 X ray crystallography Xray generated and sent through protein crystal the beam diffracted to detector and computer translates reading Determines the precise 3D structure by taking tangents to atom Chapter 4 Basic Molecular Genetic Mechanisms 1 RNA versus DNA structures and chemical differences Which is more stable DNA or RNA Why see Fig 46 0 B form helix versus A form versus Z form helix Fig 44 0 Bform DNA has a major and minor groove This is the DNA found under normal conditions in the cell Aform DNA can be produced from Bform under 70EtOh very low humidity 0 Z form is arti cially created in the lab however many gene promoters are G C rich Z form is characterized by alternating G and C in the two strands 2 How do most proteins bind DNA 0 Most proteins bind to DNA in the major groove via H bonds and van der Waals interactions 3Melting dsDNA What is the Tm What does the GC content of DNA do to Tm Fig 47 0 Tm of dsDNA is dependent on the base composition the higher the G C content 3 H bonds the higher the T111 4 What is supercoiling What introduces supercoils in bacteria Fig 48 0 Supercoiling occurs in living cells to pack all of the genetic DNA into a very small space Gyrases direct supercoiling in bacteria Example questions Question number 6 below doesn t list the structures but you get the idea 1 The cell theory has four parts to it Who stated All cells come from cells Omnis cellula e cellula A Lodish B Schleiden C Schwann D V irchow E Watson 2 Which one of the following choices is FALSE The archaea are important for the study of cell biology because A they diverged from bacteria after bacteria diverged from eukaryotes B they diverged from eukaryotes after eukaryotes diverged form bacteria C they share many similarities of both bacteria and eukaryotes D many of them afford us the chance to see how life survives in harsh environments 3 Which one of the model organisms best serves to explain the differentiation of the human immune system A The bacterium Echen39cia 001139 B The yeast Saccharomyces cerewsiae C The round worm CaenorIabditt39s elegans D The fruit y Drosoplzila melanogaster E The mouse M115 1111150111115 4 Of the macromolecules within the cell which class is considered to be the most dynamic in structure and function A Polysaccharides B Proteins C RNA D DNA E Lipids 5 Of the macromolecules within the cell which class indirectly regulates the synthesis of the others A Polysaccharides B Proteins C RNA D DNA E Lipids 6 The structure drawn on the left is while the one on the right is A alanine glycine B leucine lysine C serine cyteine Know your amino acids and nucleotides D valine proline E phenylalanine tyrosine 7 The most important characteristics in folding a protein into its proper three dimensional structure is A hydrogen bonds that form between seiine side groups and glutamine side groups B the energetically unfavorable effects between hydrophobic amino acid side groups and their surrounding aqueous environment C ionic bonds that form between glutamic acid and arginine residues D the addition of ubiquitin to the protein followed by proteosome interaction E disul de bonds that form between cysteine residues 8 Which one choice below is FALSE Phosphoglyceiides A are the principle class of phospholipids within biological membranes B consist in part of a glycerol backbone and two long chain fatty acids C consist in part of a phosphate group coupled to a polar or charged head group D are said to be amphipathic E contain a ceramide linkage We actually have not talked about this yet 9 Which one choice below is TRUE A Enzymes increase the equilibrium constant Keq for a reaction B The equilibrium constant Keq is the same as the initial forward reaction rate C Equilibrium for a particular step in a series of biosynthetic reactions is the same as statesteady D pH pl when half of the weak acid HA has dissociated into H and its cognate base A E pH logOH 10 Which of the following is used to buffer the cytosol within cells A Hydrochloric acid HCl B Acetic acid CH3COOHw C Phosphoric Acid HzPO4 D Formaldehyde CH20 E Ammonia NH3 Sucrose Gradients to Analvze Polvsome Proi Thick black line Normal Protein Synthesis Thin black line Protein Synthesis Inhibited What would the profile look like if we knocked out a nucleolar protein required for normal ribosome assembly Absorbance Units A253 nm 46 81012141618 Fraction Number DNA Replication Two Models in 1958 eselson and StahlThis was a Pulse Chase Experimel ta Predicted results Conservative mechanism Semiconservative mechanism Parental strands A synthesized in 15N HH HH New New A Old Afterfirst B doubling in 14N After second doubling in 14N i C HHLLLLLL LL HLLH LL 1 Actual results Density Density y Generai n Meselson and Stahl s Classic 0 to i L 5 1958 Expt 03 7 id Determined the true W i l B model for DNA replication lt 10 i 7 Semiconservative model 15 4 a C Matt Meselson still qurte active 19 Advisor to Congress and the 25 J White House on Bioterrorism 30 J lmpressive speaker 41 i lt Dand 119 J Danau J L mixed Fig 429b LVL HL HH LrL HL HH DNA Synthesis Starts with an RNA Primer Primer 5 3 I 3 OH on the last nucleotide is needed for condensation synthesis Nucleophilic attack on alpha phosphi WIIIIIIIIIIS Template strand Primase lays down the RNA primer DNA polymerase reads the template strand 3 to 5 to synthesize the daughter strand 5 to 3 Leading versus Lagging Strand Synthesis 539 y Point of joining Lagging strand Okazaki fragment Parental DNA duplex Short RNA primer 5 Direction of fork Leading Strand movement Topoisomerases relieve torsional stress excessive supercoils 3 introduced ahead of the replication fork F39g 43930 SV40 s Large T antigen works as a helioam a SV4D DNA 39 39 lUrK Pol 01 extends the RNA primer POL delta is the main polymerase thm roliferating Cell C PA 335 Nuclear Antigen quot DNA a PROCESSIVITY Rfc Replication factor C V RPA Replication Protein Fig 431 b PCNA Double stranded Fig 431b PCNA a homotrimeric protein fo 5 a clamp around the new helix to ensure the grocessivity of DNA polymerase 6 c RPA Single stranded DNA Replication Protein A a heterotrimeric protein maintains the ssDNA template in a uniform conformatiOI to ensure rapid replication Displaced by DNA pol alpha and delta F39g39 461C The SV40 circular chromosome has one Ecolll Origin restriction Slle EcoHl gt Circular viral c romusome l i l site where DNA replication begins Called the origin AT rich Replication bubble grows larger over time as replication forks move in opposite directions Fig 4 32 l Replication bubble i Time of replicatlon Origin of DNA Replication Bidirectional Still describing SV40 and Large T Helicases ORC and the helicase called llCll are used in eukaryotic chromosome replicali Unwinding ATPhydrolysislo unwindwhile RPAproleinsbind I Leadingstrand primer Synthesis Primase and DNA Pol or I I Fig 433 W PCNARfcPOI llLeadingstrand extension delta lU nwinding Fig 4 33 AA v El Leadingstrand extension Primase and Pol alpha synthesize Lagglngstrand prlmer lagging strand synthesis primers First Okazakia fragments Fig 433 l PCNARfcPol6 I I extentOkazaki IlLaggIngstrandextenSIon fragments Strand ligation DNA ligase seals Okazaki fragments Fig 433 Questions to think about 1 How many times will an Oriqin start DNA re Iication in typical cell cycle of a diploid cell Ans 7777 2 What would happen if DNA Pol made mistakes 5 Fingers 5 Thumb Fingers Thumb G rowing strand 5 y Template strand Edin39on dcampany 1 in 104 initial errors versus 1 in 109fina errors due to the proofreading function of DNA polymerase Nature is Incredible Types A Base excision Repair Prior to DNA replication B Mismatch excision repair just after DNA replication C Nucleotide Excision Repair Xeroderma piqmentosum a hereditary disease linked to skin cancers melanomas and squamus cell carcinomas D Repairing broken ends nonhomologous end joining EHomologous Recombination to repair doublestrand breaks recombination between sister chromatids Even more incredible is DNA repair Several examples A E Also normal dC C C N Deamination Hlil 1 o cTCH o cTc 2Deoxyribose 2 Deoxyribose d U SMelhyltylosine Thymine 5 3 5 3 5 3 539 3 Me C G Deamination G Replication Fl quot TA C G gt gt Baseexcision repair 3 5 3 5 3 5 339 5 Wildtype Mutant Wildtype DNA GT base pair DNA DNA Figure435 MoleculurCeIIEiolagySixthEdilion m I A cc 2008 w H Freeman and Company lAPEI endonuclease O I l l l39 J L AP lyase part of DNA Pol B A Base excision Repair Prior to DNA replication DNA Beta 1 A DNA glycolase specific for GT or GU mismatches removes T 2 Apurinic endonuclease 1 APE1 then cuts the DNA backbone DNA Pol Q iri iE Jll G DNA liga aenmlu slt aJJJ I deoxyribdseim r aia miavieli iulvgn ixm Edm39on n9 Mai macaw H Fran anand omvan f wildtype DNA 4 While DNA Pol beta B Mismatch excision repairman I H just after DNA replicati o r 39 Errors in the newly synthesigggfgfggg fd daughter strand are MLH1 endonuzlease repaired PM DNA helicase DNA exanudease Mutations in genes encodinsg 39 FFI I FI FFI TI I I I l I I I I3 MSH2 and MLH1 are IInkeday I 5 A z I 539 to colorectal cancer Gap repair by DNA polymerase and ligase WHICH IS THE DAUGHTER STRAND quot Deoxynbose N c o C Nucleotide Excision Repair CE H 9 o H CH3 Recognizes perturbations gt bulges in normal Bform quot quotquot DNA Tm ETRCH W0 ymme res as Can be caused by chemicals binding to 122am the bases chemical adducts 0 H Deoxyvihnse nltfi N o or by UV radiation CH3 which causes Thymine dimer formations 0 neaxyribose Ngt co cc H cH3 Thyminethymine dimer residue Flgum a Maluulnr CellBinlngMSV39nh mm H 2Lon H rmmau m Lompany n Initialdlmiye 39 n rmrmio xvrndxwa a endnnmklxu nl quotarequot musmax 5 339 s39 339 39l 39 quotITl39 3 l I A A I 5 a mu palymensl Ming 539 339 I 3 A A I 5 wildlypeuul Figure us Multwlnr all Biology Sim Edition mums w H Freeman and airman C Nucleotide Excision Repair XPC and 233 recognize DNA perturbation TFIIH unwinds helix XPG and RPA further unwind and stabilize XPF with XPG cut out damaged DNA Gap filled in by DNA Pol and ligase Xeroderma Qigmentosum a hereditary disease linked to skin cancers melanomas AlaA Doublestrand break D Repairing broken ends nonhomologous end joining DNAPK EgKusoKwo Could be within a gene heterodirner usually a loss of base pairs mutation Could be broken ends of separate I Other proteins chromosomes translocation E Homologous Recombination WEE to repair doublestrand breaks recombination between Ligase sister ch romatids Fig 442 page 152 Viruses in Cell BiologyCryoEIVI 10nm l Fig 444b Capsid protein coat surrounding the nucleic acid genome b A large icosahedral virus V Nucleocapsid capsid coat plus nucleic acid genome Fig444b so Inn Eaderiophager Avian influenza virus mm w Murmutullu39vulummnm Envelope phospholipid bilayer with embedded proteins Confluent layer of susceptible host cells growing on surface of a plate Plaque Assay Add dilute suspension containing virus after infection cover layer of cells with agar incubate Plaque i 77 Each plaque Is a clone derived from a single infectious virion Each plaque represents cell lysis initiated by one viral particle agar restricts movement so that virus can infect only conti uous cells 9 Fig 445a Bacteriophage D51 infecting Pseudomonas fluorescens cells Plaque Figure 45 mmwmceu My sum warm 1ch w diva nun and cmrlpsw Fig 445b The Lytic Pathway wl Bacteriophage T4 i as f Lysisand l Ew release 239 Adslolrptio39n CIIromI7re E i m 7 MTar NA J r proteins 957 fr w 3 fag 3 52 3 Viral proteins Replication of viral DNA Expression of viral late proteins my MeierumelialoiagniinhEdmm Fl 9 asz H Freeman and umvnrw I Rabies Virus A Nasty RNA Virus Rab39es v39rus gt v 39 O Nuoieocapsro proteln k L39P39d b39layer Ji Matrix protein Genomic RNA 7 3 A Receptorrbindlng glycoproleln t r 0 Viral RNA ol merase nun quot V Adsorption Budding Y r u 3 539 Virus receptor Cell membrane 1 23 4le 4y1111Y 1Y YVVY M39H 3 yquot 4 I if quot V Association I r 7 r Hmong at membrane 5 39 cymsol 3 Endocytosis f Progeny capsid Golgi assembiy 39 N quotV Endasnme 1 Q Transport nucleocapsid synthesis Fusron of Viral envelope Virai with endosome membrane W mHNA Replication Glycoprotein 3 4 V I K synthesis Transcription W I Fig 4 47 Release TEM of enveloped viruses emerging from an infected cell Measles virus a RNA virus that replicates like the Rabies virus Retroviruses enveloped contain two copies of genomic RNA RNAs are templates for DNA synthesis Reverse transcriptase an RNAdependent DNA polymerase reads viral RNA as it synthesizes singlestranded DNA then reads the singlestranded DNA to make a complementary DNA strand to yield doublestranded DNA Double stranded DNA then integrates into chromosomes called a provirus Provirus expresses more RNA some retroviruses contain oncogenes mice and birds Human Tr nll Illmnhn39l39rnnhir Iiruc llITI Il a Life Cycle of a Retrovirus Genomic 55 NA R Reverse tra nscriptase Retrovirus n Host cell proteins chromosomal DNA Budding FUSIon Transport to nucleus and integration Fig 4 49 Mobile DNA Elementsl Transposable Elements Can jump around genome from bacteria to man molecular parasites F Crick called them selfish DNA slow to be eliminated from the genome thus they accumulate over time first discovered by Barbara McClintock 1940 s two major categories of mobile elements 1 Transposon 2 Retrotransposon retro u Transposons v tral Ia bi DNA transposan Retrotransposon EH quoti 1 Donor DNA Donor DNA Flanking DNA RNA Former polymerase transposon 7 siie Donor DNA W 4 RNA intermediate Note the Donor DNA Reverse DNA transcriptase V Insertion 39me39me i39ates f Insertion lnte rmed lat site site Target Target DNA DNA Number Of cop39equot 39 Now you have two copi i of this Bad Boy remains bUllbtdl39 still a single copy Figure 53 Molecular Cell Biology Sixth Edition H E WHrlreemnn and Company Transposed mobile elements Fig 68 Bacterial Insertion Sequences lS s over 20 IS s known for E coli a typical IS IS eiement a i2 kbi JL 1 3139 5139 5 to 11 bp Protein ceding 5 50 bp direct repeat regien inverted repeat Characterized by 511 bp direct repeats 50 bp inverted repeats encodes 12 proteins Transposase Fig 69 A Mechanism of IS trans osItIon WWW TB39QE DNA p 5 3 5 3 3 5 3 5 Nonreplicate cutandpaste Srbntarse sile mechanism 1 zzfssaakzzzirzgzzii Notice that it is a staggered t M E39QS DNA 5 ISM 3 3 5 That is one copy pops out 5 5 elm gt3 of one place on the med 3 5 chromosome bases Transposase quotgates Isw and hops Into another spot Lgfa39rjg sg sgandedends The number of copies 5 3 5 3 gt vgt remaIns constant 3 I 539 39 C Hul rDNA l m r nd Notce that the Donor DNA Is ginsengan j a jiafgi 5 Kane 2quot Sta 39ens Blunt cut and the Target DNA is 8 Staggered cut IIIIlIlII IIl direct reBaats Fig 610 There s a regiicative model for IS transgos a replicated copy ends up in a new site 7 So copy number can increa 2b 3 7 a gwm s36 l Hui mm m a m mmnm Some bacterial IS s carry an antibiotic resistance gene Transposon 2600 hp ilt 750 bp 9k 1100 bp gti 51 Chloramphenicol 5bp resistance gene direct repeat Eukarvotic transposons Barbara McClintock and Indian corn Maize Activator element Ac a fully functional transposon Dissociation element Ds a defective tranqugmgg Needs help From the AC to be able to jump Mechanism is that of nonreplicative transposition Pelement in Drosophila transposable element that hops by nonreplicative mechanism now used to create transgenic animals Aan Spradling and Gerry Rubin the first to transform a higher organism Drosophila Viral Retrotransposons abundant in yeast called Ty elements abundant in Drosophila called copia elements LTR retrotransposon 6 11 kbl l 3 5 L Y J LT R Proteincoding Ta rgetsite 250 600 bp region direct repeat 5 1oabp Lonq terminal repeats LTRs Shown in qreen This is the hallmark of viral retrotransposons encode 34 proteins 2 mechanism of genome insertion is similar tEt aFal Quick Review ofthe Retrovirus life cycle chromoaumnl a DNA Rwavlnn prolalns Reverse Irnnsc plasa naan Namquot Huntcall LTFi Codingregion LTFi Host cell DNA f H r H E Integrated I retroviral Symw DNA Sta rt site PolylA site RNA polymerase ll Primary 5 NW jgruxf i a 3 transcript RNAeprocessing enzymes PolylA polymerase 3mm 39 A Fig 613 genome Transcript lacks complete LTRs Transcript encodes reverse transcriptase and integrase Mechanism of producing retroviral dsDNA is very complex g L ements are expressed when cells are stresse39 opia viruslike particles containing RNA accumulate within the nucle What s in your nucleus Nonviral Retrotransposons Lack LTRs Moderately middle repetitive elements Think of the CoT Two classes 1 LINES long interspersed elements 6000 7000bp 39 10 classes found in protozoa insects plants 39 but very abundant in mammals 2 SINES short interspersed elements 300 bp 39 one major class Alu found primarily in mammals L1 LINE elements 900000 copies in the human genome 21 Of the genOI long interspersed element UN 6 kb ATrich Proteincoding Targetsite region region direct repeat Figure 616 Molecular Cell Biology Sixth Edition 739 ZODB W H Freeman and Company Features of L1 short direct repeats two open reading frames ORFs 1 ORF1 encodes an RNAbinding protein Function 2 ORF2 encodes reverse transcriptase Fig 616 L1 LINE element L1 insertions cause mutations how it was first discovered Transposes by an RNA intermediate retro but not by the complex LTR mechanism shown in Fig 6 14 rather Fig 617 next several slides Most L1 s are defective can not transpose by themselves Fail to encode functional proteins But need only one functional L1 elements to provide proteins Defective L1 elements could then jump This will provide the transposase ORF2 protein Chromosomal DNA LINE RNA AT rich tarqet site ts sta ered Nlckmg 959 99 MEte Nic site 539 5 3 Nick site Nick site a Priming of reverse transcription by chromosomai DNA AAATACT 339 AAA Fig 617 5F 5 AAATACT Reverse transcription of LINE RNA by ORF2 AAATACTID gt 3 Wm TGA 539 Fig 617 AAATACTr gt 3 WTTT TBA 5 5 339 LINE RNA SI AAA AAA 339 3r TTTATGA 5 NE DNA Fig 617 LINE RNA EIAAA AAA 3Ir 339 TTTATGA S NE DNA DNA by ORFZ a Copying of chromosomal TTTATGA 5 Fig 617 TATGA celluar enzymes Insertion completed by a l 339 539 AAATACT MAAA gt 3 3 TTTATGA VWVVTTTATGA 5 Fig 617 5 AAATACT 39 3 MAAA i r3r 3 4TTTATGAWWTTTATGA 5 1 LINE DNA 5 3 AAATACT TTTATGA W AAATACT WW TTTATGA 3 I i539 LINE DNA Direct repeats I 0 what does the ORF1 protein do in all thi Inquiring minds want to know 1 Fig 617 Alu Family of SINES in Humans 300 bps in length 11 million sites in the human genome 10 Of the genc Alu sequences are similar to the 7SL RNA 7SL RNA a component of the signal recognition particle Particle used to direct secretory proteins to the rER membrane 7SL RNA predated the Alu sequences Supposition Alu s originally derived from 7SL RNA Other Alulike elements shorter found in intergenic spacer sequences and in introns Alu Family of SINES in Humans Contain direct repeats Contain no LTRs Nonviral like but Alu s have AT rich regions like LINES mechanism ofAlu transposition thought to be similar to that of LINES perhaps LINE proteins mediate Alu transposition Alu s are transcribed by RNA Pol III But Alu RNAs do not encode proteins unlike the L1 elements Alu Family and LINES in Human Evolution Function for Alu elements Dr Prescott Deininger Tulane U Medical School Function for LINES Dr Mark Batzer LSUBR Certainly both played a role in genome evolution sites for recombination See Fig 618 and 619 next slides How did mammalian primate genomes evolve differences between human chimp etc Molecular Evolution of the Mammalian Genome Three Examples of Exon Shufflinq r Alu Aiu Gene 1 m33 Gene2 39 I i Au Aiu Double crossover betweeen Aiu elements Il E Li EI I r E lll Exon shuffling introduction of a new exon by Alumediated genetic recombination Fig 618 Molecular Evolution of the Mammalian Genome a DNA transposons Gene 1 lTransposase excision from gene 1 Egaj Insertion site Gene 2 E J39 j C lTransposase insertion into gene 2 jIII E 5 Exon shuffling introduction of a new exon by transposonmediated transposition Fig 6193 Molecular Evolution of the Mammalian Genome Weak poyA Gene s polylA b gnal Mgnal l Gene 1 LINE ix 3 exon lTranscription and polyadenylation Remember that at end of downstream exon LINE have RNA WAAAA Insertion Site Gene2 l E i l l ORFZ reverse transcription and insertion W zzz Exon shuffling introduction of a new exon by line elementmediated transposition Fig 619b Big Gear Change Packaqinq of Genomic DNA In E coli 20000 HNS proteins bind the E coli chromosome to package DNA Package referring to folding in some One HNS protein per 400 bp of DNA In eukarvotes Chromatin 1 Genomic DNA 2 Five types of basic histone proteins have a char in equal mass to the genomic DNA 5 his Arginine and Lysine rich 3 Nonhistone proteins regulate transcription replication etc 4 nascent RNA still being transcribed Histone proteins H1 H2A HZB H3 and H4 Two copies of each core protein 8 total form a nucleosomc charge on these basic proteins interacts electrostatically w the charge of the DNA PO4 backbone Basic chromatin structure is remarkably conserved in eukaryotes because H2A H2B H3 and H4 are highly conserved H3 and H4 are the most highly conserved proteins known from veast to man the few amino acid substitutions that do exist are conservative That means substituting an Arg for a Lys H1 linker histone sequence varies in different organisms least conserved of the five classes variants like H5 in nucleated RBCs 2 ma ecu les each of HZAQ H285 H35 and H4 Mus 146 bp of DNA wrapped nearly Micew x I 891 it Sim X y Kygfia lgrphy g ca m mi Pr iMutU m Sitwccjiwcsg Remember eukaryotic DNA is supercoiled No gyrases in eukaryotes So how Answer One nucleosome introduces one negative supercoil as it wraps 146 bp of DNA nearly twice Transformation Uptake and expression of foreign DNA First discovered by Frederick Griffith 192 In Streptococcus pneumoniae Avery McCarty and McLeod showed that DNA is the transforming material 1940 s Today we transform E coli with recombinant DNA gawmckq g Hasmidu vector fll DNA fragment 1 to be cloned Cut and paste to build l zm fllill zm recombinant plasmid film th Recombinant 1 J h plasmid E l l eih jd Introduce recombinant p m QLEEEri ggmfhcgg n into E coli 1211 By transformation chr my i m 39Ef a quot quot9 PM Antibiotic resistance gene 7 Q 3 allows only transformed E clt 1235de quot ll ii gi s dm to survive and replicate Non lplasmidreplfQZTpmm39quotmm transformed cells can t grow Typical Antibiotics W Ampclllln in presence of ampicillin Colony of cells all containing Tetracycline Cell multiplication J Chloramphenlcol multiple copies of the same pig A 39 Kanamyci recombinant plasmid 1 39QOQQD Emmsggeesagtme 7 g same recombinantplasmid a Fig 514 So why does Eco RI not chop U E coli s own genomic DNA K Unmethylated 539 G A A T DNA 339 c T T e EcaRI will not cleave methylated DNA it Change gears cDNAs complementary DNAs made from tissuespecific or cellspeci c mRNAs classic test gug on since cDNAs are made from mRNAs mature cDNAs have no introns cDNA sequence provides the protein sequence ie the protein sequence is deduced cDNA libraries are speci c for a given tissue or cell type represents the 10000 different mRN expressed in the one tissue type mRNA How do you make cDNAs TTTT alts W Ans Start with mRNAs and oIigodT J Tm affinity chromatography am fm Tm RNA QIIQOdT mam Recall that cellular RNA consists of Mummwasmmm mRNA 2 tRNAs 3 3 19 db J E39 unneep V TTTT w TTT39l39 Most mRNAs have polyA tails 5 AWN TTTT Wash away tRNAs and rRNAs imsg maymm ll 15sz ffer Elute mRNAs Repeat 2X for purity FWW takes ALL day and better not have any RNase around 396 Puri ed mRNA preparation Actually making the cDNA mRNA 5 my 3 po ytA tail Hybridize mRNA with Oligo39dT primer n oligodT primer T T T T 539 1 OligodT is made synthetically quot by a biotech lab GeneLab m 7T T T T 539 reads the RNA template to quot synthesize first cDNA strand 1 a 339 1 T T39T T 539 Fig 515 Actually making the cDNA continued CZZZZijT 5 Remove RNA with alkali Add polydG tail 7 Singlestranded 3 G 3661 T TT T 5 cDNA V 5 V Hybridize with oligo dC primer 3 G GGGITTTT5 Fig 515 Actually making the cDNA continued 5 3 GGGGITTTT5 Synthesize complementary a strand DoubleStranded5 3 cDNA 339GGGGITTTT5 Protect cDNA by CH methylation at EcoRI sites 3 9 3 3 G GGG39TTT T 5 CH3 Fig 515 Actually making the cDNA continued CH3 Synthesize or Why 339GIG39ZG1GTITTI T 539 Purchase linkers 3 EcoRl linker Ligate cDNA to restriction Q6 A A T Til site linkers r 39r a A A 1 Es G G GITT r r i391llc 1r 139 m1 Cleave with EcoRI 1 A I TlG ammo GUGE T TIr EE Ier quotr T A A Sticky end Figura 54 5 part 4 Molecular Cell Blalogy Sixth Edition 2008 W H Fireman and Company Fig 515 Actually making the cDNA continued 1 111 LEII39 31 I a Dmxamammlle 1 i1 1 Stillquot 9 n I Ligate to plasmid m Transform E coli Individual Select for amp Plasmid with sticky ends Figure 515 part 5 Molecular Cell Biology Sixth Edition 2008 W H Freeman and Co pany But rememBer 40000 different mRNAs some in few copies thee ony at 5 others in many copies cDNA library is comple thee mgo n gst How do I pick Filter Hybridization Individual colonies Master plate of E coli colonies lace nitrocellulose filter on plate to pick up cells from each colony Nitrocellulose lter Incubate filter in alkaline solution to lyse cells and denature released plasmid DNA Hybridize with labeled probe 1 h r II as x o l J c l quot 1 V quotquot 39 e39 1 Perform autoradiagraphy Sig nal appears over plasmid DNA that is complementary to probe Figure 516 Molecular Cell Biology Sixth Edition 2006 W H Freeman and Company Bound single stranded DNA Filter Hybridized complementary DNAs Wash away labeled DNA that does not hybridize to DNA bound to filter Perform autoradiography Picking out the E coli colony clone that contains your cDNA of interest Principles of AgBrcrystal Photographic emulsion Specimen labeled celll Support 3I39i39Ilhymidine to label DN Emission is a Betaparticle 3HUridine to label RNA 3 high Speed e39ectmnl 3HAmino acid to label protein FYI mostly Chemistry of making an oligonucleotide in GeneLab All done by automation just type in the sequence let it run overnight Better yet order online and Mnnamr mum 2 1 5 Else 5 a 5552 HO k mo 5 3 A M20 mm prHnQ tweak may we am mm supvnn o i o MCH0 7aasn n Oxvdalinn by r Dnnwiarmn by man the oligo arrives the next morning Ho Notice that in Vitro synthe is i a i a New o is backwards using a 5 0 Repeat Process mm monomer mums m Inboundmid Shuttle Vector for propagation in E coli yeast URA3 is a selectable marker POIYImker ene Ura339 yeast cells require E coli Fig 517a Making a genomic library g1 mmmamwmamm Yeast genomic DNA Shuttle vector Partially digest 1 Cut with with Sau3A WHY quotit v M A m quotlt Fig 517 Overlapping Restriction Fragments Sau3A and BamHI have have compatib sticky ends They are iden caL Instead of a bacteria plasmid could also Use lambda phage Why Partial digestion with very low concentrations of Sau3A leaves overlapping genomic fragments Important for later use Of overlapping clones Called Chromosome walking Why Sau3A ArmtnaSnau3A dSBamHl leave the same compatible sticky ends 2ukb 39 partial 39 L Sau3A 39 S fragment 1 A genomic library is different from a cDNA library in what respects What s included in a genomic library that you won t find in a cDNA Iihranl9 Making use of a genome library Transform E coli Screen for ampicillin resistance plasmids from 105 transformed E coli colonies Assay yeast genomic library by functional complementation l Isolate and pool recombinant cdc cell division cycle mutants Leland Hartwell 2001 Nobel Prize Mate haploid s of Conditional mutants in this case temperature sensitive permissive temp 23 nonpermissive temp 36 Test resulting diploids for a tern peratu re opposite mating types and carrying different recessive temperature sensitive cdc mutations Mutant Mutant ItVPeaJ ttvpeal 39 Mutant Mutant type 3 type all F cchcdcY cchcch type attl type au at permissive Plate and incuhate 1 temperature Replicaplate 23 LC 7 Genetic ComplementatitlLL Analysis to determine alleli INTERPRETATTW Mutations either complement not allelic or they fail to complement they are allelic 39 39 d h t sensitive 4 cp EI39IO YPB l and incubate at nonpermissive eukv temperature quot2 4 f r 94quot f k 36 C Growth 35 3C N10 9mm PHENOTYPE Wild type Mutant Growth indicates that mutations cch and cdcY are in different genes Absence of growth same gene FlVl X771 El Respective windtype Both alleles alleles provide normal nonfunctional function indicates that mutations 2ch and cch are in the l Leland Hartwell a geneticist generated many cdc mutations in yeas 1 cdc mutations block cell cycle progression at various points used Genetic Complementation to determine which mutations were alleles of each other But what do the wild type cdc genes encode How do tl protein products regulate the cell cycle Super Hero Paul Nurse to the rescue Literally Functional Molecular Complementation Screening Library of yeast genomic DNA carrying URA3 selective marker 23 C J r cquot c e3quot 3 wquot 51 739391 a I I 7 u w J l e 1 quotIx I Selectable URA3 Marker Genla Transform yeast by treatment with LiOAC PEG and heat shock Tern peratu re se nsitive odemutant yeast ura3 requires uracii permissive temperature a wiid type CDC gene 1 Plate and incubate at Oniy colonies carrying on medium lacking uracii are able to grow Only coiom es carrying a V UHA3 marker gt are able to Replicapiate and grow 23 DC incubate at non perlmissive temperature 36 C Paul Nurse shares 2001 Nobel Prize with HartwelFig 53918 Once Nurse complemented rescued the cdc mutation he reisolated the red plasmid from the rescued yeast cells sequenced the red gene ligated into the plasmid deduce the amino acid sequence to identify the protein Voilall Cancer research takes a maior step F I Imperial Cancer Research Fund Lond yeast geneticist molecularcell bio now president of Rockefeller Universit started his career at Guinness Brewe wwwnobelsemedicinelaureates2001lindex DNA restriction fragman 6 Change gears Q x DNA Gel Electrophoresrs 1 Wall a e Cut insert out of vector I Gel pam cle Resolve on an agarose gel v wares Electrophoresis Molamles move 1 uh Dares in gel at a me vnverwly proponinnzl m heirchain lenglh Sujbiang zul i39r liography a 0 mm ye Stain with Ethidium bromide v Fluoresces in UV light NOT SAFE TO HANDLE Large DNA fragments move move more slowly than do smaller fragments Signal wrrespcndin to DNA band Typical agarose gel Polyacrylamide gel Clones kbps 3 4 5 M 2 Good for 0020 1500 b5 600 7 E HellofaIot betterthan Fig 51501 I x w Radiolabeled fragments det cted by autoradiography Stained WIth ethldlum bromlae PulseField Electrophoresis on and off at right angles Current turned on and off at right angles Resolves huge DNA fragments Genomic fragments 20 kbp 10 mega bp cut the genome with u 2 3 Restriction Enzymes Provide a Physical Map a Compareleontrast 10 unit length of DNA W mm quotP Enzyme only Enzyme l only 06 04 09 01 l I l l lb 10 Double digest I Enz Enzl gazilmemand Enzl Enz II 01 05 04 Either 06 03 4 l I I7 E 44 01 I7 0 Run an agarose gel to measure the size of the DNA fragement Polymerase Chain Reaction PCR Probably the most powerful molecular technique ever devised Developed by Kery Mullis 1983 Nobel Prize in Chemistry 1993 Amplifies replicates specific DNA sequences but any you choose Forward and Reverse Primers select the specific sequence of DNA to be amplified Primer Oligonucleotide 2 total Primers are in vast molar excess compared to template Exponential increase in target DNA Extremely sensitive Dacia nnll kinlnrul rnonarnh c I 1 Denaturation of DNA 0 W e i Annealing ofFPrimers everse 77 Forward Elongation of primers 72 Special heat stable DNA polymera Fig 5 23 Denaturation of DNA cydez Annealing of primers R903 Primers are in vast q excess Fig 5 23 c le 3 Denaturation of DNA yc Annealing of primers Fig 5 23 L 7 7 Cycles 4 5 6 etc Fig 5 23 Design your primers to include restriction enzyme sit Region to be ampli ed I 5r 3 Continue Ior 20 PER cycies 5 Cur wuh reslriclion 5339 Prime 1 DNA synthesis Enzvmes rrcaAs39 339 Round 1 TEE 3 539 339 i 539 ii Sticky end Slinky End Ligaxe with piasmid veciur 539 339 5 Primer 2 with sticky ends um gymnasts Round 2 5393 a39 It 4 5 539339 Prime DNA syniliesxs Round 3 339 i i 539 5393 i BamHl sire Hindi site Fig 524 DNA Sequencing Three techniques 1 Maxam and Gilbert cumbersome end label then chemically cleave not used much anymore 2 Sanger s dldeoxy technique now the most popular technique sequences up to 500 bases per run uses dideoxyribonucleosides triphosphates ddNTP Ingenious Fred Sanger received the 1980 Nobel Prize in Chemi again for the second time 3 Next generation DNA Sequencing Maxam and Gilbert DNA Sequencing Technique 5397 339 mmmnm Ti 5 mm i m My mmquot mm A 5mm Dimming mm Mariam wands Wm Lia van m numtadinwanii Exuhsu m mmpr m uiiiamm I g immicai reunmmsmu mm i w my mum m a A i n c x c a 3 x Gasman Dramas mm mmquot m wnduse an avng m w A c mm D2 mm m mamquot masks mm mm mm 5 a v c y mum Iawasammw in Humans Mum indlcslc hen lti lt4 4m mon ium n 3 Varailel Gel siuumnharesis Ind aummmugmuhy DNA Sequencing Three techniques 2 Sanger s dldeoxy technique now the most popular technique sequences up to 500 bases per run uses dideoxyribonucleosides triphosphates ddNTP Ingenious Fred Sanger received the 1980 Nobel Prize in Chemi again for the second time 3 Next generation DNA Sequencing 2 Sanger s dwdeoxy chem termmauon techmque So what s a dideoxyribonucleoside triphosphate A 0 o o 3907P0 39040 3907P0 c 5 A 3420 maize 3 A oo or2o OH OH H Ribonuclmia Deoxyribonuclcoside Didnoxvribnnuclaosid39 Ilipholphln Iriphosphlle niphosmm INTP thTF lddNTPl No 3 hydroxyl means no further Fig 53920 Sanger s dideoxy technique actually fun to do a Primer Oligonucleotide is the 5 end labeled with P32 5v 3 5 Low DNA pulvmerasa concentration deTPsi mDuMi of dNTPS Note differences um ii iMi quot9 u 13 Many fragments u of different size lt mm and separate by eleclmpharesm gt Once the dideoxy is incorporated extension stops QLZ39Q 3965 3992s1ue19mmomq 9pp u Bulpue Ile SIUSUJBBU 1 S OOL SPISM UODDBSJ 9quot 9 OS quot39VViiDDODliVlOVVDVVOLDVDLOVDOLV IE EH33OVVLVDDLDilDVDlOVDlOBVl39dzE5 quot39VVilDDDDiiViOVVDVVOLDVEJLOVDOLV E ElOlOllBVOlOVDlDBVl ze9 39VVLLDDDDLLVLOVVDVVDLDVDLCJVDCLLV S EIVOLOVBLOBVLdzg9 nuasajdam uouenuaauoo M0 ugM spuzus a2dma dLLP amp 39dlDP dJVP 1 Spuzsnmu aseJeuJAlod VNG sml OMEN quot39VVllDDDDlLViOVVDVVOLDVDLO f f f OLOVD CI uonoeeJ 9 am asn 0 mam penemp v VDDLV E LOEJVl39dzs 9 Electrophoresis to resolve the numerous fragments A typical dideoxy run using radiolabeled DNA Now automated to sequence genomes Fluorescenttagged nucleotides are used and detected in real time as fragments move past a fluorescent detector next slide Vast sequence information Leads to Bioinformatics Computer technology to handle the vast information GenBank at the NIH Sequence Data Base at th Both avanab39e European Molecular amine i Biology Lab EMBL Compare nucleotide and deduced AA sequence Automated machines sequencers detect the fluorescent nucleotides u A A A AA Am i m D l M ll bill WVN 5 Mn l l i ll i ll ll With automation the entire human genome has been sequel Many other genomes as well Drosophila yeast C elegans Molecular Evolution Fig 521C l 1 MM l l l H l i ii i l ii is 3 Next Generation DNA Sequencing a Billions of sequencing reactions all at the same time on a solid suppoVLW linkers annealto primers Looking atjust one reaction b Ligate linkers to ends of DNA fragments n H c Perform PCR but with both primers ML attached to a solid support primers anneal to linker DNA i l d Left with 1ooo identical DNA mil fragments tethered to a solid sup but in a very tight microcluster mm Fig 523 I Cut one DNA strand denature and wash leaving singleastrand Add new primer men fiuorescenlly labeled dNTPs nne dNTF binds wash away excess Flucrescent imaging to determine who dNTP bound Use a special uorescence microscope to see the microcluster Computer records co ors n l the repeated reactio Chemically remove bound fluorophcre h C stays in place providing 0 3 OH for next uor dNTP Cut 1 Melt DNA osc ere 6 Ar 043 nscluster Ci 6 Results in billions of short 100 bp sequencing runs fragments Repeat until DNA strand is replicated How do you assemble these short DNA sequences into a set of Unknown genomeofinterest chromosomes a genomic sequence Create aligned Create random linkbrarv of CDNA library 039 cDNA one fragment to the next F T Z J sequence ordered Sequence unordered newem fragments What s wrong WIth Flg 525 5 E 255 f Head seq ce ln order Align sequenced dictated by clone map clones by computer W 1 Genomic sequence Fig 5 25 Another powerful molecular technique mom Southern Blots Deal wl DNA l Cleave with el restriction enzymes Edward SOUthem G lt1Electrophoresrs Filter Nitrocellulose Capillary action transfers DNA from gel to nitrocellulose N Cut genomic DNA to completion with say Eco RI run a gel soak gel to denature DNA fragments transfer 9 u ssDNA to paper 0 Nitrocellulose Filter Nitrocellulose paper Gel III a me so Io Capillary actlon transfers DNA from gel to nitrocellulose neutralize pH and salt Fig 526 Special paper is Autoradiograph Nitrocellulose y Autoradiogram Hybridize with labeled DNA or RNA probe Xray film or use a phosphorimager bu quicker Fig 526 In a Southern blot DNA is blotted to the nitrocellulose 5 5 kb In a Northern blot RNA is blotted to nitrocellulose 2 kb 1 kb N 8gobin 065 kb mRNA Differentiation of Eryth roleukemia CelFl39sg 927 Southern Blots allow you to characterize the genes in the genome 1 how many copies of the gene are there 2 determine the restriction or physical map dependent on sequence of the gene Northern Blots Similar technology as used in Southern blots RNA is blotted to paper instead of DNA to detect a particular mRNA wi a cell type its relative abundance eg compared to actin mRNA its tissue type expression the induction profile of the gene encoding the mRNA Joe Gall the chromosome guy with graduate student Mary Lou Pardue now at MIT invented In Situ Hybridization RNA Gall s original technique used Singlestrand RNA probe labeled with 3H a radioactive isotope of hydrogen RNA probe specifically hybridizes anneals with mRNAs inside the cell 395 39 cell in situlthat have complementary sequences Radioactivity localized by a 3 liquid photographic emulsion at Rnn that time Appears as silver grains in the light In situ hybridization today Locating a particular mRNA in both space and time Singlestranded DNA or RNA probes containing modified nucleotides anneal to the target mRNA Antibodies directed against the nucleotides are tagged with a reporter enzyme like peroxidase Yields a purple aproduct b c m 1 landlord K m39r39 nzquot F39Q 53928 153 DNA Chip Microarray Technology HT wt b39s sed 9 to determine changes in gene 39 I patterns mRNAs are prepared from V two sample groups serum 1 lsolatetotalmRNAl gamequot aye Reversetranscribe EV L cDNAs are made from the mRNAs ltocDNAlabeledwithl a fluorescent dye usmg deoxynucleotides tagged With fluorescent dyes green or red Mix DNA h b d d Hybridizeto DNA Two groups of cDNAs are then mixed oilisfirisllgfgg lmlcroarray together and annealed to the 8600 wash 1 Measure green and red fluorescence over each spot If a gene spot shows red then the genequoti was induced turned on upon serum addition Array of 8600 genes 5 o a gene Shows green then 3A lfaspotis greenexpression ofthatgenedecreases In cells after serum addltlon there s a lack of red cDNA meaning ifaspot is redexpression ofthatgene increases in ll ft dd39 39 that this one gene was turned off down quot5mm quot39 quot in the cells treated with serum quotSit gl i f f n i iquot Fig 529 Fluorescence Microscopy to record the color and intensity of each spot Yellow means equal hybrid izations of red and green cDNAs Blue Flgnve 519 Il loluulnr all lvlognsixlh Edilv39un mum H newquot and mmmn l Sophisticated algorithims Time Each column represents a different gene at times after addition of serum fl 1 Flgnm 530 Mukwfar feliaicingn xm 5mm qoasw H Freeman m mum BED E DNA Cloning For Ex ression Pur oses E coli expression of cloned DNA to make the pi a akme Bgalactosidase lac promoter IacZ gene IPTG IPTG IPTG is a derivative of lactose IPTG can not be broken down by metabolism so it constantly induces the lac promoter Fig 5 31 Cut out the IacZ gene and ligate in the cDNA of your choice Induce with IPTG to express your protein ibi G CSF cDNA What if the gene product is lethal gene to E 00 Problem here Transform lac promoter IS leaky E can cDNA Plasmid expression vector IPTG IPTG Ammo 5 Pin 3 E Z Thls Is ExpreSSIon Screening Jam 39 1 Similar to hybridization screening Except 1 cDNA is made to express its protein product 7 2 Correct clone is identified 6 using an antibody that 1quot quotquotquot quot quot quot could be radiolabeled or tagged with peroxidase i this is 3 none radioactive way Pymins mu m AirmailHon Incubate mm m pm amith Wash iur m we Dacuaiuw Plus on mm lawn 3 Autoradiography for mm mm m mdiniaheted radiolabeling histo 31W 5K chemical staInIng for 722 WWW m m mde spaci npiuquas pe rox Idase 1 Wm WWW my im a Transient transfection In mammalian cells CDNA A 1 Viral origin of r replication ieARS Would you use the lac promoter in mammalian cells Why No bc it is unique to bacteria We do not have the right proteins to Induce that system Notice not 100 Transfect cultured cells by lipid treatment or electroporation Protein is expressed from CDNA in plasmid DNA Fig 532a Introduce plasmid DNAs into eukaryotic cells Plant and yeast cells challenge here due to cell walls Mammalian cells grown in culture use one of several transfection techniques 1 CattDNA precipitate DNA enters by pinocytc 2 Iiposome fusion Lipid treatment on previor 3 retrovirus delivery upcoming slide gt 4 electroporation brief electric shock makes cell permeable to external DNA Think of neuropharm he Select cells that took up the DNA Drug resistance gene in the introduced DNA example neomycin G418 kills all cells that failed to take up the exogenous DNA Mammalian cells are very sensitive to this If they take this up then they will be insensitive G418 BLOCKS POLYPEPTIDE SYNTHESIS IN BOTH PROKARYOTES AND EUKARYOTES THE NEO GENE GIVES AN ORGANISM RESISTANCE TO G418 b Stable transfection transformation cDNA Promoter Vector 02 To make all the cells iquot Transfect cullured transfected use 3 cells by lipid treatment selectable marker or electroporation ene 1 Select for 6 418 resistance G418resistant 69 neo l clones Now 100 are transfectec A cell line Protein is expressed from cDNA integrated Into host chromosome In most cases a random F39g 53932b Freezing mammalian cell lines 1 Freeze gradually slowly in 10 dimethylsulfoxide DMSO and 90 fetal calf serum protein rich These are cryopreservatives Prevent the formation of ice crystals within the cells 2 Store in Liquid Nitrogen 340 F 3 Thaw quickly but very gently at 37 C 3Example of a Retrowral emu vector lentiviruses 39 quotquotquotquotquot quotquotquot All three plasmids are transfected Q together A Vector plasmid contains gene of interest selectable gene quot And LTR that directs retroviral RNA synthesis RelrnvianNA 0 Packaging plasmid contains all lentiviral genes except the major envelope gene Viral coat plasmid contai s a gene that encodes a certain viral envelope protein Choosing this protein allows you to select the type of mammalian cell to be infected Fig 533 DNA introduced into eukaryotic cells by transfection could 1 randomly integrate into chromosomes transformed Most popular HOMOLOGOUS IS A RARE EVENT 2 undergo homologous recombination with normal endogenous gene 3 remain as an extrachromosomal episome


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