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39io iiiiv ie il i i i 154m 195 NJer iSTirWo Oe ie irrZUU Je Genetics and Genomics How Did We Get To Where We Are Today DM2001 lecture 17 Jeremy Martinson Dept Infectious Diseases amp Microbiology Tuesday October 7th 2008 wani two Wm WM 7i wars Genetics and Genomics lectures How did we get to where we are today 0 Where are we today DNA and genome structure Comparative genomics of pathogens Genomewide analyses The Eukaryotic nucleus Infectogenomics Hostpathogen interactions 186934913 O39s 19155 19403 1 9503 1 9603 197054 sacs TQQWBOOGS What is a gene Pedigree 39io iiiivie il 13W mo 195 19605 lSTirtSo Oe Outline 1860s 1900s The Gene as a discrete unit of heredity 1910s The Gene as a distinct locus 1940s The Gene as a blueprint for a protein 1950s The Gene as a physical molecule 1960s The Gene as transcribed code 1970s 1980s The Gene as an Open Reading Frame ORF sequence pattern 1900s 2000s The Gene as an annotated genomic entity The present The Gene as 18603719003 wani um 195v w w 18605 19005 Gene as a discrete unit of heredity 1859 Darwin publishes On the Origin of Speciesquot 1865 Mendel presents his work on peas in Austria 0 Work was largely ignored until 1900 1870s DNA Nucleinquot first extracted o It would be several generations before any connection was made between nucleic acids and heredity 1875 Darwin publishes The Descent of Manquot and proposes the concept of gemmules as a means of inheritance 1882 Walter Flemming discovers chromosomes and observes mitosis iaeowgoo was i 94 3 mid leans l970549805 iggosezooos Prasgnr Mendelian Genetics Mendel studied inheritance in peas Easy to obtain through seed merchants Can easily contol self and crosspollination w Vlllnw 0 amquot sled m mm g R Green or yell1w unrine m 7 K x l Purple or whilt mm I In ated a mum llne nods mu 91 annual owus Emu or you min 1860s71900s 13W ism 195 1900 i dTJ 71900 i dsurriwt r iii fiil Mendel s Laws of Inheritance o The hereditary determinants are particulate in nature 0 Each adult has two particles in each cell for each character 0 During sex cell formation the particles separate equally into the sex cells 0 Each sex cell carries only one particle for each character 0 Union of sex cells to form the gamete is random wrt the particles present Mendel s first law of inheritance The two members of a gene pair segregate from one another during formation of sex cells such that of the sex cells carry one of the pair and the other carry the other member Mendel s second law Different segregating pairs of genes behave independently 18603719003 73M 12 195i l dx r39i Rediscovering Mendel The Mechanics of Inheritance 1900 De Vries von Tschernack and Correns rediscover Mendel39s work while performing a literature search for work similar to theirs 1902 Sutton suggests that paired chromosomes may be the carriers of heredity and that Mendel39s particles may be located on the chromosomes 1905 Bateson coins the term genetics and shows that some characters are not independently inherited leading to the concept of linkage and the need for gene maps 1905 Wilson amp Stevens propose that the X and Y chromosomes determine sex 1909 Wilhelm Johannsen coins the term gene to describe the unit of inheritance within chromosomes 1910s The Gene as a distinct locus o 1907 Thomas Hunt Morgan begins working with fruit flies o 1910 Morgan proves that genes are carried on chromosomes and demonstrates the existance of sexlinked genes 0 1913 Morgan39s student Alfred Sturtevant constructs the first gene map 0 1918 Herbert M Evans determines that human cells contain 48 chromosomes 0 Unfortunately they actually contain 46 idrilier J Jt 19103 M 1545i WW3 WW 71900 le xrrlw l TH Morgan and Drosophia o Sutton39s 1902 observations of chromosome behavior during meiosis suggests that they may be the carriers of heredity 0 Morgan began working with Drosophila in 1907 at Columbia University why these flies o Discovered a single mutant male fly with white eyes instead of the usual wildtype red eye When mated with a redeyed female all F1 progeny were redeye The F2 progeny were all 31 redeyedzwhiteeyed BUT all the whiteeyed progeny were male and the redeyed progeny were 21 femalezmale This sex linkage is consistent with the observed behavior of the sex chromosomes in Drosophia i8603i 191 Os i 195303 f 197034 l Present Morgan and his students 0 Students in Morgan s lab laid the foundations of much of modern genetics 0 Alfred Sturtevant while an undergraduate student with Morgan deduced the foundations of linkage analysis one night instead of doing his assigned homework Calvin Bridges studied chromosomal nondisjunction designed the fly mutant nomenclature still in use today and provided most of the data correlating Drosophia genes with banding patterns 391 3 l it 19403 1951 1940s The Gene as a blueprint for a protein 1926 Hermann Muller discovers that Xrays induce mutations in flies 1500 times faster than usual 1928 Frederick Griffiths studies transformation in bacteria 0 1938 Proteins and DNA are studied using Xray crystallography The term molecular biologyquot is first used 1941 Beadle amp Tatum develop the one geneone enzyme hypothesis 0 1944 Avery MacLeod amp McCarty determine that DNA is the transforming principle 0 This is skeptically received as DNA was though to be too simple a molecule to carry complex genetic information o 1947 Barbara McClintockfirst reports on mobiue genetic elements 0 Again this receives a skeptical reception ascwguns was WAGS wscs V9603 whywmvs Griffith s transforming principle Rm Huxx hISs39lnm IndhnRSnIm 3 0quot a 9 3 0 xju kE lt13 9 99 l M NR kW 77 S v 3 7 Km vNaa lt4 MW MW MW MW maus zm P1952 n lM leGHO E 19408 l Biils lg algs Q7U w lCJSUE 199 SrJriMlS P 9 i Avery s work on transformation Avery MacLeod amp McCarty fractionat heat killed 8 strains of Streptococcus into lipid polysaccharide RNA protein and DNA fractions Only the DNA fraction can transform R strains into 8 strains This was received skeptically as it was already known that DNA had a simple composition of 4 bases compared with the 20 amino acids known to build proteins Kill RNA Protein D A l 0 Polysaccharides l G prlds l l 0 l l t l 0 9 w 73W iz L L 19503 l dm 7391 1950s The Gene as a physical molecule 1950 Chargatt39s rules established amounts of Adenosine and Thymine are equal as are the amounts of Gyanosine and Cytosine in DNA 1952 Hershey and Chase show that bacteriophage viruses introduce DNA and not protein into bacteria 1953 Watson amp Crick publish the doublehelical structure of DNA 1958 Meselson amp Stahl demonstrate the semiconservative replication of DNA 1963 John Cairns observes replication forks in DNA 880549005 was was tgsos teem 97054980 Fwesem Hershey amp Chase 1952 Eanzrinyhage Phorphprus labzlzd sulfur Labekd DNA mquot pmtnn cnpml 3Ccntril39ugalian No sulfur deremd m cells Phorphoms dmmd m 115 R 5mm damned m m phorphamx supzmalanl dzucyzd m upemmnnt The Hersheychase Experiment quot900 39i i i W 19503 33 w NJer i riSo e Significance of the HersheyChase experiment Although there was already good evidence from the 1944 announcement ofAvery MacLeod and McCarty at the Rockefeller Institute that DNA could genetically alter the surface properties of bacteria its broader significance was unknown The HersheyChase experiment had a much stronger impact than most confirmatory announcements and made me ever more certain that finding the threedimensional structure of DNA was biology s next most important objective The finding of the double helix by Francis Crick and me came only 1 1 months after my receipt of a long Hershey letter describing his blender experiment results Soon afterward brought it to Oxford to excitedly read aloud before a large April meeting on viral multiplication James Watson Hershey obituary 1997 mama 9005 l9l 0 mm lesos 36C a lu Pi 959m Solving the structure of DNA Several lines of evidence were brought together by Watson amp Crick o Hershey amp Chase s data 0 Chargaff s rules on DNA base composition amount of T equalled the amount of A and the amount of G equalled the amount of C 13505 19005 19105 19405 19505 IQGOS 19708 19805 19905 20005 Present Watson amp Crick s paper doublestranded helix antiparallel strands phosphodiester bonds holding the chains together hydrogen bonds and hydrophobic interactions holding the two chains to each other G pairs with C and A pairs with T It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material 18608 19005 19103 19405 19505 19605 19703 19803 1 9908 20008 Watson Crick predictions The daughter DNAs produced during DNA replication will have nucleotide composition identical to the original parent 0 Confirmed by Kornberg using DNA polymerase in the late 19503 O Progeny DNA synthesized in the same GCAT ratios DNA replication is semiconservative the daughter molecules will contain one parental strand and a newlysynthesized daughter one 0 Confirmed by Meselson amp Stahl in 1958 A x o D lt mama3 Lemwmthaw mmy of Mt Meselson Y l t Meselson b 1930 1mm Funk n w Sub ht 1929 Presenl igws i950 iSSHs 705498 DNA replication possibilities oa DNA New DNA Semlrconservallve Conservatm Dlsperslve i givea39mus m 18 lc VICKIJCIS 1 quot Wampum anti ae one neen reporter ny usury in 1 9 5 0 s Succharamyces cerem39siae and by Peaseu in Proteus uulgorio l c Weibull sympoainm soc Gm Mic10bwi a 111 1956 I J Lederhel39g these Pnocaamsss 42 574 1956 K MeQuillen Bimini et Biophyx Arm 27 41quot 19581 39 I K McQuillen Symporinm Sac Gen Microbiol 6 127 19511 s Spiegelman in The Chemical e I I I U E Basis of Hwedity ed W Dr MoElroy and B G s Baltimore ohm 0p in Press 1957 pp 23H 39 39 and A Thomas Jr that Pnnca 11 s Shapiro Noonquot 180 151 1957 390 Nee Biol Zoom 75 268 19511 A A Eddy and 1 11 Williamson Nature 179 1252 iary R Emerson in mm 39 Our mutant otroin Em 11200 been shown to he r 3 t hi e an ginger and an unnamed mutant ironr Dr J 11 s Fincimm University oi Leroeetor puhlishel descriptions it is likely diet these are also allelic m the cu quot mutent ot Dr H Kuweno Cytotogr o 132351 3 Mory B Mitchell end 11 K Mitchell these Pnoenenrsea as 442 1952 1 Mary B Mitohelh H K Mitrrhell end A Tiasierea these Pneeeenrnns 39 606 1953 r Pillmlmsndl g n unlit mull mum OaunwIivl mudI SemiquNmiv moons G W Bea warmero in N maroon g H n tile and E L Tatum theee Pmcnnnmss 27 4129 1941 v The hominellnlase prepamtion was obtained fmln the Nutritional Bioohemioal Conromion U h Clevelenol 23 Ohio who iniorm he thnt it is a eeneentnne nt mierohielngreal o ing e Navy rum to cellulose gumose and maltose in addlllon to he eatedstrongehiti 39 39 1 is J rigi o tain nrioellulase A eingle test in our lohoratory indi nu twlty a mu m J Lederberg and Jacqueline St Chair J BacteriaL 75 143 1958 n Phyllis Pease J Gen Microbiol 17 64 1957 NUhyhlid inns AND RELLIN LABORATORIES or mmms m f AND NORM N W CHURCH Luoku39onr or crummy BIOLOGY GALH ORNIA mmww ow monomer swam CALIFORNIA THE REPLICA TION 0F DNA IN ESCHERICHIA COLI By MA39I J HEW MEsELsoN AND FRANKLIN W STAHL own I I u Communide by Max DeHN ck May 14 1958 M l IntroductionSmdies of bacterial treurrfornintion rind haeteriaphege infection L strongly indicate that deoxyribonucleic acid DNA can carry and transmit ileredi Ismosnnn E tary informal ion and oan direct its own replication Hypotheses for the mechanism of DNA replication diHer in t e predictions they make concerning the distribution among progeny rnoleoules oi atoms derived irom parental molecules It 315 mirage dioi 39 e15 have been employed in experiments bearing on the distribu ron of porenta atoms among pro 39 geny molenrrlea in several organem H untieipateol thet a a el whieh imparts to the DNA rnoleoulo on increase or i 4 1 re a 1 L r u is i i i r i i mlg amln ni y end Ll method wits developed for the detection of emu density differences among e d density To this gt1 1 L 1 n 1 L L Origin 9 mmlcauan Fawn DNA WWVNWWWWWEAWWWWNVMWWVNWWWMWAVWWWNAVMV Dr n or ladlca on NVNAV MVNWWWWWWWWWWVMVNWW WNDNM W mmuummh mmmmmmwif Nmmmmmmv EWJVquot mammme Flgun 2m mp An aulmadmgmm and gt15 mcmvcm uman M a ammung E mlmmumnsumc Cmnesy gum cm Cam 591mg Hamnr Libmalorv wars FBi i iMOL 1545i 19603 WW 4i 19605 The Gene as transcribed code 1958 Crick described the central dogma of molecular biology information flows from DNA to RNA to protein 1959 Jacob amp Monod establish the principles of gene regulation coining the terms repressor and operon 1961 Nirenberg discovers the first codon UUU encodes phenylalanine 1966 Nirenberg Mathaei amp Ochoa complete the cracking of the genetic code ISGOSIQOO IS I Os i940s 9505 19605 Present i99OSM20005 l9TQs i9305 Cracking the genetic code Gamov postulated a 3letter code as 43 is the smallest number that is gt20 and it was known that proteins are composed of 20 amino acids Later confirmed experimentally by Crick et al Nirenberg synthesized a polyU sequence and obtained a transcript composed solely of phenylalanine By altering the sequence of the synthetic RNA Nirenberg was able to deduce 54 of the 64 possible codons Remainder were deduced by Khorana who shared the 1969 Nobel prize with Nirenberg and Holley who deduced the structure of tRNA for this work Henseisou isms igm lQEUS 19605 107 srle us Piemi The Central Dogma Genes Crick 1958 postulated that the flow of information in gene expression was l i from nucleic acid to protein V m Immediately realized that there were quotWquot 395 exceptions 539 a some genes encode rRNA tRNA m RNA viruses Protein The molecular view of the gene in the 1980s was of a code residing in nucleic acid that gives rise to afunctional product VlO lill i iH J Jt 39i Biii lMOL 1545i WWI 19703719803 layleUG 3 eight 19705 19805 Th Gene as an Open Reading Fram ORF sequence pattern a 1972 First gene to be sequenced bacteriophage M82 coat protein 0 1976 First genome to be sequenced bacteriophage M82 o Computational tools now enabled genes to be inferred from DNA sequence rather than mapped 0 Concept of the nominal genequot emerged 0 Most genes in sequenced genomes are now identified as annotated ORFs 9105 194153 19505 19805 19705719805 it New insights into gene structure Sharp amp Roberts 1977 discovered that eukaryotic genes were split into exons separated by introns Gene DD Cl ED The gene IS no longer a stngle J Cf D discrete entity alternative 5m ow am I I I Eu Allernalive splicmg spltctng could produce multiple mm products from the same gene Some genes were found to overlap challenging the operon model 19703719803 The biotechnology revolution 1970 Discovery of reverse transcriptase 1972 First recombinant DNA molecule 1975 First call for a moratorium 1976 Genentech the first biotechnology company 1977 First manmade gene used to synthesize a human protein in bacteria somatostatin 1978 Recombinant human insulin produced 1978 Discovery of genetic polymorphisms mammals iglos lydus 19505 rams imam raw Fiesem 1980s Towards the genome projects 0 1977 Sanger and Maxam amp Gilbert devise rapid DNA sequencing methods 1982 The Genbank database established 1983 Mullis invents PCR technique 0 1986 First automated fluorescence DNA sequencer 1989 James Watson heads the newlycreated National Center of Human Genome Research to oversee the 3bn Human Genome Project 39io iillvie ilt 13W iMOL 154511 iShG 19703719803 lay57311110 Genome Project milestones in the 19905 1990 Human Genome Project HGP launched in the US 1991 First US Genome Centers established 1994 HGP39s genetic mapping goal reached 1995 HGP39s physical mapping goal reached 1995 First bacterial genome sequenced Hemophilus influenzae 1996 First human gene map released 9 05 1340s 9505 9605 1970919305 1990920005 Genome milestones w 1290 99 waz was we V935 was 997 was war 20m 20m mscussmn and uebala m sdenlmc mmmumly we vepan aammgauume Mung an H nu s E 9 50 5 camvvsra suquencmg c elevansaequaumn l a mss mgaw sequencmg mum Sequencm Mmmsamuues Fhvs cm maps E a F H n h 5 EDNAsequnmng Esrs 7 u a 9 39 Mmmsa ewlea SW MW a nwmmm I wan g cnmnmmm 7 Chmmuiamn 2x new 7 team 73W tLl L L lift 1900 19703719803 l d xrrZUUG Human Genome Project Inaugural meeting of sequencing groups Bermuda 1996 Genome sequence data was to be released immediately on generation 0 Data was to be freely available in the public domain not restricted to private consortia HGP was a consciously international undertaking Crosschecking and thirdparty analysis of raw data resulted in a very low error rate 1 base in 100000 in certified data Raw data alone is not that useful the draft sequence completed in 2000 was not as useful as the annotated finished version completed in 2003 86054 9005 Sequence Mb 9 0 mm 9505 9605 1970a 9305 0905720005 Human genome sequence data I Fwnished I Unfinished draft and predran F wesent 1880549005 13105 TWHE 19505 WQbO 1970849808 199057400 Presem Another week another genome may team 73W MM 1545i ieo l l dx i 4900 19005 20005 The Gene as an annotated genomic entity The current definition of a gene still relies on the sequence View 0 a DNA segment that contributes to phenotypefunction In the absence of demonstrated function a gene may be characterized by sequence transcription or homology Human Genome Nomenclature Organization As genome sequence data became available many more sequences that satisfy this definition were identified Much interest in gene number in different organisms the Gene Sweepstake Overemphasizes proteincoding genes 19903720003 lit 915 r Moore s Law of sequencing the cost of sequencing has g 33 a 039 P31 2 ill l ii i airlift i If 39 as iquot i V i l i l 3 i 1 50 ll qr S 21 St century challenges halved every 22 months since 1990 o The five largest sequencing centers in the USA alone can now produce 150 billion basepairs of sequence each year Bioinformatics approaches to gene identification are now more challenging than the process of obtaining sequence 19908 20008 Postgenomics sequencing is easy identifying genes is harder understanding complex interactions between gene products is harder still ll i l My ll EEEE EETTEEE EEG TEEEE EEEE EEEEEEEEEEEETE EEETTE I I39I I High 1QDE 253D Tdtilhrrs l TBr l UM 1545i WWI WW 71900 le vrrlw l Present The present The Gene as o The more we know about the nature of genes and genomes the more difficult the definition of a gene becomes 0 Distal regulatory sequences 0 Overlapping and spliced genes 0 Parasitic and mobile genes 0 Large amount of junk DNA under selection 166 i900 raw 1340 WCUS 19605 iWUerdUs warsews Present What s hot right now Comparative genomics to identity functional elements conserved between species Medical sequencing eg of cancer patients ENCODE ENCycIopedia Of DNA Elements HapMap database of genomic variation CaseControl study resources GWAS GenomeWide Association Studies Ancestry Informative Markers AIMs CopyNumber Variation CNV io t Hv team 13M MACK 1545i 1900 i d xrrZUUG Present i371 71900 Key online resources httpgenomeucscedu UC Santa Cruz genome browser httpwwwhapmaporg Haplotype mapping consortium httpwwwgenomegov NHGRI homepage httpwwwncbinmnihgov National Center for Biotechnology Information httpwwwensemborg EMBL genome database httpwwwwtcccorguk Wellcome Trust Case Control Consortium Li39emisu y Her aii Kiwi s Genetics and Genomics DNA and genome structure DM2001 lecture 18 Jeremy Martinson Dept Infectious Diseases amp Microbiology Thursday October 9th 2008 Di39vemrsh y War me 5 Outline DNA chemistry DNA damage and repair DNADNA interactions ry DNA Chemistry CMmsW Nature Apni 25th i953 Watson and Crick again We wish to suggest a strudure for the salt 0 Xylibose nucleic acid DNA This strudure has novel features which are of considerable biological interest llll39igllVJ Raw Nucleic Acids are polynucleotides Chemistry 0 Nucleotides are composed of o a cyclic 5carbon sugar ribose in RNA deoxyribose in o a nitrogencontaining base attached to the 1 carbon atom o a phosphate attached to the 5 carbon atom o Nucleosides are composed of the base and the sugar nucleotides are sometimes termed Nucleotide phosphates NH till l9 NH AN ll N L 2 l l gt quotk gt r N N u N N o o n N N inf 07W O PnED39CH2 foepeciefeoepeoecH 0 393 0 r 9 o o n 0 y B H 0H OH OH Deoxyadenu5me9 Deuxyadenosine 39 diphosphate amp Hiphnspham warp Demysuenosmesu munopnospnaie dAMPl Chemtstry Hepah ngncs Nucleotide structural features Free rotation around the bonds in the phosphodiester groups Free rotation around the Nglycosidic bond Bases are planar ring structures magi N 2 L 171 ltNMH 0712707 H o t 4 tr anaemic bond chemmw Purines and Pyrimidines Fumes Purlmmlnes y H H H N N fj H u N H l n A If u H H Anenlne quot Eulnslne I H N H I H H i Hch H Llrncll N N 39lt H I I A u H Eunnlne nme Flg Structural turmulns n the Durlnes and Durlmmlnes Chemmry nepaw u H39MENCS Polynucleotides mm 15 A W ynuc emme chain ounsisw 09 a series at 5 4 sugu phosphma hnks mm arm n backbone lrnm which he bases pvelrude lt Nmm nmnl i quotmum me OHX Fume bus Chemlslry Hepalr PurinePyrimidine pairing Figun s the doub a quotsix maintains n canslam us AT ec Klneucs Chemistry Repall39 Kinetics Base pairs lie perpendicular to the backbone o In suguv phosphna bankhnne P PHth Chemistry Repair Kinetics DNA structural features Fulfilling WatsonCrick predictions Xray diffraction data showed a regular helical structure making a complete tu rn every 34A 0 Distance between nucleotides is 31 so there are about 10 nucleotides per turn a DNA density suggests two polynucleotide chains 0 Constant diameter due to purinepyrimidine binding not purinepurine or pyrimidinepyrimidine Chargaff s rules 0 CG and AT base pairs Chemistry Repair Kinetics It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material Chemistry Repair Polymerase Chain Reaction 0 PCR uses the same basic mechanism as DNA replication In VIVO 0 Heat is used to separate the DNA strands o A heatstable DNA polymerase enables the process to be automated Chemistry Repair Kinetics Dideoxy sequencing La mm mmmum mu synmuls 9 w MumII way my rmmmmmnny wwwmmmv 1 mm mm mmquot mm o dmnmn Imam an m mama a nimunumqm mm m a um up by m m mumquot nm mum Gnacvanms s mum 5 3 anpkmlnuuy mm mu mm m humnu s m 1 m m m nmuamv um 23 V AEEEGEtTTEEGECEATCEEEAAEB u AGEETTB zsau Chemistry DNA structure variants 0 Everything covered so far describes the B form of DNA 0 Commonest and most physiologically important form but other types can be produced 0 Differ in key parameters such as nucleotide spacing number of residues per turn etc Chemistry bulge quot minor gruave very deep wove dgm handed hem le handed mlx Chemistry Hawaii Ni will 3 BDNAZDNA junctions o Junctions form dynamically o Juxtaposition point accompanied by the breaking and flipping out of a single basepair o Suggests a low energy barrier to transition consistent with the dynamic nature of 8 2 transitions Nature 437 1097 8 amp 1183 6 2005 Function of ZDNA o ZDNA is a higherenergy form of the double helix stabilized by negative supercoiling o Often generated by transcription or by the unwrapping of nucleosomes Sequence motifs favorable to ZDNA formation PuPyPuPy are often found near transcription start sites 0 ZDNA binding proteins have now been identified 0 RNAediting enzyme ADAR1 o interferoninducible tumorassociated Z DNA binding protein DLM1 0 viral virulence factor protein E3L The CSF1 gene promoter requires ZDNA formation for expression ZDNA cannot enter nucleosomes so keeps DNA region open for transcription kiiiwfi DNA damage and repair Rep air Kim DNA damage and repair 0 DNA in the living cell is easily damaged by many mutagens o Radiation gamma rays Xrays ultraviolet light 0 Oxygen radicals produced during normal metabolism 0 39 39 39 hemicals ld wbuns 39 39 quot 39 products 0 Chemotherapy agents used in cancer treatment 0 Failure to repairthis damage leads to mutations 0 Currently over 130 human genes are known to participate in DNA repair 0 This is probably an underestimate Repair kiwi Types of DNA damage 0 Modification of the bases 0 deamination of C leading to U 0 Base mismatches introduced during DNA replication 0 Breaks in the DNA backbone o singlestrand breaks SSB o doublestrand breaks DSB o Covalent crosslinks between bases 0 bases on the same strand intrastrand o bases on opposite strands interstrand 0 Many anticancer chemotherapeutic agents are crosslinkers o cyclophosphamide cisplatin Repair lltli ten Repairing damaged bases 0 Direct chemical reversal of the damage eg dealkylation o wasteful process requires a specific enzyme for each type of damage 0 Base Excision Repair BER 0 DNA glycosylase removes damaged base then deoxyribose phosphate backbone is removed the correct base is inserted DNA pol and the break is ligated o This happens 20000 times a day in each cell in the body 0 Nucleotide Excision Repair NER 0 Several nucleotides are removed creating a bald patch 0 DNA polymerases 6 and e fill in the patch 0 NER proceeds most rapidly in cells that are transcriptionally active and on the DNA strand that is serving as the template for transcription Repair Base mismatch repair 0 Corrects mistakes in basepairing failure to maintain normal WatsonCrick pairing 0 Uses many of the enzymes used in BER and NER as well as some specialized ones 0 Requires the recognition of the mismatch by MSH2 0 Removal of the mismatch involves many proteins including MLH1 o Mutations affecting MSH2 andor MLH1 predispose affected individuals to some cancers eg colon so these are tumorsuppressor genes 0 MMR system is also used to enhance the fidelity of recombination by ensuring that only the homologous regions of two chromosomes pair up during crossover Repair Kit mu Repair of DNA strand breaks 0 Singlestrand breaks are repaired using the same enzymes as the last stage of BaseExcision Repair 0 Doublestrand breaks can be repaired in two ways 0 Direct joining of the broken ends by NonHomologous End Joining NHEJ 0 errors in NHEJ result in chromosomal translocations seen in some cancers eg Burkitt s lymphoma Bcell leukamia o Homologous Recombination using information from o sister chromatid if the cell is in G2 phase 0 homologous chromosome if it is nearby in G1 phase 0 same chromosome if there are duplicate copies of the gene Repair NHEJ and the Ku proteins 0 Repair of DSBs requires many proteins 0 Ku70Ku80 complex 0 XRCC4DNA ligase IV complex o DNAdependent protein ki ase o The Ku complex supports th broken ends and postitions the helix in phase across the junction making end processing and ligation poss Nature 470 607 614 2001 K i run f 3 l zermstry Repair Kinetics DNA repair by recombination The D88 is enlarged or resected by exonucleases to produce single stranded termini One of these strands invades the homologous region of the donor DNA Heteroduplex DNA formation creates a displacement Dloop The Dloop can then become a template for DNA synthesis itself Two joints are produced that must be resolved by cleavage Izmlmw vi minn inmth Chernisii y Repair Kinetics The Holliday Junction Depending on which strands are cleved a Holliday Junction can be resolved in one of two ways A patch in which a small amount of DNA sequence is moved between the chromosomes A splice in which the ends of the two chromsomes are joined to form a recombinant chromosome Resolution of Holliday junctions x gt QC l 39 X 7 e a gt 391 Q i I Repair The whole process dsb Nonhomologous end joining DNAPKcs KU70 KU80 MREllRADSOXRS2NBSI Homologous recombination RADSlRADSIBRADSICRADSID DMC 1RAD54AIRAD54B DNADNA interactions Kinetics and genome complexity Chemistry Repair Kinetics DNA denaturation and reannealing Fielative absorbence at 260nm Temperature 0 Using heat to denature DNA the change in UV absorbance can be measured easily 0 A260 dsDNA1 00 A260 ssDNA137 A260 bases160 o The temperature at the midpoint of the transition is the melting temperature or Tm Chemistry Repair Kinetics Factors influencing DNA melting GC content why Ionic strength of the solution temperature 0 time DNA concentration DNA fragment size sequence mismatches each mismatch lowers Tm by 1 C 0 DNA sequence complexity Percent guanine plus cytosine REFS Kinetics DNA sequence complexity o This is not the same as length or GC content 0 It is the number of basepairs of unique nonrepeating DNA in a sequence 0 Consider a 500bp length of DNA 0 500bp of unique random sequence 0 10 copies of the same 50bp sequence 0 The unique sequence is more complex Cl ten ll gtil DNA reassociation kinetics 0 Less complex DNA reassociates faster than more complex 0 The association kinetics are second order where the rate is determined by the concentration of DNA targets W nucleation zippering gt gt W 2nd 1st order order slow fast Denatyred DNA A short duplex Renatured two single forms at a region NA strands of complementarity two strands In duplex Quantifying DNA reassociation 0 DNA is sheared to an average length of 4 500bp heatdenatured and reannealed o The extent of renaturation is measured as a Cot value Cot Virus 539 can Yeast I measuresthe dissociated DNA as a function of the initial concentration on and the expired time t 0 Got is then plotted against the proportion of ssDNA Singlestranded DNA 0 5 Paws u Eukaryotic genomes are mixtures of complex and simple sequences 3 Highly Repealed 5 DNA 97 Moderately Repeats DNA I 05 lrtermedlal E Unique E DNA u Slow 100 out gt initial DNA concentration x time for reassociation Hrsrail Kinetics Summary DNA is a polymeric molecule composed of nucleotide subunits The doublestranded structure of DNA gives rise to its replication mechanism 0 This can be exploited in the lab eg PCR sequencing Many mechanisms exist to repair damaged DNA molecules 0 These are common to many biological processes DNA structural complexity varies greatly between organisms o Eukaryotes have more complex genomes than prokaryotes The Cytoskeleton 3 building blocks Microtubules Intermediate Microfilaments filaments rrn niruu ir Irhm mnin mlmeLiquot Tubulin Vimentin Functions O Scaffolding produce overall shape 0 internal framework for positionally organizing organelles O apparatus for moving things inside cell 0 locomotion O translation initiation 0 signal transduction Microtubules I m EM of negatively stained mt Outer dia 24 nm Wall thickness 5 nm Length 1 nm Functions of Microtubules Internal scaffold roadway for transport of vesicles and organelles motile elements of cilia and agella spindle structure for chromosome segregation at mitosis Diagram of longitudinal and cross section Heterodimer of oc and 5 tubulin is building block quot Tubule is asymetric end to end 35 x 40 A subunit Polarity reflected in organization and function resists compression and bending Heterodimer a 91939 oc tub 31g Acub 21g umb gig 57m pig a tub 91g B Cub pig 171mb pig rub 21g ucub pig irub Sequence comparison of 06 to B 39l39llocp TZ loep H1 82 H2 83 r 1 10 20 30 40 50 so 70 so 90 MRECISIHGGNACWELYCLEHGIQPDGQM SDKTIGGGDDSFNTF FSET GKHVPRAVF PT IDEVRTGTYRQLFHPEQLITGKE MREIVHIQ AKFWEVISDEHGIDPTGSYHGDSDL QLERINVYYNEAAJ KYVPRAIL P MDSVRSGPFGQIF RPDNFVFGQS H1 39 H2 H2 TS laop N loop ms laop H3 H 3 S4 H4 S 5 H5 0 10 110 120 130 140 150 160 170 180 NYARGHYTIGKEI IDLVLDRIRKLADQCTGLQGFSVF SFGGGTSGFTSLLMERLSVDYGKKS KLEF PQVWEPYNS ILTTHTT AG NWAKGHYTEGAELVDSVLDWRKESESCDCLQG FQLT SLGGGTSGMGTLLISKIREEYPDRIMNTF vvps PKV PYNATLSVHQL H3 T5 locp 17 1091 ss H6 H7 H8 37 H9 y y 200 210 220 230 240 250 260 270 280 290 LEHS DCAFMV EAIYD ICRRNLDIERP T R EGQIVSS 1TAsLRFDG VDLaEFQTNLVPyPRGHFPLATYAPVI SAEKAYHEQLSVAEI VENTDE I YCI EALYD ICFRTLKLTT P G H SATMSGVTTCLRFPG ADL LAVNM VPFPRLHFFM PGFAPL I SRGSQQYRALTVP EL y s 8 H1 0 s 9 s 10 w v 300 310 320 330 340 350 360 370 380 TNACFEPANQMVKCDPRHGKYMACCLLYRG DVVPKDVNAAIATIKT KRTIQFV39DWCPTG VGINYEPPTWPGGDLAKVQRAVCMLSNT TAIAEAW TQQMFDAKNMMAACDPRHGRYLTVAAVFRGRMSMKEVD EQMLNVQNKNSSYFVEWI PNN TAVCDI PP RGLKMSATFIGNS TAI QELF l 119 39 H1 1 H 1 l 39 H 12 390 400 410 420 430 440 ARLDHKFDLMYAKRAFVHWYVGEGMEEGEF SEARE DMAALEKDYEEVGVDSVE G EG EEEG EEY G EGE DEA KRIS EQFTAMFRRKAFLHWYTGEGMDEMEFTEAES NMNDLVS EYQQYQDATADEQ Hll39 Nature 391199 1998 Taxol a potent anticancer natural product with activity against a number ofleukeamias and solid turnouts in breast ovary brain and lung in humans brevifolia Nutt in 1971 and the structure elucidated by Wani and Wall The initial iological activity Was related to the biological activity of taxol Was in fact unique It Was shown to have a complementary effect ie binding to polymerised tublin stabilizing it against disassemny and consequently inhibiting mitosis Retraced loop between H1 and 2 of B tubulin J M01 Biol 313 1045 2001 Microtubule Associated Proteins Micro ubuiciA suciawd Proteins Molecular Proteins weight H Source MAFIA 350 Nerve tissue MAPIB MAPS 325 Nerve tissue Vesikin 295 Nerve issue MAPZA MAPZB 270 Nerve tissue MAP4 200 Widespread MAP3 180 Widespread Dynamin 100 Nerve fissile MAPZC 70 Newe tissue tau 505 Nerve tissue The EMBO Journal Vol21 No12 pp 2923 2935 2002 Shigella deliver an effector protein to trigger host microtubule destabilization which promotes Rac1 activity and efficient bacterial internalization Sei Yoshidaltz Eisaku Katayamaa trigger profound membrane ruf ing and macropinocytosis Asaomi Kuwaequot Hitomi Mimurol Isberg and Tran Van Nhieu 1994 1reton and Cossart Toshihiko Suzuki12 and 1998 The former type of invasion represented by Chihiro sasakawa l Yersinia pseudomberrulnsis lsberg 199i Isberg and Tran Van Nhieu 1994 or Lixleriu monarymgmes 1Department oi Micmbiulogy and immunology and 3Department of Cossm and Lecuil 1998 hemquot and Cossan 1998 is Basic Medical Sciences Institute of Medical Science University of Tokyo 461 Shirukanedai MinuloKul Tokyo ills863 and med aiedPy lPWr I ke m hm m lth cases is IPRESTOV Japan Science and rruchmlugy Carpomion HST Japan bacterial lmEl39nZLllL UOl l event 1S limited tons oyvn uptake 4C d m by the host cells The latter class of 1nvasxon event orrespon mg an or Y mail sasakuwaimsulokyoacjp represented by Shigella exnen or Salmonella nphxmm ium allows uptake of other particles together with the The delivery of effector proteins such as IpaA IpaB IpaC IpaD ngD and VirA through the type 111 protein secretion system from Shigella into and onto host epithelial cells is a prerequisite for triggering largescale Mr membrane ruf ing and macropinocytosis envelopment 39 of the invading organism Target ell membrane Ky 39 l n a Type III Host contact activated translocation Shigella deliver a subset of effectors into the host cell via the type III secretion system that stimulate host cell signal pathways to modulate the actin dynamics required for invasion of epithelial cells Here we show that one of the Shigella effectors called VirA can interact with tubulin to promote microtubule MT m ruf in Under m vuro con ltlons 1r 1 I It polymerization of tubulin and stimulated MT destabi lization Upon microinjection of VirA into HeLa cells a localized membrane ruf ing was induced rapidly Overexpression of VirA in host cells caused MT destruction and protruding membrane ruf es which were absent when VirA was co expressed with a dom inantnegative Racl mutant Indeed Shigella but not the VirA mutant stimulated Racl including the formation of membrane ruf es in infected cells Importantly the MT structure beneath the protruding ruf ing was destroyed Furthermore druginduced MT growth in HeLa cells greatly enhanced the Shigella entry These results indicate that VirA is a novel type of bacterial e ector capable of inducing em rane r mg oug e s m on o esta 1 za Ion Wham ruf ingmicrotubuleShigella invasionNirA Time course of a Hela cell micro injected with VirA ElVlBO J 212923 2002 Proports to show extrusion of sheets of membrane giving the cell surface a ruf ed appearance VirA does not bind to polymerized tubulin C TaxolMT TamiW Vim U39irg n SPSF SF i H 139 TamlMT r Vim EBB staining In Vitro binding assay of taxolMT and VirA A 10 ml aliquot of 35 mM taxolMT and 30 ml of 12 mM puri ed VirA test TaxolMT VirA or 30 ml of PM buffer control 1 TaxolMT were mixed gently and incubated for 10 min at room temperature At the same time 10 ml of PM4M buffer containing 35 mM taxol and 30 ml of 12 mM puri ed VirA were also mixed gently and incubated for 10 min at room temperature control 2 VirA Each mixture was layered onto a 40 ml cushion of 60 glycerol containing 10 mM taxol and sedimentated for 10 min at 100 000 g at room temperature After the centrifugation the supematants total 80 ml were added to 20 ml of 53 SDS sample buffer while the pellets was dissolved into 100 ml of 13 SDS sample buffer For SDSiPAGE analysis 95 ml of each sample were run on the 10 gel and the proteins were stained with CBB Little VirA was pelleted with taxolMT P pellet S supernatant Fig 4 Xrhodamine itubulin assay The structure of the X rhodamineMT almost completely disappeared when incubated with VirA for 30 min arrowheads VirA It was still intact when incubated for 30 min with PM buffer alone Bar 10 um 5amp5 1 40 FL N315 3 5 or when 2240 4 incubated 22quot m m 224277 w1th N224 224 315 4 lubulmbinding domain VirA regulates MT stabilization and membrane ruf ing A I I MTActin Vector FL ViIA C057 cells transfected with plasmids FL WT vir N224 truncation leaving n terrninal 224 residues Shigella invasion occurs at sites of membrane ruf ing Transfnission Actin I Microtubule Microtubule VirA Disruption of MT occurs where bacteria are invading B The local cytoskeletal architecture around the invading bacteria in a high resolution photograph The actin tubulin and VirA were visualized by rhodamineiphalloidin a mixture of antioc and antiBtubulin antibodies followed by CySantimouse IgG antibody or the antiVirAFL antibody with an Alexa 488anti rabbit IgG antibody respectively Arrowheads indicate the bacteria The boxed portions of the MT network in MT and MT VirA are shown enlarged The VirA secreted from bacteria was detected mostly around the destroyed MTs I Fllopodla bulin I Jquot MT destabilization O Vinculln J l 39 Raci v I Ac n Lamall39pwia depolymeriza cn Macropinocytusls Fig 9 Cytoskeletal rearrangements induced during Shigella invasion of epithelial cells Shigella secretes the effector proteins such as IpaB IpaC IpaA and VirA into host cells IpaB and IpaC are integrated into the host membrane and IpaC modulates Cdc42 dependent filopodial formation which in turn may cause activation of Racl and lamellipodial formation IpaA binds vinculin and the resulting IpaAivinculin complex promotes depolymerization of actin filaments which is thought to be required for modulation of lamellipodial formation VirA binds ocBtubulin heterodimers and induces MT destabilization VirA induced MT destabilization would in turn lead to MT growth and stimulation of the Racl activity and thus evoke the local membrane ruf ing Microtubules are intracellular highways Molecular stepper motors move cargo along lament highways Bmdmg We MT and kinesin co localize Completely inaccurate cartoon Three families Kinesins Microtubules Dyneins Microtubules Myosins Microfilaments Kinesin Attach Move Detach Reset Binding site Four steps in cycle light mam Actual motor composed of 4 chains Huxxbtn hinge 2 Heavy chains 2 Light chains Heavy ChaIHE Hmd i 7 TM Movement towards the end of the MT a parked u pawn a Parked Needs ATP direction only 900 nmsecond 8 nmstep outward flow Much less inaccurate cartoon See Nature V01435 308 2005 The Intracellular Fate of Salmonella Depends on the Recruitment of Kinesin Emmanuel Boucrot1 Thomas Henry1 JeanPaul Borg2 JeanPierre Gorvel1 St phane M eresse saimaneua entelica causes a variety or diseases indading gastroenteritis arid typhoid fever The success of this pathogen depends on its capac ratew host cells I n ound mm artmen nesrn Bacterial e ector ed to the host cell by a type III secretion system anta onistica re ulated this event Amun these effec tors silA targete SKIP a ost protein t at ownregulated the recruitment of kinesin on the bacterial vacuole and in turn controlled vacuolar membrane dynamics Intracellular replication of Salmonella enterica takes place in a membranebound compartment e S mone lacontzining vacuole SCV and requires the type III secretion system TTS S encoded by the Salmonella pathogenicity island 2 SPlZ SifA is required for the formation of Salmonellainduced laments Sifs 4 5 and is necessary to maintain the integrity ofthe SCV ZOMAV zoos VOLEDB SCIENCE wwwseieneemagnrg B sifA psifA2HA SKIP SifA and kinesin interacting protein SifA recruits SKIP on Salmonella Containing Vacuoles and Sifs Salmonella induced laments HeLa cells expressing moderate levels of myc SKIP 5X to 10X endogenous level were infected for 14 hours with either a S typhimurium strain sifA psifAZHA secreting SifAZHA a sifA mutant strain and immunostained for SifA HA green SKIP myctagged red LPS blue Scale bar 5 pm B I wt ctrI RNAi El sifA ctrlRNAi I wt SKIPRNAi r T L T i a Q o 9 2583 9 xv 30 0 04 SKIPRNAi treatment of cells strongly reduced the ability of wild type S typhimurium to induce the formation of Sifs decreased the stability of SCVs resulting in the release of wildtype bacteria into the cytosol redistributed SCVs away from the nucleus Phenocopied the effect of the sifA mutation Thus SKIPdepleted cells are unresponsive to Si A and SKIP acts as an essential mediator of SifA functions Kinesin red Bacteria green LAMPl blue O 3164 kinesin MelJuSo BMM Vacuoles containing the sifA mutant accumulate kinesin during the course of an infection The percentage of kinesinpositiVe SCVs at different time points of an infection of HeLa cells was scored SCV positive for kinesin 30 O sifA39 sifA psifA The ssaV mutant which is defective for the secretion of all SPI2 TTSS effector proteins were seldom positive for kinesin thereby indicating that the absence of SifA per se is not sufficient to induce the recruitment of kinesin Rather this phenomenon requires the translocation of other effector proteins Via the SP1 2 TTSS Thus the formation of the kinesin coat on SCVs requires a Jnctional SPI2 secretion system and is negatively regulated by SifA A dynamic process of kinesin recruitment in Salmonella infected cells is mediated by the secretion of SPI2 TTSS effectors and is downregulated by the SifAmediated recruitment of SKIP on membranes S lyphz murz um is thereby able to fine tune the SCVassociated kinesin motor activity by regulating the secretion of its own effector proteins The consequences of an increased kinesin motor activity associated With SCVs may be an excessive formation of outgoing tubules and vesicles eventually leading to SCV disruption The identification of SKIP as a SifA interactor and the demonstration that it acts as a negative regulator of kinesin motor activity provide a key to understanding the molecular mechanism of SifA action Dyneins gt1000000 d composed of10 chains 3 Taildcmaln Moardumain I quotquotquot11IrLIZL P1 P2 P3 P4 135 p6 Heavy nam mnawu L gm chams w ATPase Associated with cellular Activities A MTbindine c Cargo Attachment Journal of Cell Science 113 25212526 2000 Background Recent iterative methods for sequence alignment have indicated that the 380 kDa motor unit of dynein belongs to the AAA class of chaperonelike ATPases These alignments indicate that the core of the 380 kDa motor unit contains a concatenated chain of six AAA modules of which four correspond to the ATP binding sites with Ploop signatures described pre viously and two are modules in which the P loop has been lost in evolution Mocz and Gibbons Structure 993 2001 ATP only binds at a site between D1 and D2 Structure Vol 9 93 103 February 2001 How motive force is generated by ATP hydrolysis The catalytic head must coordinate three cycles Stepping of Dynein Snml Generating a mechaniCal motion Atme Binding and hydrolysis of ATP and release of products D Binding and release of its track 8 12 16 20 Time seconds Cell 126 July 28 2006 2006 Elsevier Inc 243 There are movies mu llLIIL KIM1 mm Imcmumm SW1 pgu pm y Hum PI V M 39l H39Hum 4m In Icglm AAA 1 39I39IV W A a m Linn Ldrumm l 4H0 l 21 l l all l E l ml E l l O H l 5 El l ll 03 Proposed modular evolution of the dynein heavy chain 1 the premotor AAA domain was 2 duplicated once then 3duplicated again 41nteractive domain and 2 additional AAA domains and then 5a neck and tail lt3 I I I I lt4 I I I I 0 I I 5 I I I I 0 I I Fig 4 m 2 r 39 39 m 5 The addulon onne neck blue and tail yellow Simplified view of carriers An ongoing tug of war between dynein and kinesin occurs at the Golgi level Golgi scattering has been observed upon disruption of dynein function presumably because the plusend directed motor is still active In contrast the disruption of kinesin function causes the collapse of the Golgi apparatus Journal ofGeneral Virology 2001 82 26912696 Printed in Great Britain SHORT COMMUNICATION Molecular basis for the interaction between rabies virus phosphoprotein P and the dynein light chain LC8 dissociation of dyneinbinding properties and transcriptional functionality of P Nicolas Poisson Eleonore RealZ Yves Gaudin1 MarieChristine Vaney Stephen King Yves JacobZ Noel Tordo2 and Danielle Blondel Laboratoire de G n tique des Virus CNRS 9 198 Git sur Yvette France 3 Laboratoire des Lyssavirus lnstitut Pasteur 25 rue du Dr Roux 75724 Paris Cedex 15 France Department of Biochemistry Connecticut 060303305 US The lyssavirus phosphoprotein P is a cofactor of the viral RNA polymerase and plays a central role in virus transcription and replication It has been shown previously that P interacts with the dynein light chain LC8 which is involved i minus end directed movement of organelles along microtubules Coimmunoprecipitation experi ments and the twohybrid system were used to map the LC8binding site to the sequence 139RSSEDKS TQTTGR S Sitedirected mutagenesis of residues D 3 and Q quot to an A residue abolished binding to LC8 The P LC8 association is not required ior virus transcription since the double mutant was not affected in its transcription ability in a minigenome assay Based on the crystal structure of LC8 bound to a peptide from neuronal nitric oxide synthase a model for the complex between the peptide span ning residues 140 150 of P and LC8 is proposed This model suggests that F binds LC8 in a manner similar to other LC8 cellular partners Members of the genus Iussunim l1sz 2 cinoladrnlal University of Connecticut Health Center 263 Farmington Avenue Farmington A anisms involved in axonal transport of the virus remain unclear Lyssavirus ribonucleoproteins RNP contain the genomic RNA tightly encapsidated by the viral nucleoprotein N and the RNA polymerase complex consisting of the large protein L and its cotactor the phosphoprotein P Emerson amp Wagner 1972 Both L and P proteins are involved in transcription and replication During transcription a positive stranded leader RNA and five mRNAs are synthesized The replication process yields nucleocapsids containing fulllength antisense genomic RNA which in turn serves as a template or the synthesis of positive sense genomic RNA V P protein is a noncatalytic cotactor and a regulatory protein it associates with the L protein in the polymerase complex and interacts with both soluble and genome associated N proteins We have demonstrated previously the existence of two N proteinbinding sites on the P protein one located between amino acids 69 and 139 and the other located in the carboxyterminal region comprising amino acids 268 to 297 Chenik el 111 1994 We have shown also that the maior Lbinding site resides within the rst 19 residues of P Chenik cl nl 1998 in addition four other aminoeterminally truncated products PAZ PA PA4 and PAS translated from P mRNA Luuh quota AU J ehA E J a r f I II n u The lyssavirus phosphoprotein P is a cofactor of the viral RNA polymerase and plays a central role in virus transcription and replication It has been shown previously that P interacts with the dynein light chain LC8 which is involved in minus enddirected movement of organelles along microtubules Coimmunoprecipitation experi ments and the twohybrid system were used to map the LC8binding site to the sequence 39RSSEDK5 TQ ITGR S Sitedirected mutagenesis of residues D145 and Q 7 to an A residue abolished binding to LC8 The P LCB association is not required for virus transcription since the double mutant was not affected in in transcription ability in a minigenome assay Based on the c tal structure of LC8 bound to a eptide from neuronal nitric oxide synthase a ning residues 140 150 of P and LC8 is proposed his model suggests that P binds LC8 in a manner similar to other LC8 cellular partners Protein Sequence CeHIulrar N05 peptide D T C 1 Q v Mychsz 1quot D T Q 1 Q L Dynrmrirl D S 1N L Q v Bim D K S T Q T Vira 1w P D K 5 T Q T Kuhn rms WM D K S T Q LH Cul mrkr mvirtas lquotI Z D R V LIEfl Q L Fm39r39oz Ems lv FE D R V UM Q L JOURNAL or VlRDLOGY Oct 2003 p 10270 10279 Vol 77 No 19 llUZZSJSXOJEUSIlOit DO 101128IVL77J9l0270 l2792lt3 Copyright 5 2001 American Society for Microbiology All Rights Reserved Exploitation of Microtubule Cytoskeleton and Dynein during Parvoviral Tra ic toward the Nucleus Sarina Suikkanen Tuula Aaltonen Marjukka Nevalainen Outi Valilehto Laura Lindholrn Matti Vuento and Maija Vihinen Ranta Department of Biological and Envimnmenml Science University of Jyudskyl FIN40500 bums9111 Finland Received 21 April 2003Accepled 30 June 2003 Caninc parvovirus CPV a model virus for the study of parvoviral entry enters host cells by receptor mediated endocytosis escapes from endosnmal vesicles to the cytosol and then replicates iu the nucleus We examined the role of the microtubule MDmediated cytoplasmic traf cking of viral particles toward the agents viral capsids were unable to reach the nucleus The nuclear accumulation of capsids was also reduced by microiniection of an antidynein antibody Moreover electron microscopy and light microscopy experiments demonstmted that viral capsids associate with tubulin and dynein in vitro Coprccipitation studies indicated that viral capsids interact with dynein When the cytoplasmic transport process was studied in living cells by microiniectlng uorescently labeled capsids into the cytoplasm of cells containing uorescent tubulin capsids were found in close contact with Ms These results suggest that intact W5 and the motor protein dyuein are required for the cytoplasmic transport of CPV capsids and contribute to the accumulation of the capsid in the clus Nontreated Nocodazole Vinblastine Taxol l l 1n um E Parvovirus tubulin Control Nocodazole Vlnblastine Taxol of cells showing nuclear fluorescence Antibody Capsids Control Ab Antikinesin Ab Antidynein Ab In Vitro binding MTs Capsids With PNS Without PNS post nuclear supernatant PNS contains dynein In living cells anfzi ra 7 quotm 339 S I 3 Coimmunoprecipitation PRECIPITATED WITH CONTROL IMMUNOBLOTTED CPV Control Ab Antidyneln Ab Antndyneln Ab 1 2 3 4 CONCENTRATED VIRUSES NONINFECTED CPV CELLS Western blot probed for VP2 MTOCs Microtubule organizing centers Centrosome Centriole Centrosome Many microtubules converge on this site Polarity is always the same Microtubule Regrowth after Depolymerization Treat with nocodazole wash out allow regrowth Mts radiate from centriole with their minus ends localized at the centriole and the plus growing ends radiating away Ci 0 Photo is fixed and probed for Mts a 5 a with a fluorescence antibody 30 039 quotquotquot 339 min after recovery from drug Mex w 9 K 043 dimers added to the growing end Relationship of endomembrane system to microtubule network GMAPZ 1 O and Golgi positioning Cell 118271276 2004 A Fericentrosnmal Golgl rlbbon B Linking adlacem Golgi cisternae I 9 mm o rlubuhn Centrosome C Membrane capture and mum engagement Attachment LLleHiH Y 39 GMAPZ 1 0 Y tubulin Very low concentration relative to or and B tubulin double stain of cell for B tubulin and y tubulin Where two protiens coincide in space the fluorescence glow is orange Role of39Y tubulin in MTOC Postulated to act as a template within the MTOC 9 that initiates tubule assembly ytubulin Microtubule dynamics 1 min growth caught by injection of a tagged biotin tubulin monomer that got incorporated into the growing ends of microtubules Fluorescent antibodies that react with biotin reveal the new growth Dynamic instability Bleached um 3 I09 EEI 392 i lt1 Na b q 39 1 1 391 3939 H B subunit of Monomers binds GTP Essential for incorporation into a tubule GDP i GTP Cilia and agella Plasma Inner membrane dynem arm Outer dynern arm Central sheath Nexm budge Outer doublet A tubule Radlal microtubule spoke Central pair of microtubules surrounded by 9 doublets Basal body anchors cilia in the cytoplasm Cllium Basai body Centrai Centrai Radial Outer mlcrotubule Sheath spoke doubiet J l quot quot 39qu 3 building blocks Microtubules Intermediate Microfilaments filaments Vimentin nn nmu in Emualumnus mmm Tubulin Vimentin Actin 3 building blocks Intermediate Microfilaments filaments Vimentin rhnxln minus 39 nunrestuiu wrmmm Vimentin Actin Intermediate Filaments Intermediate Filaments 0 Solid smooth unbranched lOnm dia 0 Approx 1 of protein 0 thicker than micro laments thinner than microtubules 0 animal cells 0 a heterogeneous group of related proteins gt50 genes Properties and Distribution of the Major IF Proteins Component Polypeptides Type of IF ns Cellnlnr nation Nuclear iamins lamlns A B and C nuclear lamina of eucaryotlc cells ssooo 75ooo Vimentlnlike 39 Mquot quot proteins often expressed transiently during development desmln 53000 muscle bnllary audit glial cells astrocytes and protein 50000 Schwann cells penphenn 66000 neurons Keratlns type I acidic 40000 70 epithelial cells and their derivatives type I neutralbasic cg air and nails LOUD70000 Neuronal neuro lament proteins neurons intermediate NFL NFM and NFH ents 641000130000 Structural similarity ahelical rod domain amino terminus keratins i 7 vimentin n 4 neuro lament proteins H nuclear lamins W u 1 LJ I Q LJI 41 regions containing heptad repeats O B COOH uhelical region coiledcoil dimer NH NH Proto bril staggered tetramer of two coiledcoil dimers COOH Lack of polarity in the lament Assembly and Disassembly 0 Highly resistant to tensile forces 0 stable to chemical disruption 0 yet dynamic as shown by the incorporation of new subunits into the structure at many scattered sites IF network O 12 life is 12 hrs 0 Mediated by phosphorylation 0 not apparently involved in any vital cellular processes IFs provide tensile strength to cells New subunits added dead keratinizsd layer uf squames quotm grammar ce l layer prickle cell layers basal ca layer H basal lamina connective tissue of dermis A man pm Keratohyalin filaggrl39n crosslinking and compacting keratin filaments K5 and K14 Epidermolysz39s bullosa Simplex Skin sudace W 45 Stvamm comeum i Granular layer 53 r C is u swam g 9quot keratinlilamanls 1 madeothande gs 4 Basal layer 39 W keratin mamems W made 01 K5 and K14 1 zasemem membrane g l 39 K5 mutant lament disrupting t Null Minor mukallon mechanical stress Gleaner mechanical stress Dsmus mice exhibit skin blistering on mild mechanical trauma K14 different mutations severity of blistering correlated with degree of perturbed assembly
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