Science Magazine 5855 2007-11-30

Eee)The Nucleus INTRODUCTION Journey tothe Conte ofthe Celt NEWS Return ofthe Matrix REVIEWS. IAL SECTION Toward a High-Resolution View of Nuclear Dynamics TinkleMulcaty and. amore Blucring the Boundary: The Nuclear Envelope Extend Is Reach CL Stemart, Kj. Rou. B. Burke Crossing the Nuclear Envelope: Hierarchical Regulation of Nucleoeyoplasmic Tensport LJ Tem, € 8 Shows, 5.R Wente COVER The cell nucleus houses the genetic Dlueprin, butts also a dynamic organelle that interacts with the ret ofthe cell on many levels. A special section beginning ‘on page 1399 focuses onthe cel biology ofthe nucleus. mage: Chris Bickel Science 1399 1400 1402 1408 1412 Seo Scone # SIE mater p. 1388 ort wscncemes ogpceWenarden! vawuscioncomag.org SCIENCE VOL318 ON Volume 328, Issue 5855, DEPARTMENTS 1343 1345 1351 1354 1357 1359 1396 1482 1484 EDITORIAL 1349 The Quality of Public Dialogue by Katy Ses Science Online Thi Week in Science Editor’ Choice Contact Science Fandom Samples Newsmakers AAAS News 8 Notes New Preducts 1368 NEWS OF THE WEEK New Estimates Scale Back Scope of HIVIAIDS Epidemic Tense Meating Produces Some Hope for Flu Sharing Deat Hominid Herems: Big Mates Competed for Smal ‘Ausraloplthecine Females SCIENCESCOPE Smithsonian Struggles to Strike a Balance th Sponsors Pilot NSF Pragram Files int Sif Community Headinds Germany Finally Picks a Nationa Science Academy NEWS FOCUS Should Oceanographers Pump iron? MIT Engineer Shakes Korean Academia t Its Core Camel Scientists Ask: What's Sinking the Ships of the Desert? ‘Conele Antes Nake Waves Columbus Iniects Science Int Stet LETTERS (Global and Local Conservation Priorities _Dinestein: FR. Scarano; . Choudhary: A. Mitermere etal. Response)? Rodrigue et al CORRECTIONS AND CLARIFICATIONS CONTENTS cont 30 NOVEMBER 2007 1360 1361 1363 1363 1364 1365 1367 1368 aan 4372 1374 1377 1382Science SCIENCE EXPRESS MOLECULAR BIOLOGY Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation 5. Vasudevan ¥. Tong, j.A. Stete |Athowhtey kit trsion ining els kayo microRNAs an bind Othe untried gional mesenger RUA ard acivatetansaen upon cee aes. IO.L12Sicienee, 1149460 ASTROPHYSICS Stellar Feedback in wart Galaxy Formation 5. Mashchento, J. Wadsley, H. M.P. Couctnan Simatons show tha stellar wigs ond material exes kom supeevae ater the gratatonl patent of a gales, perapseplaling thei eark mater coves YOLI2slcience. 1148666 BOOKS é7 AL The Stuf of Thought Language as aWindaw into 1384, Human Nature 5. Pinker, reviewed by. Lakoff ‘Supercontinent Ten Bilion Years in the Life of 1385 (Our Flanet ied, reviewed by K. Burke POLICY FORUM Climate Assesment: What's Next? 386 Rees ond R Swart EDUCATION FORUM Preschool Program Improves Cognitive Contol 387 A. Diamond, W.S. Barret J. Thomas §. Nunto PERSPECTIVES The Leaking Mantle 369 D.R Hilton Paradigm for tite 390 1.0. Mcinerney and 0. Pisani Suiface-Conducting Diamond 301 CE Nebel ‘Metal-Based Therapeutics 392 We Hambley SCIENCE CONTENTS L ASTROPHYSICS. Willion-Degree Plasia Pervading the Extended Orion Nebula 1M. Gael, KR. Briggs, - Montmerle a. Auda, L. Rebull, SL. Skinner ‘ior -dege as ils he Oxon Nebel, implying tha stokheated gas om sell cuton conan ner Gala, 26icien 149926 Ecology Climate Change, Deforestation, andthe Fate ofthe Amazon Y.Malhi JT Roberts RA. Betts, TJ. Klleen Wi, CA. Nobre Te fleets ft Amazonian forest ame onthe basphere andthe atosphere spend on uur nancial and pata responses to ciate change TOLL L2s\science.1146961 Precision Without Entanglement 1393 JL O'Brien [-Defersin Repertoke Expands 1395 JR Dorin and I.) Jackson >> TECHNICAL COMMENT ABSTRACTS Comment on “Decagonal and Quasi Crystalline tings in Medieval Islamic Architecture” E. Makovicky Response to Comment on "Decagonal ard Quasi-rysallne Tilings in Aedieval Islamic Architecture” PJ). and }.Sweinhaide BREVIA BoTany Plants Tolerant of High Boron Lovels Kina etal ‘An rcbidpes protein ennancsthe borate etx tem rots; Incorporating its gene inte cops may hele ingrove their gromth ‘nave si containing exes boon. RESEARCH ARTICLES GENETICS {Af-Defensin Mutation Causes Black Coat Color in Domestic Dogs ‘entcaton gene causing black coat clr in dos revees £2 surprising nen rl or protein Fmt rewously pistes in the body's eens agaist microbes CHEMISTRY Charge Tansfer Equilibria Between ‘an Aquecus Oxygen Electrochemical Redox Couple ‘he puzling nigh elects conduct of ame chaond surfaces Is procuced by electron varserto agen in wate as coating te CONTENTS cont VOL318 30NOVEMBER 2007 1339REPORTS PHYSICS Coherent Control ofa Single Election Spin with 1430 Electric Fietds KC Nowack etal Te sin sat fa single elecion ona quantum cet canbe maniou- [ated by apatiation of local elec ld, imprevieg the cont of mle cts over what is achievable witha magrticfiea. GEOCHEMISTRY low of Mantle Fluids through the Ductile 1433 Lower Crust: Helium Isotope Tends B.A. Kennedy andi. van Soest tn hittin areas the western United States, heli ‘yourewaterinaeatean lxot gas tom the mane, imag that Aelrmationhas enhanced permedbiiy, > Pespectiep. 139 MATERIALS SCIENCE In Situ Determination ofthe Nanoscale Chemisty 1437 and Bohavior of Said-Liquid Systems 5.K Esucramoorthy J. M. Howe, 6 Murlidharant AAmotiied tansmission eleven microscope operated wake hating asamp reveals metastable pase boundaries nt chemical composition cress a sli guid intra. PaYSics Fluctuation Superconductivty in Mesoscopic 1440 Aluminum Rings N.C Keshnic, H Bluhm, M. E. Huber KA Aller soni scaning microscope reveals magnetic and superconducting fctvatins of nanometesizedalurinum ings ‘mat approach te coherence se of tae phenomena ANTHROPOLOGY Extended Male Growth ina Fossil Hominin Species 1443 CA Lockwood et al. ‘large sale of fail bones oman eat homie Parenthropus roosts shaws that mates mature ater than females andimay have rrenopoltes gaups lfm, >» ws ser». 2269 PLANTSCIENCE Bororonicty Tolerance in Baley Arising rom 1446 Efile Transporter Amplification T Sutton etal. Wie barey stains ha are usually tleran a boron have ona Coies ola gene that codes orator anspor GeNENcs GGencme-Wide Experimental Determination of 1449 Barriers to Horizontal Gene Transfer Soret et al. Characterization of gee faiesthat cannot cloned inthe commen -nctesaEsheichi ct suggests that inceasee dosage and expression of era genes at toric te host. >> Perspect» 1290 GENEncs Altolanocotin 1 Receptor Allele Suggests Varying 1453 Pigmentation Among Neanderthals C Lolueza-For eta Neandertal carried variant a stn ell ects o that in modern Eurooeuns, suggesting that ter eigrentaibn may have ber similar saab CONTENTS L IMUNOLOGY 5'Triohosohate-Dependent Activation of PRR by 1.455 [Ras wth Short Ster-Loops S.A, Nollagatla et a ‘A pathogensesing peceine he inrateamune ten ecogios the -tiphesphate structures of single standed RNA preset in an bacera ae vses BIOPHYSICS Direct Observation of Chaperone-Induced Changes 1.58 ina Protein Folding Pathnay P Becttieftot a Singlemmoiecule measurements eval how the chaperone Sec affects the folding pathway of maltose bincing poten toast ts ttanslcation acrss the cytoplasmic memérane in bara, STRUCTURAL BIOLOGY Carbon Dioxide Activation atthe Ni,Fe-Clusterof 1467 Anaerobic Cerbon Monoxide Dehydrogenese JH. Jeoung and H. Dabber Stuctuesof a carbon manoiide dehyrogenase enzyme in three ‘ier xtiation sates shaw bow cern luster actos seduces carbon die BIOCHEMISTRY Solvent Tuning of Electrochemical Potentials inthe 164 Active Sites of HiPP Versus Ferredoxin A. Deyetal ay absoption Ais show tat hydration the ace ite etetraees eter on-air casters election waster protelas react enieatialy or edutvely GENETICS The Obesity-Asociated FTO Gene Encodes @ 1469 2-Oxoglutarate-Dependent Nucleic Acid Demethylase T Gerkenetal {A gene that acts aningivoua’ ret for obey cede for pete that reroves etl groups from DNA, althcugh how this fanction regulates best is under NEUROSCIENCE {Expression and Function of Junctional Adhesion 472 Molecul-C in ayetinated Peripheral Nerves .Seheiermann etal. ‘nice tat ormspartetary ght junctions between mestinal ot endothelial cls aie seals he ends ofthe myetin wrapping around ADVANCING SCENGE, senna YOCtETY absense Selimpaeccahecananie Sec ea een cece ots enero wt Senger ence Printed on recycled paper. 30% post-consumer CONTENTS continued >> vawuscioncomag.org SCIENCE VOL318 30NOVEMBER 2007 1341“angled webs, SCIENCENOW musciencenoa.org DALY NEWS COVERAG Untangling an Artistic Spider Web ‘Aeaige spiders eave complex patent ai! prey, ‘eathey py a price ‘An Environmental Conteminant Invades the Wom’ Mothers may pass hei eects of arsenic exposure cntother ering, lyme Disease’s Unusual Suspects Mice, wall blamed forthe cisease, turnout to be cy one of many els. SPECIAL SECTION The Nucleus SCIENCE’SSTKE twnsthe.og. SIGN ‘on en i EDITORIAL GLIDE: Focus sue—astoring Nuclear Dynamics NR Gough The nuceuss connected to cjtopsmic sgnaling networks ands itso comples signaling enrerment, PERSPECTIVE: Genomic Maintenance— The p53 Poly ADP-ribosyUation Connection .Alvarez-Gonzalez In esponse to DNAdamace, 953 is madiied by pMADP-rbose) polymerase PARP-1) and acumultes inte nucleus. PERSPECTIVE: Nuclear Localization of Growth Hormone Receptor —Another Age of Discovery for Cytokine Action? 5. Swanson and }. J. Kopchice Transecation ol the goat hoxmonereceptr to the ruceasis Linke mt tumerigoness ‘Scientists salaries in Europe, SCIENCE CAREERS: un sclencecaeers.org CAREER ESOUNCES FORSCIN) EUROPE: To Have and Have Not A. Sworup Salaries fo European sie vary grey by country and region. NISCINET: Big Science at a Small College P Shulman avert Andrea Morris the fst smal-cllege faculty membsr toni an NI Corer Development Ava, US: Teating Up—Overeoming Conftias inthe Lab and Beyond D.Jersen I you face cont squarely and calmly, you can transfor them {ia beter eationshis and pssona gent. US: GREAT Expectations B.Berderty ‘nes oxgariaton aims to focus national attention on sues facing meical poste, SCIENCE PODCAST. Download the 30 November Science Fodcast to hear about itoa festiliation experiments, and more won cer emag sco e. sexual dimorphism in male fossilhominins, hot plasma - outfiow from the Orion Nebula, —_ ‘Separate individual or nstttional subscriptions to these products mey be required for ful text access, wuwuscioncomag.org SCIENCE VOL318 30NOVEMBER 2007 1343EDITED BY STELLA HURTLEY AND PHIL SZUROM! << To Fold or Not to Fold... Breathing Conductivity into Diamonds ‘hamond san excellent electrical insulator, but For neatly 20 years, researchers have puzzled ‘ver observations thatthe surlace of hyerogen- terminated diamonds becomes conducting after exposes tai. The phenomenon has recently been applied to device design, but a com pling explanation ofthe underying chemistry remained elusive. Through careful electro chemical measurements, Chakrapani et al. (p. 1424; se the Perspective by Nebel) show that the postive hale catiers responsible for Conduction arise from electron transfer to oxy gen cssolved in aqueous films onthe surfaces. Similar interacial charge trensfer equilibria ould be ative in other semiconducting sais. Controlling Spin Electrically Manipulation of the spin of asingle electron stored on 2 quantum dot is a strong contender a the basis for quantum information processing Coherent manipulation of the spin has been demonstated fora locally applied magnetic File, but scaling up to laiger ensemble systems of spirs ane the individual manipulation of spin th magnetic field preset sigelicant exp mental problems. Nowack et el (p. 1430, pub- lished online 1 Novernber now show that coher ent manipulation ofthe spin state can be achieved using a locally applied electric fet to the quantum dot. They argue thatthe observa wawesciencemag.org SCIENCE VOL318 30NOVEMBER 2007 tion of coherent Rabi oscilations arises from electrical modulation ofthe spin-orbit interac tion experienced bythe confined electron Helium Tracing of Rising Mantle Fluids ‘Mantle helium has a high "He*He rato but is diluted by He from other crustal isotopic processes. Kennedy and van Soost (p. 1423; see the Perspective by Hilton) Investigated the «geographic distribution of helium isotopes across the Basin and Range province of North America. A gradient in Here corelates with rates of active deformation ofthe crust ane the highest ratios occur where the extension and shear stain rates are oreatest. This finding Inleates that deformation enhances the pe: rmeobilty ofthe lithosphere and alow uid from the mantle to penetrate even inthe absence of local ‘magmatism. Loca spots with high isotope ratios may also pinpoint areas of high ‘austal permeability that maybe valuable for geothermal energy development, Snapshots of Crystal Growth When compler alloys crystallize, partitioning of elements can be expected between the liquid and solié phases, but measuring these Molecular chaperones not only prevent misfolding of proteins but also play an active role in protein folding pathways. However, how protein folding pathnays are affected by chaperones remains poorly understood. Bechtlutt et al. (p. 1458) used a combination of experi- ‘mental and computational approachesto study the effect of the chaperone SecB on the folding and unfolding pathways of maltose binding protein (MBP) atthe single- molecule level. SecB retains MBP in @ molten globule- Uike structure so that no energy wil be needed to disrupt stable tertiary interactions during translocation across the cell membrane. ‘changes isa challenging experimental task Eswaramoorthy et a. (p. 1437) used energy dispessve, ray spectioscopy during in situ transmission electron microscopy observations te allow crystallization of an AL-Si-Cu-Mg alloy used in automobile and aerospace applications. They heated nanoscale particles to different temperatures and observed stable and m stable phase boundaries, as well as measured the chemical composition in each phese. This technique may be of use n clarifying the growth mechanisms of nanowires and related siretutes, Parsing Extinct Primate Dynamics Infetrng the social characteristics of early homiains iseffcult, in part because fossils are fen, scattered, and rarely reveal aroup dynam Jes, One more rl: able indicator may be the develop: ‘mental patterns between males and females and, by inference with other primate specie, wat such Findings might imply about socal and repro ductive strategies. Lockwood et al. (p. 1443) used a large sample of facial fossils from Poronthropusrobustus 1 show that males of this specie, extant about 2 milion years2go, Deparnet of Flog and Eveutenay ily, Preston Une nce BUSA. *Conigonteg author onal aber@ovdtitest ae Reference and Notes 1. GG, eh, Mapping ceserton inet: nascent sty nding ain Aes {nd ibe Coates” abot Sips Poy, ashing, . ec 2. Padre, 8 Simones A Pl MA. Ma Came fc 29,569 0009. 3. figun Kit Rogue in kk tin tends a ep anars o.20, 140 208) 4. Theses tne of Wei eed (aout sestou Ihekat Conergy (masta gatoaaerts) Wl Cononaten Sty mses agree) 2nd Canenasen tomate tps oceans CME ou, 5. Gn Nace ta, Meta 405, 393 2000 GG Pent, Senne 296 904 20029 CORRECTIONS AND CLARIFICATIONS Reports: ‘R‘slent’priymarpisn in the MOR gene ‘ranges subsate pecticebyC ine Safety tal. 26 January p-525). Based on anne rom xk Konia, teach’ wish to daily that te ete sequence was ‘tain rom a etaied mass pctramotc tidy pe- formed atthe Hanae WeracherityFacity (AMF) by miocaplay reverse-phase HPLC ano decor ia ‘em assspucvomety. HF eormec oth chymotyptic jee iz ee Sa Faae teres oe DRE SONOVEMBER 2007 VOL3I8 SCIENCE SESE ieee wanuscioncemag.orgLETTERS i ‘and pronase digestions ofthe pron In al, 82 peptides (ereserting 379% ofthe Pap sequence by arin act ‘cun) were senile and sequenced Gee Supporing Onno Maer a wa sienceag xfegcontony ald BIBSBSSM3BLOCL. Each Tes sequnas wasident ‘alto the eevee of apletpePahcoprokn. Meee ‘sever ile pee ede by Iesyneryous SNP (GABSC=N, which the key polyno ne tote funcral hangs in Pep, nee sequenced and ound to be undhange. In addon, the analysis of codon usage, table Stine eriginal Suppoting Online Materia we sewage 13S208/0CL/) contains Fer each cece around the te polymorphisms the fe- ‘quency ef tis cadon per 1000 codors in the human ‘genaenstac of SCU ales, atte inthe ex These ‘luce were obtained from the cecen wage Wed ste (oniaasa.orjpeadoagbinshoncolonceP specie Nomossapirse%s5 tarP65D)-Figze 1, panes Dt ‘has esse presence of gelxpen ACCA), et (G/C,andcated in he by ol he Bg. ri con- recy ated in te lecend. Tes darfeatns co net ae the condusrs athe page TECHNICAL COMMENT ABSTRACTS Comment on “Decagonal and Quasi- Crystalline Tilings in Medieval Islamic Architectur Emil Makovicky Lu ad Serhart Reports, 23 Febrary 2007, p. 1106) claimed the discove'y ofa lage, potentially quasi ‘stare lacing in the Dart imam shrine but teguc the ene Naragha lrg. evs exe bed as ‘quasperindk, asa smal olated met We comonsrate ‘hat the arb Imam pater i prec anc that te ‘quas-caline is sperinpoiee on Latte 3 Aervatins f the Magna pate. Full text at wen scencerag ogiegitomtentul318/ s8s9/383a Resronse 10 CoMMENT ON “Decagonal and Quasi-Crystalline Tilings in Medieval Islamic Architecture” Peter). Lu and Paul) Steinhardt ‘ur tudy showed that bet Gurka i Kabud and Dar Imomtesltons belong to» sequence of lami tings that esl ieto common sof gi less loca int lates re expected However, histo acurateecor- structions stow tat Darb imam unique, the only Tron exanpte ta does rot pent perl an that ‘gaya al-a randormatin enaning con vation 2¢nintum to a pret eashestaine pte Full tt at wwmscieceragorgtg/cortentful’316/ Sess/1383b Teena og BN ul dp » e @ Tackling Reproducibility Issues Join our panel of expertsin a LIVE online seminar on December 5, 2007 Participating Experts: feces (Srvc o Montel techs Tinothy Veerste, PLO. AC rece, Ine Rattonn Canecr nse atrredek Frederick MO ToniWhistier Ph.D. (Ghent Vit Bstases Branch Cners fr Disease contol, gadPreverton ‘Alona, 6A eee eee eee in Mass Spectrometry-based Biomarker Discovery Mass spectrometry is an extremely powerful and Sensitive technology and can detect very small ‘changes in expression levels. Some of these changes may stem from the bialogical differences related to adisease or treatment of interest. Others. however, may reflect the heterogeneity ‘of patients across multiple sites, the inherent biological complexity and diversity ofcifferent ‘sample types, and even small cferences in sample collection, processing, handling, and analysis techniques used by multiple operators actoss multiple locations, As a consequence, data may be tainted by ite-, study, population-, ‘or sample-specitic anomalies an¢, therefore, not be sufficiently robust for biomarker discovery. + Join our panel of experts ina live, audience- driven Q&A as they discuss hovr to overcome these reproducibility issues to generate less biased and more reliable results. » Questions for the panel can be submitted live through your viewing console. Register now! For more information and complimentary registration online visit www.sciencemag.org/webinar Brought to you bythe Science ARAS Business Office RYAAAS Webinar sponsored by BIO-RAD eee eee coe ooo1384 PSYCHOLOGY Learning from Words Robin Lakott ge sees tury touts, oo patoay an pacona dea. bat pak haparvay? Comms tas fil sane orwell oer unease tne sage should some fe wor oily hpi il id peau and elk dation in Steven Pitkers new book The Sf of hows Langage onto oto iano Sena, ake cog ne ach pitt laren Un Complex might terse be off-pting Hiscnerl hemes the vay in wich ings ‘our brains are wired. Ina sense, the work of Pinker and his col- Feagues is continuation of Chomsky’s revo lution ofa half-century ago. Chomsky. after all, famously called a winden’ into the minel a statement Pinker's subtitle echoes. But where Chomsky justified his theories inuuitively,creatinghisexample sentences and testing their grammaticality in his own mind, Pinker and his colleagues are empiricists, proving their claims by experimentation Pinker discusses language at all levels from sounds, 10 words and phrases, to lager units (sentences and beyond). There isa set ‘oF English words that begin with gl: glare, low, glass, and. glimpse (among many others). Is the similarity due to mere ‘coincidence? Pinker says that all such words have as part of their meaning the suggestion ‘of “emission of light.” Even The want (© banish from the domains of orderly reason like fourclewer words, fans re works according. to ‘general principles. Pinker cites the work ofthe fictitious linguist Quang Fue Dong (an alter ego ofthe very real linguist James D. MeCawley), sho raved against the common assumption that “Fuck you!" is an imperative. Ta a true imperative. a seeon-person diteet object Te reviener sa the Deparnent of tings. 1203 Davrtle Wall Unvesty of Calemla, Bereey, CA 9620-2650, USk Ea ak erey cs 30 NOVEMBER 2007 Pn Alice finds Humpty Dumpty very clever at ‘explaining words. must be reflexive: yourself, not you. OF course, this discussion (inthe chapter “The seven Words you can't say on television”) 004 Pinker deepens the discussion to illustrate important properties of Tanguage. The fun aspects of li istic theory are inseparable from its deeply serious nature. his necessary connection between depth and wit is echoed in Pinker’ style. Where the discussion might bog down ofits own ‘weight, he leavens the dough with ‘humor: quips, anecdotes, jokes, and cot strips. Chomsky ancl other cognitive theorists are often cited. as we would expect; but so, 0 good effect, ate Groucho Marx, Lewis oll, and Dovethy Parker language: the way we use it inte choosing to be direct or indirect, polite or rude—that is, o behave as proper (oF not) social beings. Just as language is rigorously rule-governed at the sound and word level, ‘our brains impose predictability on our deci- sions at this more abstract level 2s well, How do you get someone to lend you money? Some of us might adopt a strategy of indirect pessimism, “I don't suppose you'd be able co mney?” Whereas others naraderie: “I know f ean count on you to help me out with some ‘Cultures, and the individuals within ave preferences, depending on spe crs’ assessments of the social and psycholoz ical contexts in which they ae trying to meet their needs. Althouzh Pinker’s diseussion of this top is generally informative and eng e place where he migh nd the fields in which he claims expertise, Interactional socialinguists like John Gumperz, Deborah Tannen, and Deborah ‘Schiffrin have had a great deal of imerest to say on these topics. Likewise, the work of William Labov ina great many areas of soci linguistics would provide Pinker with additional evidence of the rationality and logic of linguistic choices, But the book is curiously lacking in any discussion of socio= Linguistics whatsoever Albough he author generally shows aper- fect intuition for how much to say about com plex topies, occasionally be falters, His dise cussionof meaning and naming inthe chapter “What's in 2 name?” while usefil in leading n ofnaming con ventions of all kinds, goes into too much for: detail, atleast fre my tae. On the other hhand. despite its length his discussion of politeness and indirectness, as noted above, ‘oversimplifies the qu But the: cussing such an inviting and important VOL318 SCIENCE wnsciencomag.orgbook. Everyone with an interest in language and how’ it gets to be how it is—that is, everyone interested in how we get 10 be human and do our human business should read The Stuff of Thought waarbrcece. 150867 GEOSCIENCE Dancing Continents Kevin Bute G iets ec saloon isthe Pa seca Tactsaonc dra mening be ranon aia cee ares fara esti me thc slept ae el withthe epening al hn ety yout ape Tse Wi pone! ta because similar analyses are impossible for tes fort age tc oe ca fn tebe we cand ve inepe te ole feologial ecole tims of eee (nw Eiadtviton ela) of zope snlecosngatecen bash Ths wey of Svein fea ica Lorlaces ice beter inp ben dopa wikis rene at eat Bane opens and closed Ted Nii magnon has ben tsb on pet of hese cits, Me wes The Eas Tad meses oe loted nn al Ghodrile tat geological Siperanen Chee be gn esol tic patrs ono inv mo moycnvonome ede toteabb acept tat thos dh tat tei supersonic boa eng inthe lar Nel i ltr fing Soy ot Conon’ msgs Genco hs "sponded tobi bok y win Dok thao inne anf comraing He has tha el sia arden ba touching cn shige dee of tp, Hs reso fu Ea wih ge per Content ved ye saceush om rin inn iu scans fr hs ks a Mon blo finland posed ‘ome of the world’s ocean basins, like The rene i at he Departmetof Geosciences, SA, Unvarty ef Hotsten, Heaton 12 77208-5503, USA. mae Kola Biber he summsclencemag.org continents as well as their colorful and, in some cases, disreputable proxagonists, Nicld relates many subjects that are currently major foci of research in Earth history to his theme. For example, he provides clear and well informed treatments of evidence fiom Australia forthe existence of abundant conti- ‘ental material slbout Four billion years ago: evidence fora snowball Earth at the end of the Proxerozoic: and evidence forthe. very likely related, ori= igi of complex life forms after the end of that extroondi episode of cooling “Alheugh Lenjayed the book and learned much from it, I remain puzzled about how t9 recognize a supercontinent [came tothe book assuming that supercontinents contain al the ‘continental crust ofthe Earth, The illusirated future example, boldly constructed by Roy Tare Livermore, contains ll continental meterial in Rodinia reconstruction 4 single roughly elliptical body, and Nield describes Pang as a supercontinent that con= tained nearly all continental ens. In ‘surprise when I read: “Pangaea consi ‘nwo smaller supercontinents joined at the hip in the region of the Equator: Laurasia in the ‘Northem Hemisphere... and Gondwamatand inthe Southern....” Iwas left asking what a supercontinent is Ida not think its simply something assembled from smaller continen+ tal blocks, because that would make all cont- nents supercontinenis, Even Greenland (only two million km?) is crossed by two sures, ‘which indicates assembly from three smaller BOOKSe continental blocks. I suggest a testable prop erty for identifying supercontinents would be contains nearly all extant continental crust” Rodinia has been suggested to have been & supercontinent assembled through collisions of various fra already ‘one billion years ago, although my. estimate ofthe area ofits assem: bled blocks mikes it only about half as lage as the younger Pangea. Nield writes a jot about Rodinia, mostly emphasizing its breakup, but does not include a ‘map oF perhaps because there Js ag consensus about what Rodinia looked like. Assembled ‘continental fragments have been arranged around Laurentia in a variety of ways: the figure shows version drawn by Trond Torsvik (2) ‘The difficulties in recognizing ancient supercontinents involve not only estimating hhow much of then-extant continental crust ‘was incorporated into the newly assembled ‘body but also obtaining high-resolution ‘age determinations and distinguish- ing other convergent plate bound- ‘ary phenomena from continental collisions. An even harder prob- Jem is determining where the objects involved in an assembly ‘vere, not only with respect each fother but also with respect to Earth's spinanis. Ancient latitudes and longitudes are essential. There has been progress in estimating. ancient latitudes using paleo: maignetism: thus a few continental blocks now have credible poles for as ong ago as one billion years. But resolving ancient longitudes ‘snot yet feasible, Nield has no persuaded me that there is yet a ease forthe existence fof ancient supercontinents. (other than Pangea) that contained all ofthe E tinental crust-let alone evidence of cycles ‘of Supercontinent assembly. Nevertheless. | think that Supercontinent is a good book because ois ively and stimulating perspective on many topical aspects of Earth's hise tory. A robust foundation is not, after all, essential fra good book: Proust's master- piece rested on no more than the smell of a pasty f References 1. J ilo, fae An Phils Soe 12,309 968. 2. To sence300, 1579 2003. r2eiecenis1202 SCIENCE VOL3I8 30 NOVEMBER 2007 TAL { 13851386 AU) CLIMATE CHANGE Climate Assessment: What's Next? Frank Raes'* and Rob Swart? he Fourth Assessment Report ofthe I Intergovernmental Panel on Climate Change (PCC) (/) is a milesone for climate-change seience and policy, It con: chides that global greenhouse gas emissions must peak and decline within the next dovade tw keep the increase of global mea tempera ‘urebelow limits accepted by some parties. By 2000, there should bean ay proceed with emission cuts afer the Kyoto Protocol's first commitmes 2012, Is the IPCC sil sulfic to support the Us ‘Comention on Climate Change (UNFCCC) in directing the required action? ‘The IPCC is not a UNFCCC body, but the UNFCCC's Subsidiary Body on Scientific and Technical Advice (SBSTA) considers the IPCC a main source of independent informa- sion. We believe that comprehensive IPCC reports every 5to 6 years (which also require Aaditional years to Filter through the SBSTA) are not sufficient to adequately inform policy. In addition, key questions are likely to cu across the boundaries of the eurrent Working Group (WG) structure of the IPCC: WGI deals with the scientific understanding ofthe climate system: WG2 with climate-chang impacts, adaptation, and vulnerability: and WG3 with mitigation of climate change. Topies such as large-scale biofuel use and the regional and global eosts of adlepting to cli= mate change will require better integration among the natural, economic, and social sie fences anid hence, among WG, “The IPCC could lear from an assessment process that is fser and mee ietegrated and ‘that supports the U.N. Convention on Long: Range Transboundary Air Pollution (UNCLR= TAP) (2). Boththe UNFCCCans the UNCLR- ‘TAP went trough an inital phase in which so ence was mainly used provide the foundation for requesting action by the decision-makers ‘The UNCLRTAP has moved to the next sige, using science to support the ientificaion and design of policy responses, whilethe UNFCCC ispresently meking dha cransition ‘state for Enviroment and Sutlabiiy, Decorate Genera Joint Research Cente, Crapeen Commisen, 21000 spre (VA, aly. "ANP he. Netherlands Envicrmerta sessment Agency) 3720 AH Bithoven, Nethotnds “Autor or corespondence. Eom: tankraes@yct ‘The UNCLRTAP was signed in 1979 afier the observation of lake acidification in ralinavia caused by polluiants emitted cbewhere. The first Sulfur Protocol (1985) imolved a flat rate 30% emissions reducticn, based on economic and technical feasibil rather than on scientific, However 1 justify further emission reductions, a quantitative analysis ofthe link between emissions and acidification was require, as well as scion fic basis for developing new targetsandost- effective measures, A policy-orieneed research and assessment community was formed around formal work ing groups, task forces, and centers under the UNCLRTAP. reporting annually to its subsidiary bodies, They developed simplified descriptions of atmospheric transport.ecosys tem and health impacts indieaors, and infor ution on emission contrls, feeding into integrated assessment models, Pragmatially, the number of models and data souroes was epi limited. Cutting-edge research was not included, but procedures for review and reso- Jution of disagreement were developed that involved scientists and relevant stakeholders This extended peer review broadened the quality of the assessments from purely scien tific w “fit-for-purpose: During the 1990s, 2 parallel community in atmospheric science made advances, bat without structural Tinks 10 policy-making groups. Asa consequence, new tools, knowl edge, and aktemative views were not system atically ineliaed for consideration, Now. the zap between those Focusing on poliey and on basic science has started 0 shrink. The UNCLRTAP’s scieniifie support bodies are ‘more open to partnering with the scientific community. In particular, the Task Force and Centre for Integrated Assessment Modeling has been key ( fostering integration and stakcholder interaction, Simultansousy, basic research programs ae increasingly bringing new problems to the attention of policy smukers, such as hemispheric transport of air pollution, and presiding a sciemitic basis for addressing policy questions (3). ‘The PCC does not havethesume relevance to climate policy as do the seientfie assess ‘ments for air pollution policies. Cross-cutting issues cannot currently be addressed compre= in a timely fashion becaus the divided working group structure of the Future International slenific climate change assessments should be faster, move integrated, and more directly linked to policy questions. IPCC. A systematic comparison othe costs and benefits of different levels of intervention vwas wchled outside the IPCC by the Stern review (#). However it lacked global letti~ macy, coming from oaly one counay. znd did not hnave the sciemifie credibility of IPCC's reviewed assessments, aly all forspe= ‘ial reportswitha 2- to 3-yeurproduetion eyeke A faster procedure mig be achievedby creat- ing a group dedicated to integrated assessment that marries the more immediate policy relo- vance of the UNCLRTAP scientific bodies with the scientific credibility of the IPCC. It wou itera dtectly with the SBSTA and vwouldaddress specific issues simultaneously needed, covering the diverse prortiesand con- ‘ems ofall courties and stakeholders. Fully comprehensive science updates will still be needa! wo inform policy. although per haps les frequently Here, 09, it is possible to lear from other organizations. The Mille nium Ecosystems Assessment developed an approach linking different geographical scales, The Technical and Economic Advisory Panel (TEAP) of the Monireal Protocol involves private-sector stakeholders better than the IPCC does. With the emphasis shifting to response ‘options, the IPCC cout collaborate mare systematically with international on asin the area of development, economy. and tech nology. The scientific community bas already ‘organized itself within the Eurth System Science Parinetship (5) © underpin such assessments with the required integrated sei- emi knowledge. Reference ad Notes 1 cme Crane 207, fouth esc Hper erin Group eps 1 I FC, Cambridge Ue Psy, Cumbadgn, 2007 wipe 2. Sige tbe, Clay fe Ae 25 eof the Comontin a8 eng operand Patton Utes nasen, Wes Yor, 2080) 2. an | Hor Ee, ocvorte UibroGurton [ACHAT ePisttokey ben Sythe CCEM Ppt (tee, wee, ay, 200 wacarter ripiesiencen ep {N.S efor Cae Chong (abridge nw Pes Cambri, 2007. 5, Eat Syoon Sane ano nae 5. Teeth mat wa arr ae Bt te ‘nd Ro ae UU) Ty testing (5 amet. Te ea ety teats acs el everest 30 NOVEMBER2007 VOL318 SCIENCE ymwsciencemag.ora Vor NalONNI 0) 01 THE EARLY YEARS Preschool Program Improves Cognitive Control ‘Adele Diamond,** W. Steven Barnett? Jessica Thomas? Sarah Munro recive fintons (EF, also called inschol and Me Aon sl ata tng thy eb. The Tok ofthe Mind (ool) cur 0 section ole setae ee (acho at minelogese Coe EF re (Onto cont sting hab topo races holding ang ai Cedetng to change) 2 Significance EFs are more strongly associated with schoo! readinessthan are iteli- _gence quotient (IQ) or entry-level reading or math skills (3, 4), Kindergarten teachers rank skills like self-discipline and attentional cconttol as mote critical for school readiness than content knowledge (3), EFs are important for academic achievement throughout the school years. Working memory and inhi tion independently predict math and reading scores in preschool through high school [eg (3. 4, 7), ‘Many children begin school lacking in EF shills (5), Teachers receive litle insrection in how to improve EF and have preschoolers removed fom class for poor self-control at alanming rates (8, 9). Pr pis 10 ‘improve children’s EF have offen been costly and of limited success (0-12). Poor FFs are associated with such problems ay ADHD, teacher burnout, student dropout, drug use, and crime (2). Young lower-income children have disproportionately poor EFs (13, 14). They fall progressivel behind in school each year (15), The Study The opportunity to evaluate Tools of the Mind (Tools) and another curriculum arose when a low-income, urban school district, *eparmert of Pohiany,Uivesiy of ertsh Colurba, Vanco, V6 2A Chien Hsp Yano, BC Caroda "Nara tse fr Ey Edcation Reseach (NEED, Rage Une New rs MUSA “Autor tor copenderce Ema: ate damerd@vtcca agreed coranlomly assign dren to these two curricula, Our study included 18 classrooms initially and added 3 ‘more per condition the next year. Quali standards were set by the state. AI class rooms received exactly the same resources and the same amounts of teacher training and suppor: (2). Stratified random assign “Buddy reading” Two preschoolers engaged in Toes activity. The ear Ene rang held by one guides her atention (2. ‘ment of teachers and assistants minimized confounds due to teacher characteristics, EF-iroiningenrriculum: Tools, The Tools (16) is based on Vygotsky's insights into EF and its development. Its core is 40 EF-promoting activities, includ= oneself out loud what one should regulatory private speech”) (17), dramatic play (18), and aids 10 facilitate _memory and attention (/9). Tools teachers spent ~80% of cach day promoting EF skills. Tools has been refined through 12 years of research in preschools and kinder- zgartens. Only when EFs were challenged and supported by activities throughout the day did gains generalize w new contexts 2), Districts version of Balanced Literacy curriculum (BL). The curriculum developed by the schoo! district was based on balanced literacy and included thematic units. Tools and UBL covered the same academic content, but MBL did not address EF development. [For eacher taining and fidelity. see 2] Participants. Data are reported on 147 preschoolers (62 in dBL and 8S in Tools) in Cognitive contro skis important fr success in sehool and ifeare amenable to imarovemeat in at-risk preschoolers without costlyinterventions their second year of preschool (average years in both) who reesived UBL or Tools for | or 2 years, Those who entered in year 2 had attended other preschools fora year. All ‘came from the same neighborhood and were randomly assigned t© Tools or dBL with no self-selection into All came from low-income with yearly income “$25,000 2). After year 1, $0 convinced were educators in one school that ‘Tools children were doing sub- ly better than BL chile dren that they halted the experi ‘ment in their school, reducing our sample of AB chikires Measures of EE Outcome measures (the Dots task and a Flanker task) were quite different from what any child had done before, These measure appropriate for ages 4 through adilts, assess all three EF con ponents, and require prefrontal cortex (20-2/)- They were admin stered in May and Juncof year 2 In all conditions of the Dots 20), a red heart or flower appeared on or left. In the congruent condition, fone nile applied ("press on the same side as the heart”). Dots-Incongruent also required remembering a rule ("press on the side oppo e the flower" plus it requited inhibition of the tendeney to respond on the side where the stimulus appeared. In Dots-Mixed incongrt- entand congruent rials were intermixed (tax ing all three core EF), Children were given a Jocof ime o respond [over five timesas long as preschoolers usually take (20) “The central stimalus for our Flanker task vas a eirele or triangle, Memory demands ‘were minimized by a triangle atop the right handkey andatthe bouwom rightof the screen, with similar aids for the lefi-hand circ response. The image to focus on was he small, shape inthe center: the distractor (or larker) tobe ignored was the larger shape sarrounding it. Congruent(eg., Dinside 0) and incongrie ent (et, 8 inside ©) trials were intermixed, Next came“Reverse” Flanker, where children had to focus on the outside shape, inhibi attention to the inside, plus flexibly switching task the Fi wwnuscioncemagorg SCIENCE VOL318 30 NOVEMBER 2007 13877 EDUCATIONFORUM. 1388 ‘mingsets and attentional focus. The rules were still “press right for and left for" Again, children were encouraged to take thei time and pot torush, Independently, NIEER administered academic measures to Tools children only: These ate described in 2), Results We report accuracy rather than speed because, for young children, accuracy He is the more sensitive mea sure (23), We conducted multiple regression analy ses with age, gender, curriculum, and years in eur riculum as independent variables, Inteaetion terms ‘sere insignificant and were dropped, On Dots-Congn ent, which had! minimal EF demands, children performed similarly regard lessof curriculum, year in a curriculum, or gender, though older children performed beter ‘When an inhibition demand was added (Dois- Incongruent) Teolschieren significantly outperformed AABL children (se the figure, left of above), Dots-Mixed taxed al three EF skills and was too difficult for mest dBL. children: Almost twice as many Tools as dBL children achieved 75% correct on taining trials (see the figure, right of above), (Our Flanker ask, like Dots-Incongru taxed inhibition (vith minimal memory or Nexibility demands) Tools children signif. canily outperformed dBL children (figure above), On Reverse Flanker, ABL children ‘med near chance (65% correct), but chikdren averaged 84% correct (see Figure, above), Thus, the most demanding. Dots and Flanker conditions showed the largest effects: those effects are socially esizeaite. Tasks that were more demanding of E's +d more strongly with standardized academicmcasures, For example, “Get Ready to Read” scores correlated 0.05, 0.32, and 0.42 with Dots-Congraent, -Incongruent, and -Mised, respeetively (2). Conclusions Some think preschool is too early to try 10 improve EFs. Yet it can be done, EFS can be improved in4-to S-yearolds in regular publi school classes with regutar teachers. Being in 30 NOVEMBER 2007 VOL318. SCIENCE Denne Tools accounted for more variance inFsthan did age or gender and remained significant when we controlled for those. These findings of superior scores by Tools children compared with closely matched peers on objective, neurocognitive EF measures are consistent wit teachers observations (24), Although play soften thou, may be essential, Tools uses mature, dramatic o Chen aig Pte) ge 8 y Tester — ey flee” Dasiaed ‘mere Deming “Tools children (blue) performed better on measures of EF than dBL ctildren (red did. (A) The dependent measure is percentage of correct fesponses. Del-Incongruert, Flanker, and Reverse Flanker tasks are Aescbed inthe tov. (B) The dependent measure is percentage of hilren fasting the pets orth task Statistics ave pred in the SON (2). play to help improve EFS. Ye preschools are under pressure to limit pla. If, throughout the school day, EFs are supported and progressively challenged, benefits generalize and transfer to new activities. Daily EF “exercise enhance EF development much as physi exeteise builds bodies (2) ‘The more EF-demanding the sk, the more highly it correlated with academic measures, Superior academic performance has been found for Tools children in other schools and states, with other teachers and comparison curricula (24, 25). EPs [espe= cially self diseipline(inhibition)| predict and aceoun for unique variance in academe ute comes independent of and more robustly than oes 1Q (2.5.20) Tools successfully moves children with poor EFs to a more optimal state. It is not Known how much it would help children who begin with better EFS. ‘No study is perfect, an! ours is no excep tion, Before and after measures of EFs,as well, as academic measures in dBL children, would have strengthened it, Strengths include random assignment and use of objective meas tues Noauthors ortestershaala sake in either ‘curriculum, Many competing explanations have boon riled ou (2). Most interventions For at-risk children target consequences of poor EFs rather than seeking prevention, as does Tools, Wehhypoth- ‘size thi oving EFs early may have increasing benefits over time an! may rel reeds for costly special education, societal ‘costs ftom unregulated antisocial behavior, ADHD and conduct disorder (2)}. References and Notes 1 A Dianon, nessa Comio Mectonss of Ciuge sik ia ES. Cl Us, New 206, p. 70-95. 2, Seer one nate x move ifrmaion. 5. C set fain. Ce. 78, 67 D0. 46 Mat mecletand FJ. L Mees fry ht es 9.13, 307 oe 5. SUERinerkaian CP M1 ox, ry cite fed. 15, 47 2000. 8 R Bel, Scr. europa 29,273 2000, 1. 5. EGaeoe Clay, rte A. Tho, ASPAC ea [Ci Pal. Paci a, $88 2009. 8. ten sear ina tighate of xpi Detoot” menor ines, 1 ay 20, 9.12 15. cil, Pendens ef Bid Dion aus te Petia Stes Male Che Sus Cre Wen Mave, 205. CA Ret, Tomb, De Meares 6 2s. 1 Snowe, |. ese, ex cone 36,161 Goon, 22, MLA Td ALA ata, canes, Secon. Pete NAH SUS 16. mebe ta, Ce S20, 464 007 KG mee ta, be 5.8.76 2033) 1 OShauneeseal, ened Soe Ec. 28.27 Goo. Baro.) eo Fete in Me esti zpos ary Cilteedesicaton Werner men Yok 2 2007, ‘tat rac inte Pye of Capt RC ndsen D. Pasibe Es. earn ‘ar, Hew 1965), 350-363. CS wre Saver sca. 7.6 98") CS vrei ane ace ie Dewees of ater Peto races wt Hes, Comore. 1978) MC Doves tt, ergs 44,2037 (008. MLR. Rudaet a, newopsyatapa 21029 (2009. Deon el Mewainge 20,2185 003. ‘Dison nim, yc Se. 36, 291 Goosn. 1. kane, al, “eeatonl eet ofthe Teas te mink arta adie (atonal est a nt ean eect Rages ‘Unies, New nsw, M2008. 5S pape pest te 178 Area coerce of Re eampean eat choos Eaton Reseach ‘secon Pape, ic pb, 2 AaB 2007. AC ovinrt. ME Psi Piya S 18.959 008. {is estar was made posse by oding A. om ELF Oe man ay eager) ek "Natranstse yOugAue OL DALPEES) % 2 Supporting Onin teat wring er/egriert AVS T8558 sa az6eceree 215088 wnauscioncemag.orgGEOCHEMISTRY The Leaking Mantle arths mantle (Ihe massive silicate shell Fiicatrcoicennantonmin important reservoir for velatles such as ‘water, carbon dioxide, and the noble gases. It combines volatiles caprured during Ean’ f ‘mation with those added later by nuclear reactions or by recycling from the surface. ‘Consequently, mantle-derived volatiles provide st arm about Earth seay history iS continuing evolution (/), Although Earths enustal carapace limits aecess wo the mantle, sampling its volatiles has been pretty siraightforward: Researchers target re where mantle-erived mels (magn) i the crus, such as mid-ocean ridges, vol arcs andhot spots, But can volatilesescape the ‘mantle without the help of magma? On pag 1433 of this issue, Kennedy’ and van Soest (2) show thatthe Businand Range province (hich ‘covers much of the southwestern continental United Staes) is leaking n a wide area, despite little meg Moreover, they propose thatthe lover crust usually considered a barrier to volatiles. shows, anenhancedpermeabilty caused bythe Pacific and North American plates sliding past one another. These observations, and their pr: posed explanation, have farseaching implica sions forthe origins of earthquakes and finding new georhermal resources. Researchers trace mantle volatiles by a- Iyzing the isotopic composition ofthe lis oble was, helium (3), Both isotopes, He and ‘He, are produced in the crust at a ratio of ~0.02 Ry (where R, is the "He/He ratio in air), Higher values indicate the presence of helium from a reservoir enriched in He. The only viable possibility isthe mantle, which stores ‘He captured during planetary accre tion, Hence, JHe*He valves between 0.1 and 3.R, throughout the Basin and Range signify manile-denived volatiles. What is so unusual bout this finding is the lack of magmatic activity in the Basin and Range, except atthe ‘xesternmost transition to the Cascades m: matic are, How can mantle volatiles traverse the ductile lower erust and brittle upper erust, without melts? Maps of the deformation dynamics ofthe vwester United States may provide the answer (4), Plate motion bewseen the Pacific and North American plates drives east-west exten- Hilton le voluiles ever he authors athe Scripps issn of Oceaneorapty, aol, 497093, USK Ena dinttongued es summsclencemag.org Zane of hallow hydrthernal activity The flow of mantle fluids through Earth's crust reveals fundamental geophysical processes and may help pinpoint sources of geothermal eneray. Crustal exension [BAR | witheut shear t Incpert mane meting Escape routes. Schematic ross section of continental rust, showing escape routes of mantle volatiles tothe surface. Zones of enhaiced permeabiiy inthe ductile lower crust, formed 35a result of shear stresses Imposed on areas of crustal ecenson, may provide an adational connection te the mantle to supplement that provided by vlcanic and mags driven hydrothermal activity. Helism isotope ratios ar in boxes sion of the Basin and Range province oeross ‘mos ofthe Central Nevada Seismic Belt east, of 242°T) atrates heoween 3 andl ram year? In thisregion, He*Heraiosdefine minimum (baseline) values of ~0.1 t0 0.2 Ry. West of 242°E, strain rates and corresponding flow velocities increase sharply (up co 13 to Lam, year), their direction changes to the north- ‘west, and baseline *He*He values also WS 3 Ry. The increase in Increase w as hi Me He values must reflest increased flow of amuntle-derived volatiles through the crust. Therefore, the pantera of increasing baseline SHo“He values toward the west is tracking the Jncrease in permeability ofthe crust inckad- ing the ductile lower erust and its transition to the source of the high ?HeHe values: the lithospheric mantle Two ingredients are required to explain this enhanced permeability. First, fractures, that can act as conduits for the mantle volatiles must be present, Kennedy and van Soest suggest thatthe shear force twisting the regional strain to the northwe: vertical faults that link the brite uppererust with the ductile lower erust. Extension alone produces normal faults that are refracted (0 rcar-horizoatal positions atthe britle-ductile transition (see the figure). Second, these pathways must remain open, at least for part of the time, to maimain a connection Derween the surface and the mantle source of the high *He*He values. Therefore, uid pressures within the conduits must be «extremely high, Continuous connection from the surface to the mantle 4s not required! Changes in Maid pressure may facilitate stowth or scaling of fractures sothat the How ‘ould be episodic rather than steady-state. “Te wea ation that helium isotopes ea identify zones of enhanced crustal perreabi ity may open new lines of nguiry. For exam ple, Kennody and yan Soest point to Helle anomalies in the Basin and Range (areas where the "He/*He values are much greater than the baseline) as reas of enhanced uhermal potential. They argue that high he isotope ratios mark localitics such as Disie Valley, Nevada (5) possessing the enhanced crsal permeability and doep aid production necessary for geothermal energy Alevelopment. Mapping helium isoxope ratios may thus be a valuable tool for Finding new geothermal energy sourves. nether prospect concerns earthquakes. Nomvoleane tremor (quasicontinuous ground vibration) has been detected! in southwest (6) snd bens (7). The Foumer ease reflects mov aqueous fluids shyouzh the male (8 later. sch evens may reveal te presence of ‘uid below the seismogenic zone (i. rl uppercrust where earthquakes occur) (7). The role of deep fluid movernent in changing SCIENCE VOL3I8 30 NOVEMBER 2007 13897 PERSPECTIVES 1390 stressesin the upper erustand triggering earth- «quakes remains unclear, yetthese wovery different tion regimes share a commoa fea ture: “He*He ratios much higher than expected for egions virwally devoid of mantle melting (8, 9). Looking more closely at the helium isotope characteristics of such regions—spatially and temporally—may provide evidence coupling the leaking mantle cards to one of our greatest natural ha References 1 RO. Fepin D Fra eu Mr Coote. 87,391 2002. 2, BM Keane Can eet Since 318,108 007. 3. I: Lele Cig Ler lent Se Let 26, 133 tase 4 Lak ee MLE Hl A. Hanes shen, See 287.2400. 5. 8 ted Can ust etre 35,26 008. XC ota, Sane 296,479 2002, RM cei, don, Sec B07 389 2005, Dish ort ect 2008 G0 AL zescence. sonia, 8 Umeha, A Mami Groh: Rs 412 810295 (207 9. 8M Kemeay eta, Seace 278, 12780997, so. n26ere 1151999 GENETICS Paradigm for Life James 0. Mcinerney and Davide Pisani that prokaryotes can swap their DNA through the processes of conjugation, transformation, and transduetion (/). This requisition of forcign genes is generally referred to as horizontal gene transfer (as ‘opposed to vertical inheritance. in which brganism receives DNA fiom an ancestor), The consequence is that each prokaryotic chromesome is & mosaic of vertically ine herited and horizontally aequired genes (2). On page 449 ofthisissve, Sorek et a. (3) report the first large-scale empirical analysis of the transferability of wenes by analyzing the inrodketion of almost niin genes from other eubac: terial and archaebacterial species into the eubacterium Escherichia col, The findings pro- Vide insight imo the likely evolutionary history I has been known for more than 60 years £s genes that cannot be transferred Therole of horizonal gene transfer in evo lution has raised fierce debate about the relo- vance ofthe Tree of Life, a long-accepted rep resentation of the interrelatedness of living things through evolutionary time, based primarily on the sequence of the genes that encode the small subunit of ribosomal RNA. (4), The question is whether this depiction should be replaced with a network, or Web of ‘he author arin he Department of Boley, Naira nvr of Feline ayo Mayet, Canty Kae, Irshad. onal moe ameneraeyQnuimis 30 NOVEMBER 2007 Life (5) seethe figure), Whereasa tree reflects evolution as a process in wich new species ise, or branch, from specificancescors a web may moe accurately portray microbial evel tion based oa the rise of variation (and new species) through the eral transfer of genetic information between distantly related species iglogenetic methods, ithas been “proposed under the complexity hypothesis that not all genes cpl affected by hore snc rans (6). ave born iene about whet ‘Kee or Web of Lt 7S Informational genes these involved in DNA replication, wanscription, and ‘ranslation—are part of more complex. protein-intrsetion ular thus less likely to be imselved gene transfer. So-called oper snes-—these involved in day-to-day processes of cell maintenance, such as genes thatcontol energy metabolism and the biosyn- thesis of nuclenides ae amino acids —may be more prone to transfer (6) This has led to the notion that a core of pontransferrd or rarely transferred genes might have kept track of the prcachesto uncover mechanisas involved in the dynamic engnizaion of nuckar stucwre and Function throughout th coll eyele. We foes on transcription, replication, and condensation of chromatin and on the regulaied assembly and isasernbly of nuclear structures during miosis, Emerging Technologies: maging and Proteomics Green fuowscent proein (GFP) and iis many ts have been major driving forces in cabling dhe visualization of nucksar saci and fianeion in live oss, There is now an entire toolbox" of Muorescent proteins with different chromatic nd structural properties reviewed in (D)). Combinations of chromatic variants can be Visualized sy the samo cell, and specific pairs permit prowin-proxin ieractions to be mee sured in vivo by fluorescence resonance enorey transfer (FRED), In fluorscence complement tion, the direst interaction of two preteins, cach fied to a nonfluorescent fragment of thao rophor, is relleied by formation of the fact fuonescent complex (2). Other interesting var- nis are photoactivatable GFP. (paGEP) and similar Muorophonss (3), Both GFP and photoactivatable wes, when combined with cis aser photobleaching or photoaetivation, respectively, allow rates of prctein movement within cells bbemeasured under a wide rings of conditions A major advantage of crescent pres is thet ovoniscan be analyzed atthe level of single cell and inthe same eel noughoue the elle. By fising FP tags to DNA or RNA binding domains, the movement of specific DNA ket and RNP compleses can be visualizes the in vivo analysis of gone expression reviewed in (4 5] A caveat with all FP-caging Tosnniques, baweve, is thatthe presence ofthe tag can alier the properties and fanetion of the moleculeboing visualize. Therefore itis ipo tut t contol for he efit of the tag in ‘experiment design an, where possible, validate the reals obtained using other methods. An in teresting parallel approach developed recently volves “ehromobodis.” Le. epitope rsogition fragments of anaboay hey’ esis Fist fa rophres that can be exes in ving cells and allow Tivecell imaging f endogenous proscins (6) ‘Proteomics. Am important goal in character iving nuclear owarization ist kleniy prove soncentated on chromatin and i cach of the diferent subnuciear domains, which ean guide future functional experiments. The metnod of choive for such analyses is MS-hased proteomics, although non MS-bass techniques have alo proved sell Tbe cae ofthe mucous, proteomic analyses have boon perfonnalon puis maceol caichad repute of iercinomatin granule clasts me slear envelope and pore complexes, and various pusitied chromain and chromesome factions 10) A roajr limistion, however tha the sling “Senie™ preteome prenidss an averaged ‘iw, which does not take flo account dynamic tus fom the fiber centers (37), These sub- nits, eriginally belivad wo remain associated with rueceolar organizing rgions (NORS) throuehout mien, were shown to transiently kave NORS ‘during metaphase. Dsoctton of proteins from the remaining nucleolar subsompariments ‘cued a a ister rae and was coincide with NE brcakdown, Reassembly of nuleol alo followed a define temporal sequence. stbough not neces- sarily inthe same onder, asthe proteins were im Ported into newly formed mle, Tinelapse FRET nai between FP-agcd nucleolar proteins has exter such fve-ell imaging expe ments {0 include information about protein pein interxtions during nucleolar reformation (G3, Both carly and leteRNA processing factors passthrough the same prenicleoar bodies, and ‘during eanslocation ofthe factors, teractions heenveen proicin partners fiom the same eRNA, processing machinery can oscur and may even induoe formation of prenuskolar bodies Altbough proteomics studies have been car. ried out on various nuclear prosin eamplexcs and cmrihed factions of inerchromatin granule chasers (8), nucleoli have proven the most amemible of the nuckar bodies w biochemical Purification and detalal protzomic analysis to dine their protcin composition in both mame malian and plant calls. Neclooli have aso been used 10 develop the vime-aps_prowcomnies” the SILAC otepe nie wate kvels ‘of mucleoke proteins were systematically eval uated by repeated proteomic analysis at multiple 30 NOVEMBER 2097 VOL318 SCIENCE time points afer weatnent of cells with inhibiars alleting either transcription, proteasome ati, ‘orspeciic kinases (7). Pulse labeing with heavy iscopes has also boon used to charcterize the dlferen tumover rats of nucteoke proteins (12). “These stucies in the mcleolus have provide the fist global characterization of the lus of pro teins through a cellular erganele. It will lealy ig. 4. Vbualiation of nuclear structures, (A) Conventional wide-field flucrescence micrograph ofa paraformaldehydefied HeLa cel stained with markers for the NPC (ve), nucleolus (green, and chromatin (blue). A resolution limit of 200 nm is imposed by the wavelength of visibie bight. (8) Fiolé-enision scanning elecron micrograph of dry fractured HeLa cell ich permits spatial esolution of structures within 3 to 5m, The fracture ‘exposed PCs (aronhead, which ae clearly Wek ble on the surace of the nucleus, while a fracture across the surface exposes the chromatin within the nucleus, The paler, brighter circular area at te top center isa reslt ofthe increased backscatter electron signal from the dense nucelar regio, ‘be imponant in the fare 10 apply simile p= proaches to study ember clear bodies and ‘efined complexes Muclear envelope and aransport. The NE 1 sssociated clear pore complexes (NPCS) provile a barr betscen the genome and the etaplasin and play a snajor ole in controling cnt and est of the wide rnge of molecules that nus. continually move beween these cellular ‘compartments [reviewed in (3) Ip ation to reuulating nuclevcytoplasmic exchange, the NE thas also bec inked to contol of bel signal if mitotic progression [reviewed in (40) event advances i imaging and single ticle tacking hive been appliod to study cargo transpon, periting visualization of singe moke- ‘ules interacting with NPCs, Cargo molecules were Shown to randomly dlluse within a NPC, ‘eviting as @ result of a single rate tinting step (4d), The sal suaesine wiphesptauase (ATPase) Ran plays a central role inthe trnsport mech asm and is ao required during mitesis when the NE isno longer present, The common feat js that Ran signals the location of chromatin within the ent of a pri oa in the rete levels of RanGTP GDP, with 4 higher RaxGTPGDP rain occaning i the Vicinity of chromatey as Ran interacts with the facior RCCL oman hydrolysis of GTP bound lated by a Ran GTPase activating n (RarGAP). This gradient defines the Airetionality of uckeoey plasmic transport during inerphase, s requied for the correct fo nation of bipolar mitotic spindles during mitosis 442), and defines the stes whore NE and nuchear formation will cevur at Uae end of telophase FRET probes have been usod to visualize the RanGTPGDP gradiva! in both ellie systems and in lve aanumaian cols (42,43), As showen in Fig. 3, Band, an import binding domain Aanked hy eyan (CEP) and yellow Mrescert protem (YFP) on opposite ends provides an ‘yan! visual wadout that iniecty reflects the relative level of RanGiTP by monitoring the RanGTP-cependent cargo reese from impor Bat diferent sts within to eel. This conveniaen assay fir monitoring changes in the Ran GTP? ‘GDP aracient upon specitic perturbations has boon used to probe the contribution of Ran to nsport and chiemosome-sesregaion mochanisms (2, Tn! eukaryoes, chromosome seuregation during mitosis occurs inthe absence of a dined relear siracine: Thus an open mitosis i ear actriz by the breakdown ofthe NE ana asso- aed stctues before association of conde 00. MD macromolecule stuctaes that form channels sparning the double lipid bilayer of the mac ‘envelope (Fig. 1, A and BY, 2) Bach NPC is ‘equipped 1 faite both import and sport of prateinsard RNAs Fig. 10 G). The cansinvent pres of the NPC, nucleoporins (Nua), serve Degartnet of el ane Dewlaprenal logy, Vardi Unveraty Medesl cer Nhviln TH 37232, USE 30 NOVEMBER 2097 VOL318 SCIENCE precise oles in NPC assembly and function (4) Structural Nups contute to the overall NPC architecture, and pore manbrane eokins (Pons) avehor the NPC inthe clear envelope. FG-Nus onsite a thind clas of Nups. they contin scrote domains with maths phenylalanine eine (FO), GLFG (L, leucine), or FAFG (%, y) repeat modi, spared by charzad or poke spooer sexuasnces (5). The FG-Nups presunably Tine the cemval NPC channel ard exten flees ‘on both the \toplasmic and nasloplasni faces (Fie 1B). Adtionaly. he unfolded nau ofthe FG domains might allow” mulipl> topological posiionsin the NPC (6-8) ‘Movement of fons, metabolies, and other small molecles thmugh the NPC ovcus via passive difsion, bat the tanslocation of cargoes lager than ~40 KD yencrally roquites specifi manspot receptors (9, 10), The FO-Nups are sonshted to play an fatimate roe in generating this NPC permeability barren, slthough oles for structural Nop in formation ofthe banir have also been reported (J, /2). FG-Nups are «essential components of the active translocation scaffold through the NPC (/3). Transport receptors are thought to in multiple, stochastic, low-afinty ineraetions. with FG: repeats during tanslocation. The series of inte actions benween ao FG-Nup and 3 transport receptor during docking and translocation are cenengy-independent, il nucleotide hydrolysis apparsly rguired only ding tenn slectionality sens (0) The biophysical male of the permeability barrier and the mechanism of translocation 1 iain controversial and poorly understood (J), Ac the come af the dchate is whether the NPC pemeabiliy barvce is physical or energsic, and recent staics suggest tat ether ox bat cou be the case (/, 8, 1-15), Asa physical baer, the FGANups would imeract to fm a gelatin meshivor throu nly stall molevules could passively diffuse (14). Translocation Hough such a meshwork Would be malited by tansport mxepiony that locally dissolve the baer and permit passage of reeeptorcaryo complexes, AS an cnergetic barrier the NPC ‘would form a repulsive gat, repelling non binding molooules (J, 8), Reospior binding FGerepeats. would eifixtively allow concen tration of tansport complexes at the NPC, overcoming the entropic arrer and increasing the probability of transport. Finally, a two 40 KD or -8 nm); smaller molecules move through independent of a transport receptor. SCIENCE VOL318 30NOVEMBER 2097 14151416 The Nucleus for eoessquences of specimen preparation. De= spite a central aqucous channel ameter of 10 rm (ZO), dae NPC efiisienly transports large cargoes such as ~1 1 3 MD (-25 nm diameter) ribosomal subumis and prot (19, 76) Aitcial substrates up to. 39 nmin slimeter, such as trasport wept cuted gold particles, ean also be tranlocstod (9). Iniret ingly, the x-ray crystal structures of mNupS8 and mNup4S incicae th these to Nups might slide slong their common e-belical itrostion surface (77). The interaction surface Is the potcatal to shit by “11 A. Moceover, wih beth mNupS8 and mNupt5 Tocatal in the central NPC channel, cxcumterental Nop sliding is estimated 10 potentially inerease the NPC ‘darntor by up w ~30 A, which might Funston to citer regulate or aecornmedte ge cargo translocation, The biophysical impact of this sliding on the incractions between other Nups | unclear, a6 are the mochanisms that could insigate these changes. Overall, there are muliple mechanisms by which ekinges to the NPC cornpostion soul! broadly alse transport capacity and pore srucun Next Stops in Understanding Transport Regulation We have outlined the importance of roguated uckeestoplasmic tllicking and have di ‘eussod how conto fs accomplished at multiple levels (Fig. 2) However, thor are unansvcred ‘questions at each level. At the mest fundamen stage, continued investigation snc to further delineate the basic transport machinery. All the receprors usa by various cellular cargoes andthe prscse signal soquences rovognized are still not fully identified. Adding 10 tbe complexiy, a sinale receptor ean bind more than ove signal motif witout a realy identifiable primary con sensu sequence (78). Wealso do n0t complexly Know ow complex capo molecules sich as mRNPS ae selected for targeting to the NPC through couplet maturation an! quality cons steps (9). Elcidating these cago signals and how they are recognized by specitic transport receptors wil roquite maltiisiplinary boinfor matic, stuctural, and cell biological appxoaches. [As those cages and recepios are prime targets for regulation, this information could provide Insight ito abroad range of biological processes Given the hierarchical rok of the NPC as the sole poral for nuckeeytoplasmic exchange, understanding the NPC translocation mechanism is a priotiy. Specially. studies are nealed to resolve the biophysical nature ofthe permeability barrier and explain how wanslocaiion process trough this barier (Fig. 3). This will abo reguie models that accommexate all the dec mented octive and dilsive transport capactics ‘of the NPC, as well as sorsideratios of bow frteratons within and among cargoes, taaspoe receptors, ax) Nups affet the sablity of the 30 NOVEMBER 2097 VOL318 SCIENCE [NPC harris. Wit tis knowledge in and, the Feld cou mace rational rections andeondct tess fr how transport would be affected by changes in Nup composition during viral infee- tion, cll evel transit, signaling cascades, oF call diferente, There are clearly emerging examples of biological conditions uncer wich eels badly alice ‘nuclear transport. This includes the imrison- 7-madiated up-regulation of mNup, the cell cyclo conttol of yNupS3, and the change in ‘cargo trafficking during ncaral stem cell dif entiation (48, 6/, 72). However, many of the imcihansms by which this oceurs are not well defined, For example, its not fully understood hhow heat sbock- induced mRNA is prerenially cexportod unde sees conditions fas summarized in (79)), Futur studies of cell Fate determination ax! dovelepment will reveal the prevalence of spaixerperal regulation of transport coping adavor, snd Nup level and the cargors whose tnalicking is allected, A further challenge fs 1 integrate knewledge of this multiored,hisrrchical epulatory scheme imolving cargoes, transport receptors, and the [NPC intoa global systems biolozy perspective of the eoowtination of ellul responses to intemal und estemal stink, 8: cenensiae References and Notes 1m Peto a, J. Ceo 1 2 crenstan, AM. tagcat, War... 1 Manns Cl il 138, 5 200. Smet eli eB. 208 57388), Lan, S Weve Cet 125, 1081 ae. MP Hoa, 5. Wee, es Ce Ba 357 (998 8 ftv ta, 1S il. 140,254 202) PD, 5.5. Pam V- Urery, AL exh eat Aca Se USA 209, 2850 (2008. Yio, Pac Aa Acad Se USA 15. 9512008. I Pate Me Kn el Bo. et 13, 425 2002). Fe ata Co MeL eS. 60,1659 003) Sha 0. Golda, Ctl 23.538 2009. CP Desa 5 AOS fle 6,121 207. Seva Mt evi Cl 8.195 2007. Scryer Deh, See 14 815 Om. 5.5. 6). Benr, M.S LF. Resch, Ce 12, 8 2007. Fe Pees Tie 6, 421 2005. Lr Pembenan 8 eh, Mf 6,287 2009. DS.ouda A WeCatet DAM eT Haren, S.kAlun es Cel 80 34,505 2300, 9, Rte Ht, Wa ee Ce BL 8, 7612007. 2), Fa. Suh 8 Gani, Cr 8181 0958, 21 8 Fetch, € Que some, E Hanon, ole, Ce Bl 26, 8697 (2000. 22. Lk Poet LA a Tai 6173 003. 23. ML. bats 5. Hason, Cl 95,749 (198) 24 Tr, 8. Man, 5. ake Go Ce 95,759 se. 25. LA. Sermo, E480 | 28, 1860 (A999 26, Mana eo Ax Ce 9, 2175 OC. 27. G5. )nees, 5H Mh I-A. Sorel GML a, Oncgere 18, 7658899. AC Nl fl Bol. Chen. 201 23585 (006. D eget Bead Cl Moet, 50 23, 40 2009. 32. Akane, EK O'S Swe 204 977 (199). SLL Yang HS, 5. als £5 ides, 5. Kombi, 1 Bil her 276, 3604 BDO BER BEEREB Se 2 » » » 3 Xe Es 2» ». a a. a. e 6. @. ry ™ m ”. WA Sot, 8, rte, F. Wong Sa, Doi, 1 Bit. Cem. 279, 2795 0t08. LG Tem tf Cl 428,341 2000. SMefate Pky AW Hem, Moen, EG Bic 307, 649 GOD8. Ac Haper, CP Genes er 37, 162 ons, ther al tre 443,254 2008, B can Tops Saboek Mle Gate, CL Baden Ct Bol 175, 15 GCOS, TEA tape aly ance es 65, 10742 2009, XV Fasano, Cal 123,249 05) oA Hang A Wee Tend, See 35, acon. AM. Aer oman J.T 5.649 5 oe, Na (el Bot 8,713 (208) 5. Wein eal, Mat Cel Bot 8668 2008. Yk np (es, 5, Sener 285, (999 F.Stae, (ule, end Cl Bol 2,329 203) Mole eta Mt Ca Bel 39,782 999. ober ets 5 Le. 497,108 (1997, MLV. Maha, W ye ane K Wes, re Nat dco 6 USA. 3, 582 0950. N Yash etl, nt Ce Bel. 9,72 2907, Reb 1G Moan, ML Sit bil 3,10 2000. RH, PA Sh. Net i acer 16 208 aN et el Chin, ance fe. 3, 3950 (205). \ Weep! Ma ate 98,472 206. 5. Ped ely Wk 89, S156 2000. Fam ta Wel 0, 9612 2007). BL Timer eal} Ca Bo 175, $79 2300, Nsom LF Pb, Ted Cl Bie. 34, 540 Goon. 1 Yang 5 Muse Gel 174 951 2606 5. cal. aepy, 6 Soh, 0. Kobe Hoare 322, 41 0906. 1 ery $8 Wet Jl ied 478,121 2007. CA. Sun Sha, M Shs, 0.5. Gel SA Were, Na Cl Bl 6,197 2008 teed a A Andon (50. athzon, Wook Call 136, 819 (2003. EF lk et a af 64 (200, key Pa CPs, 0. Achion, (LW Mosh el Bl 177,39 2007. Wolsne Ssh Oe pCa 27.389 2081, M.Sriteran, ee, Seager, Kap, 8 Edman Mo Clot 20,56 (200. RE Wvetal, Gere: Oo. 18 1945 2000) Rath JCal Biel. 163,701 03). Gay eto Ga 204 63 (200. (CE Gat, sds 95, 3812000, N Satta Pee cd 5.USA 1041853007, [reanngs 0. € tay, @ Bate, BALA Fert, Scant 295, 1523 202 pbs eee 2 Jase, 2003 10126 1067861. LERiio RA Mii | Src 2,259 aD, Beker, Sece 306, 1307 2000; plied tie 28 Oder 206410. A2rce CABO, CW Ae, MeL Bil 248,279 199. id, MW. Golder, 1. Ale, CW. ley, Fete 11,223 90. 1 Lmae Jaa, Bat, 9 Kote, trent Mot Be 337, 01 (202. LM AH Bibel Sence 35,1729 ODN Bits aly Co 126, 43 2000. Chaleeage, CA alge, C M Col, Euan, $05.10, NOW Gates, 0. leg J Ml ot 257,64 856. "he Birk embers fh Renta an iy ab cal ements manus anh autos so Ly paved iene tha ats iy. 1 Me reget at pce etiton pert cnolegeneat pyres and tame. Seppe by HH ane ROL GUSTAV 4 203-0057158 GW), Fiera enh Sic ard RSA) ST2-CR09285. IU)t) sed mesa st cuconsse eS 1.642206 ua scioncemag.orgSTN Plants Tolerant of High Boron Levels Kyoto tive,” anpe Takano Hioyuki Omori* Mosk’ Seki,” Kuo Shinezak* Tova Fujwara™™* cron (Bysan senators We ft AIBORY (Te Arabians Boetetsintrcomies iematin Renee 'Stoxic Living owanions inckaing plans, ‘must corto the B distibation to maintain ace ‘quate evel of Bin theircells, Raluced crop qual ty and yields in soils coming toxic evel ot B are a workivide problem in food production, e+ peal vari areas (). By manipulating B tans- mv miegaced “B13 HB pls that axle of BORSOFP al ee fin ach of B vn - . Bccumulation ocous boo nately and hough aiicial moans such mignon. About five mit ion ha of sols containing vce than 1Smghy™ B, above the dyeshold for nonnal plant growth, exist in southern Australia, comaponting 1 30% of the region (ww.dibe sa.gov.aulland topics roctzonetbocon im. Up 117% of the basey yk! loss in this area mated o be cased by B voxicity @), Beolenamt ‘cukivars with reid B. GenBank code NM_LO1413), one of the six BORI paralogs prosent in th ‘genome. B trrsport activi BORA geen fluorescent protein (GFP) fasion seven independont ransgenie 4 thaiona lines paling the BOR4-GFP fusion under the com tolof culflwvermowic vin $88 RNA promot TImmurxblo aralysis of @ generated tars ‘tenis ne showed tha! BORS aecurlatd in the presence of ahigh B supply (Fiz 1A) suazesting tha BOR4 is excmpr fiom the postransatioral BORI degradation system. “The supply of 10 mM boric acid was sue stantially lethal wo wiklsype plans, ut much more Vigorous root andl shoot growth with varying degroes was obsenved in all the homewy Pro33S-BORS-GFP transgenic lines grown on solid nxvium containing I mMt boric acid 52) Accumulation of BORE CGEP and jolerance of B were positively core: bued (lig $2), The B concentrations in the ots and shoots of these tansgenie pnt Fines were lower than tha inthe wildtype inthe presence of 3 mM boric acid (Fig. IC), Overall urcer Bb uptake was also reduced in the trasgonie Line 4 Aig, $3}, These rests syepes thatthe over o= duction of BOR4-GEP improved growth under ‘ondtions of B exicy timough B edu, Fusthemore, GFP fhuorsoenee derived fom BORS-GFP was stongly dette in the plasma membres ofthe distal sidos of epidermal cell in the clongaton zone of wots of te transgenic lines canying PvBOR(-BORI-GEP (Fig. 1D), Ths distal localization of BORS is likey ianpor- tt fr the dirstinal export of B ram the rots to the sil to prevent the arcumulaion of inthe xylem and growing cll. This enhanced B eM fiom the rots of crop plat is expecta to sult inimproved! erp procuctvity inthe tose sails fund in a numbsr of resins of the world. Atl 18460 and eA. thaliana sof BORE and Raterances and Notes upiake are known for © sop D OTN 6 at oa barey (3h bat brocding = é e 2. hgh 8 eg Maal at St Programs have not yet oa ‘ees 22, 261 1984) prescals actcal sus Geo 1. Age Bt, Pe 196337608 Pete Dice ea 1 | Ian ane a, 97 oD irubegs tana Ba 5: Ll, hi eo BORI, anetluvtype bo- gg E 6 K Mina) akan, TF, Ant 46,1088 (2008), fo Diemyoncriin | NELLA WE "fie Sout tenon sone Land re ee tool “Shoots Me Gears Pret so Minky opt, fiod ina biological system (4) BORI is round for the tmnsport of fom reats to shoots under con ditions of low B supply. BOR is capable of con fering high B toleznce to yeast by pumping bo Fic acid out of the cell However in plans under high B conditions BORL iS degraded via endo. cextesis (5), and overex pression of BOR/ docs not improve plant sroth in the presenae of tox keel of B (6. the area inside the box in the right-hand image. wunuscioncemag.org SCIENCE Fig. 1. Improved gronth in transgenic A. thane plants overproducing ORS under concitions of boron toxicy, (A) Immunoblot analysis of transgenic pant line 4 carrying Pro355-BOR4-GPP. Plants were exposed to (0.1 lt (8), 30M (8), oF 3 mM (+48) boric acd for 3 days, and root microsomal proteins were subjected to mmunobloting with an antody ‘gainst GFP. (B) Transgenic plants grown for 17 days on solid medium ‘ontaining 10 mit boric acid. Scale bar indicates 10 mm. (set) An enlargement ofthe wild¥ype plant (sale bar, 3. mm. WT, wildtype Col 0; 14,15, L12,transgeniclines 45, and 12, respectively, ari {BOR3-GFP contract (€) Toa B concentrations in shoots and roo. Panis ‘were grown on said medium containing 3 mM boric acd for 18 days. Means — = SD are shonm (1 = 5 to 6). Asterisks indicate significant diference (P< (0.001) ty Staton’ test DU, dry weight. () GFP fivrescence in r91s of tranigentc plant crying the PreBORT-BOR4-GFP construc. Plants were ‘ronn on sod medium containing 30 uM boric ack for 10 dys. Seale bars, 25 um (ef and 100m righ The et-handimage & a magnified view of 2 Gtr Scenic Raho Pry as she ey Ean Caer, St, Se, 2 eh legy 0 ap, an 215 Cea Cet beeline poet TF Supperting Online aateriat sooecencemag ag rerll21BSES/1 790, fie sites 1B ne 2007 aed 9 Oaber 2007 the Pro35s- “Blaedenloy Reach Cees, Uabersy of Toya Tyo 1134087 apa “HEM Pam Scene Cre, Yana 220-0065, Japan. "Sltr-Orierted Resear fr Scere snd Tecilgy, japan Science ané Tecnology AoE, “ohye 105-002, aa, “To when catespondence shoud be adsl. Emalt soulugralecutha|e YOL318 30 NOVEMBER 2007 1417A £-Defensin Mutation Causes Black Coat Color in Domestic Dogs Sophie 1, Candille,** Christopher 8. Kaelin,** Bruce M. Cattenech,* Bin Yu, Darren A. Thompson,” Matthew A. Nix,? Julie A. Kerns,*4 Sheila M. Schmutz,* Glenn L. Millhauser,* Gregory S. Barsh*t AUTHORS’ SUMMARY marked spectrum of color | J diversi of ptems dat ose in mann aries expectedly, fom vatican ho que iy, quit, and regional Gstnbaton ‘of ust 190 (ype of pigment black rin an yellow phomebinin Thc peal of una cot cobs ad potters has motivated thir seston in domesic animals providing genet ‘eis wih a mods for stating ame actin and introction that began a ‘erty ago an cruise. Mos ‘of the work fas bos caiod ot in bbhoctory ace, where sudics of exe genome and wonton that the Ginincive gore smacure of dog toon alls free gene mop ping Gh. Dogs wore domes fom wos rene than 15{00) years ago an egal ino ach ion anh the see of dg beds This poplin istry F etlstiiel fee tighten ao ss nipping of oi tis, ke black coat con tha a Rnd in lipte tmodkrn breeds. Using combine tion of patigree analysis and ass ciation sudiss win and among dog break, we identified «mut shay 10 det eects ton in Bdefersin aons, CADIS, have provid ih ito sem col that cores with black coat cobe Dinky (air gay, Wogenss ot B-defensin in 3 cillorttroeds, We confimad inact onsncesipisnntry di the mle of CBD 103 in pigment ype eon, and booncno repr inne sotching by demensrting ha te sons vb Kee the sys Velo Labador Reever doz gone causes a black ca in ‘of cucin sn phos), tamamic mise CBDIOS ka mem The later proses comely ber ofa lng fry of set poy kao x pest typesitching cs with stractires sno th i ertctil pemaiy by fe mene cin syst in which aly of protein coupled westosidntifed by vine of ther rapurse 0 makweste stimulating bomnone cr Brdefensin adrenocorticotphic hormone) Ins 8) - ‘Metr, kang to an upadned mci of been pit ne nly in pase Black Cay Coated River gs pyanant pons pethvay sca but a in coro proctic, Prodution of yellow vesus Back pigmert in dogs is controled by three genes Ge inh More suas wah beady weit regulation and xveine MEY, Aga and CBDIO. Dogs carrying wile alls aalltvee geneshave seth (ecfesin andl addon ‘eho scction In most mammaak pies 2 yellow coat resulting from Agouti antagonism of Miclr signaling in melanocytes —tunocortin weepiees reveal the pout mw woosvicing cca by Welow Great Dan top) Dogs anyinga assaf funcion mutaion at McIsharea al arentanne ewseulk percen to goes the Mecrcorin J vecepr YA Cet, regres of tet genotype a acu or C8013 elow Ladador — fedtnsns and the mkenKor 3 for (Mein ane Agnes, which ence Retriever, middle. Dogs caring wipe alles for McIr and Agout together fy mans andor nis seven tansncmbrne domain ae Vi the dominant black allele of (60203 i) have ablackcoat resting Fom the Goknsins axe hishly polymoshic in cor and he svaelsar gant, HEC betes a f-celesn and Mca Cay Cate Retievr, btom). ve scp mnt af respectively. Indoed, our cinent undcntnding of meknovorin bingy’ stems defemsin research is foswsed primarily on the inwmune system. This seras fiom the identiiction in tiwcinry mice of Meir mutations as the curse of fram the early discovery of cefenséns in phagoeyic cells and thei ante recessive yellow and gone anti 38 th cause of lethal el micrcbial propetics in vito, togsther wih more recent work demonstat ‘Cience Cock Litle, who developad many of de original laberauey ing that defensins can 1s Oe mouse sans and foundad The Jackson Labortery, was abs ene ofthe fst work indicts that B-dofensins do more than defend and sagaoss that the dog genic He rovngrized that denim inhersece of a blak coat was marked molecular and rapidly mdiatad citer in dogs tan in othr annals (J). Using chsscal linkage evolving fimily of ligands for G protein-coupled recepton 1 many amis, we walizal dt the dears Mack yon wprescated a previously different biologie sysiems, lunecngnizad component oF dhe moanacoatin pact (2) Unexecely, we fund the esponsble gem wo nook a Prleersin a sccoiad prciin previously sudo fo ts wl ity “The idetitction of dint black (onal roel on two mujer advances in dog genetics ‘elon Great Dane of Agouti and is highly expressed in dog skin. We wal biodhamicl and och fuss assus 19 show tha CBDIGS, ke Agcut binds corpettvely tothe rate in this faily supplies diver Summary Rlecoces vy ECCI Be nhc Con Con By one, ea, W857) als ofthe" lois") 5am rat me 396 0 007 a ening of He dog 4 tation oe £94 208 30 NOVEMBER 2007 VOL318 SCIENCE wxwscioncomag.orgFUL Genetic analysis of mammalian color variation has provided fundamental insight inte hhuman biology and disease. in most vertebrates, 1 Hey genes, Agouti end Melanocortin receptor (Med, encode a ligand-receptor system that controls pigment type-switching, but in domestic dogs, a third gene implicated, the K locus, whose genetic charact tics predict 4 previously unrecognized component of the melanocortin pathway. We identity the K locus as [Bedefensin 103 (CBDI03) and show that its protein product binds with high affinity to the [Mir and has a simple and strong effect on pigment type-switching in domestic dogs and transgenic mie previous) signaling through metanocortn receptors. ‘netic analysis of model systems in kb- cralory animals underlies mach of what ful, because much of the meleculr used by the pigmentary system is ciber by, or homologous to, genes used for ober phys- iologic pathways () This approach has been particularly usetal {or pigment “type-swinching,” a phenomenon in which melanocyiessyathesize eumelanin (8 black or brown pigment) verus pheomelanin {a red or yellow pigment), depending on the phase of the hair growth eye positon onthe body, and the genotype of soveral key loei (2) In most mammals, (wo genes that como pi g have been resogniged: the fa seven transmombrans syurning domain protein expressod on melano- eyes (5), and gout, which encodes a Hizand for the Melt that is secreted by specialized naling 16-9) MeIr acsivation causes exclusive prode- tion of eumelanin, whereas Mele inhibition causes exclusive production of pheomelanin (5, 10}. Thus, gain-offanetion Me 1» mutations cause dotninant inheritance of black coat, Whereas gain-of-function gout) mutations cause dominant inheritance of a yellow’ coat The Melr was fist recognized by viewe of its ability w respond to peptides derived from proopiomelanocortin (POMC), such as ‘e-mslanocyte-stimubating hormone (a-MSH) (3, 4) however, a null mutstion of Pome: has no fleet on pigmentaion in CS7BL6 mice, leading to the Sigasstion that the Metra high basal setivity and/or additional stimulatory nls Uh. “oepannents of Genetics ang veda, Sanford Un essiy, sutfod, CK USA Medkal Resa Cour (URC warmnatyn Genes Unt, Kael, Oxf, ‘0111 ORD UX ‘Depts of Chest atl Box Isty, Unrest of calf at Satta Cir (CO, ht, Ch 95068, USA ‘Dearne of nina nd Poukty ‘Sonce,Unlvsty a Sestathewan, Satan" S758. Grat “The authors conbuted equa 6 the werk. Present acerese Tred tchinson Cancer eseaeh Gere, Seat, WA 98109, U5A, Ho whom caesponence should be addesed. Era Atach@stantodecs wunuscioncemag.org . These results expand the functional role of f-detensins, a protein family i ind identity an additional class of li nds for In a classie series of papers in the ely 1900s, Sewall Wright (/2) concluded that mechanisms for color variation were ved across mammals, An ex ception, however, later became apparent from the work of Chucace Cook Little on donne dogs (13), in whieh dominant inberitan black cost was shown 10 involve a focus distinct from Mefr. At the time, Lite posted that dominant black 33 caused by an usual allele of Ago, however, using. molecular Finkage analysis, we recently demonstrated the presence of a thind gene in days that interacts swith Agent and Med, whieh we named the & focus (4. We found that the K locus Ins thyve allokes with a simple dominance order [Black 8) > brindle") > yellow (kat the A locus and vigouti behave similarly in ‘genetic interaction staies (Med? is episatie to ‘both Aang gent), and that the genetic map position of K docs net comespond to the pre- ited Iacation of any previously known pigmentation gene. We use the dist evolutionary history of domestic dogs to show that the K locus encodes @ previously unreengnized class of melanocontin receptor ligands Linkage and association mapping of the K locus. We showed previously that tho A? ‘mutation mapped to a 12-Mb interval on the distal end of dog chromosome 16 (CEAL6}, between markers REN292N24 and FH3S92 UD, We asconained additional kindreds A, and A to refine the map supporting online ext] and defined overtopping critical regions of $8 and 7.6 Mb for the A and A” mutations, respectively (Fig. LA and figs, SI Ww $4). We used an association-based strategy to narrow the critical region, Because mest broods were derived in the past 200 years from small founding populations (75), muta: tions within a brood ate expected be identical by descent and share extended haplotypes. In Boxers and Grea Danes, we identified bread peaks of significant association (Bonfermonicorecied P value < 0.08) that extended over [9 Mb and 320. kb, respectively (Fig. 1, B a9 D). Sixtoon gence hhave boon annotalal to the region of sige ofa RESEARCH ARTICLES I nificant association in Great Dans, inching 4 geno chster hat encodes 12 fedofesins (16) small anincobial peptides that ae sccroted mainly by epithchal ells (17, 19) We sequenced the mature protin-codin regione for nine members of Tie fedeTrsin sluser (hose known at in dogs crying A" andor & and ientifid seve polsmarphisms concordant with dae A all, including a 3-ba second exon of CBDIOR human DEFBIO3. th hie doltion (62 “To evaluate the polymorph more broly, we examined dows fi breeds that could be clasifed imo. wo catgorss with regard to their putive & Incas genotype [SOM text), Among 484 dows, there were 13 cases where the AG23 poly~ mnempbism did not corelate with exacobe phonotype. However, sequencing of gout and e/r reveal that each discordant ce ‘souk be explained by known episatic ine actions (19, 20) tale $3). These results incicate that A? allles in all broads are protubly identical by descent and suggest that the 3G23 polymorshism or closely linked variant in comple linkage disequlibrinn (UD) i the A muration Short range, haplotype and resequencing analysis of K#bearing chromosomes. by contrast to the patiem of LD within breeds, which affeds a posterfl approach foe aan ciation mapping with mezabsse resolution, the pattem of LD across breeds is more grained and therefore provides the opportunity for high-esolaion haplaype mapping when mutations in diffrent breeds are sentical by descent (27), We identified 28 polsnampismns [22 single-natcotde polymorphisms (SNPs) and siv indels including the G23 polymer Dhista) ina 20ekl> snterval surrounding CBDIOB that were thea usd 10 infer shox range haploiypes for 14 K%bearing and 16 bai bygods (SOM tex). We obser sx paren ta” k-bearing and five paren” A bearing chomasomes (picts in Pair (op) deletion in the the ortholog of ancestral recombination event, ih tox defined a maximal imerval for A" of 9146 bp ig 2A). Complete wscouencing of this eval (exeoy fr thee he 1°? arimals, one KR” animal, and four KK animals. fom seven breeds 1 seals! wo polymoephisms tusides AG23 tha ae perfetly concordant with A hoeus gen0- iype [S104 and S105) (Fig 24 and wie $2) “The 9}46-bp interval contains both exons of €8D 105, the fint exon of dog expressed sequence tat (EST) CX990240, and do EST CoV5262 (Fig 2) Honever several coms rations indivate that CBDIO( is, indeed, the SCIENCE VOL318 30NOVEMBER 2097 1419i RESEARCH ARTICLES 1420 K locus and that the AG23- deletion in CBDIOS isthe K® mutstion. First, the other two transcribed ekmeats in the critical iter- val are represented in the database by single EST and are not kaown to encode proteins or to ne expressed in the skin, Second, SLOM and S105 fie in long terminal repeat clement that |S 3 kb upstream of the firs excn of CD15 and have no cffect on mRNA levels. of CHDIO3 (Fig. 3A). Finally, as discussed further below, CADIO3 is highly expressed in skin, the G23 deletion affects CBD 103, protein function, and pharmacologic studies reveal that CBD103 can modulate mclanocer- tin signaling. ‘The preceding discussion has referred to A" lecus variation as though &” is ancestral, whereas A is derived: a hypothesis based on the comparative genetic diseibution of coat. color phenotypes and inheritance pattems. ‘Consideratiens fase on sequence alignments this hypothesis: Mammalian CBDI03 “orthologs that we identified from the available ‘genome sequence are each 67 amino acids in length, and the optimal sequence simitirity alignment contains no gaps or insertions (Fig. 2B), indicating that G23 and consequently tho A® mutation oscurrad specifically within the canidlincae. Expression of dog defensins in skin and in transfected keratinocytes. We isolated RNA. from the skin of a 7" Doberman Pinscher and a KA” mixed-breed dog and surveyed the expression of the 19 Bedefensin ‘genes that are clustered on chromosomes 16 for 2S by revere transcription pal chain reaction (RT-PCR) (/6). Expression was detectable only for wee pones! CBDT and CBDI03 (fg, $6). We then used quari- tative RT-PCR to measure levels of skin mRNA, from four KA? ssimples and four 4/" samples, which were all from mined-broed dogs, and found 19 eect of K locus genotype ‘on loves of CADI, CBDI03. 01 Aout mRNA (Fin. 3A), ‘Arailable antisera again’ human DEFBIO3 are unable detect the endogenous dog proxin by Wesiem blotting or immunohistochemisry: therefore, we generate epitopestagaeal expres sion constructs for cach allele (CBD1O3VS and ‘CBDIO8AG23VS) and staid thee pater and kkvels of proein expression ater transection of ‘ulure! mous keratinacyes fy cell extracts. analyzed by W blowing, 2 single fi corresponds 16 the expected molecular mass fof the tagged protein aller signal peptide cleavage: in media, an additional slightly smaller band is present, which sug: Additional processing (Fig. 38). The relative ratios of the two bands ae similar in media from keratinocytes transfected with however, the wtal amount of immunodetectable protein in media was 30 NOVEMBER 2097 VOL318 SCIENCE nificantly greater for CBDIONMG23V5 as compared with that for CBDIOSVS (P= 0.0021, Cochran-Mantel-Hacnszel chi-square test} Thus, loss of the N-terminal glycine CBDICS docs ot allcct intracellukac processing. bur allows) mere of the mature protein to accumulate in the media andior cexacellalar space, CBDLO3 activity in vivo and in vitro, To further explore the finction of CBDI03 in an imental gonelic system, we generated which a eDNA encoding cither the A or the K? alle was driven by a stoong and widely expressed promoter (22). We chose 2 genetic background that ponnally transgenic mice Lun q_Lineage grace 2:38 has Ageutichanded hairs and observed that two twansgonie founders gonorated with the CBD1038G23. DNA (the K* allele) dise played a predominantly black coat with small patches of banded hair (Fig. 4), Unexpected, the normal C8D/03 cDNA (ihe k allele) also produced transgenic mice with a black coat in 20 out of 21 founders ( more, we observed that transgen mall sham their nontransgenic litier- female transeri animals were dark ‘coat and small size; in adult mice, reduced body weight persis in both makes and fers ig, 97. mates, By 2 weeks of a Twat) Boxers bina vs. Yeon KZZZLLLZLZ |, Reginsotsinicant ‘Great Danee:bisck ve yolew ‘ocean (3 005) peyscarcnusce(ue) Fig. 1. Genetic mapping of the K locus (A) Initial fnkage studies [phase 2, (24)] defined a 12-Mb «ritica region fork ascertainment and characterization of acdtiona kindeeds narroned the interval to 3.8 i (phase 2, figs SL to $4. Assocation analysis for 60 markers in brindle (n = 12) versus yellow {= 10) Boxes, and for 5L markers in black (n = 9) versus yellow (v= 10) Geeat Danes, was caried ‘out as described in the tert. (B) Candidate genes in he 320-5 region of greatest assedation in Great Danes; this region incluces 12 f-dofensin genes (shown in red). (C and D) Significance, pltted as ~log of P values from a chi-square test of ellele count, is shown as function of distance along ‘CFALS (oniy for SNPs present at greater than 10% frequency and genotyped in a east 75% of the samples) The dashed ted line indicates 2 Bonferroni-corected 5% significance level; these regions are ingicated by hatched and black bars fr Boxers and Great Danes, respectively, in (A). Annotation {i based on the Nonrdg RefSeq track in the UCSC Genome Browser, excopt for CBD102, identified by Patil et a. (2). ua scioncemag.org‘Thee considerations suggest tee persible The CBDI03 K* allele predicts a mat mechanisms by which CBDIOS might act to peptide (ater modulate melanocontn signaling: (i) by binding amino acids tha! contains six eysteine residues to and activating the Met, (i) by binding to the and begins with the glycine that ix deleted in Mele and preventing its inhibition by Agoui the A alkle (Fig, 2B), The prodisted signal prtein, oF Gi) by binding 10 Agowti prowin sequence cleavage site is supported. by 1p its seoucsiation and! ition, biochemical studies of the omhologous human Toisinguish among these ideas, we generated protein [known ss DEFBIO3 cr human f+ synthatic fomns of CBDIO3 and tested their defensin 3 (IIBDS)]_purifiod from faman ability to ineracs with the Metr and agouti sige tissues 2H. We symtiesizad the 4S-esidue naling protein YY (ASIP-YY), a synthetic A? form and 44-residue &® form of the dog version of the C teminus of Agouti proxin protein thence refered to as CBDIO3 and that behaves as a competitive antagonist of CBDIORAG23, respectively), allowed oxide ‘u-MSIF at the Mel and Me4e (23). tive refoldin y al sequence cleavage) of 4 A complete association with KE 3623 —= Bmaeinal tone tor ! esr mutation (46 bp) Teer a n_Mexso0240 CONSE ge0103 _- Mouse i¢r1.4YLLEVFLILPLVPAPGDAFLPKTLEKFYCRIRGORCAVLNCLGKESOI ..- Human MRIHYLLPALLPLE lVEVPGHGGE INTLORYYCRVRGGRCAVISCLPKEEOI |. - Chinchilla NetHyLieAVLeLY LMPVPGEGGI INTIGRYFCRYRGSRCAALTCLPRETOI -. (GoW NRLYYLLSALLELE LUPV PGNGGI ISGLORYYCKIRSGRCALIGCLPRESOL Pig NRIHYLLFALLELY LPL PONGRI INTLORYY CKIRRGRCAVLGCLPEEEOI Opossum MRIHYLLFFFFELY LVPAPGESWLLERINNNY CRYROGRCRE IRCTSREENT Dog MRIYYLULLLPLLE LMPVPGNGGI INTLORYYCRIRSGRCALLSCLPEEEOT oe signal sequence G23 Fig. 2. Resequencing and recombinant haplotype-based mutation analysis for K-bearing_ versus k= bearing chromosomes. A) A 20-kb region surrounding CBOI03AG23 was resequenced (except for repetitive regions) in 10 dogs fiom 7 breeds, and haplotypes were infered for 28 high-recuency biallelic pelymorphisms. Blue and yellow squares represent the major ané minor alles in K*-bearn chromosomes, respective, and allow some haplotypes to be designated as “sparen,” "x parental” cr “proximal recombinart,” as indicated. White squares represent missing date. Genotypes for five Great Danes (denoted by asterisks) were detormined in a second resequencing round targeted specially for distal recombinants as described in the SOM text. Witin the 9.1-kb itewval defined by recombinant haplotyoe analysis, thee polymarpisms are completely associate ith K* versus KY, as incicated in te upper part of the figure. (B) Exon structure of transcripts within the maximal candidate interval and alignment of slectee CBDIO3 orthologs (38). RESEA\ and high-performance liguid chromatography le congener with thrce inrachain disulfide bond, [Nie 9-Pho’ Ha MSH (NDP-MSH), a potent derivative of «MSHI, stimnslatcs robust accumulation of eyctic adenosine monophosphate (cAMP) in melanocytes (25). However, neither CBDIO3 nor CBDIOBAG23 has any ‘effect on cAMP accumation (Fig. 5A), ix dicaiing tha CBDIO8 is net a conventional Melt agonist, To test for receptor binding, we transiently traisfoied human embryonic kid ney (HEK) 293 cells with Mele expression constructs 22) and used caropium-labeled NDP-MSH (Eu.NDP-MSID as & fluorescent tracer We usol saturation binding assays to fist alate the allingy of Eu-NDP-AISH for the dog Mele (Fig. SB) and then carried out die placement assays with progressively increasing 2 21L0 \doinaes at acidic conitions (1) 0, 4 2,0 | de> = 40H dominates at basic comitions The reactions in Bg. and 2are not independent, but ave related by the water elibrium, Eq, 3 1h0 == HY + O1F 3) Although electrons re not present as separate Uhr election al, i well defined and s given by the Nemst equation. The wactons in Bg Land have expressions he given, spective in ele¥) =-444 4 (-1)(41.229) + 10899 hy - =D laren!) (A) ule) = Aaa + (-1)(40.401) 2.0892 snot b (s) “ pemiro,)) Hire, (60) refed tthe vac te acy of HO i esaned wo Be ny the Kolar aposnnaion Ir wid, the temp ature 298K, andthe patil prosreof ou in ar The eleorachml pte ‘swone inthe atandad hydrogen lode coun evel fy = 20) The sind ects ic jl eons Fay a 1.29 W tnd 0401 V sent SHE, rage 2. The ttn btwn para pOH snp pons Mot-Scathy measurement (2 and the- vc comer 23) fate that fhe dlooron affiniy, EA. of hydogsrtemined Siren fo coma wit war or ost at tppoxinay "030 eV, which is TeV mene Pecave tan he vibe otal in ace (20, Besa th diamond band gp i 83, is shit pls the op a the dianond valence and Bt ogprniraty “52 eV. This etme as teed to eve fe pston ofthe band edge of Garver it Fip 1 Abo shown fn Fel ote eleeehenicl pts Br he axyasn dou Couple called fom Ege. an 8 for O21 bara pl = Ua fl = 14, Tse er cic, 46 eV ant ASS eX, rope Stdgke the etna poston of the valence band main of elmond "The band neap shown in Fe | is Igénapevemnimasd dimond in cnt Wi oe acca lene, Onygen and orn ane bah SCIENCE VOL318 30NOVEMBER 2097 14251426 ARCH ARTICLES more electronegative than carbon in contest to hydrogen, whi is. more okopositie than catbon, Therefore, srfice tration with cir ‘oxygen oF flaring ines a surface Gps thet will increase dhe ston afinity, orposite to he coffe hygeine due, The lee= tron affinity of an exitizad diamend surface is about 1 eV more positive than hydogenatst surficss (24, 25), and the effet of Huong is expected 10° be sina This lowering of te slinmond energy bands by ~ 1 eV places the vakace bared maximum a £ = ~ 62 eV, bebo the electrochemical potential ofthe aqueous phase atall pIfs Electrochemical mediated charge transfer. Water in expibeiam with ai has ap = 6 foes the naturally cocaring CO; in the ait, which ‘comesponds to an elzcaochemicl potential of ie = $3 6V from Fg. 4. Lowering the pit Towers ie Therefor, ation of hydrogenated 4, (0). and clsetroas wansfer from the diamond 0 the Eq. 1 proceeds in the forward dieeton, which results im consumption of prations. However, ‘when diamond powder equilibrated with mist ai is brought in contact with a basic solution, e(dia) < pg(ag); ckvinns tansier fom the sluion imo the diamond, which drives the reaction in Bq. 2 in the revere direction Hy- roxy ions are consumest and the solution be ‘somes mon ac, Ise are de only proossses taking place at the iterfae, the protons. cae sumed lis the hydeons ers fom ar el the number of eins tansfared fon tbe de ‘mura to the solution. Reduction in the concen. tration of electochemical acceptor, ie. the isoed oxygen, should decrease the amount of clecton trate fom the dsmond and sup press the hangs of pH ofthe sokticn, “The overall stoichiometry of the okt chemically mediied charge transfer at moderate pil can be writen as Eg. 6 (18 HOW ea} Ona + 2H:0ae + ACO2 a solution until yg (dia) = (aq). The reaction in Ahi, + AICO}; C) ig. 1. Estimated bad tine Ambient E,eV—-Diamond cee ele Ta eens! aqueaus soon. The elec 1- E trochemical potentials of the “cas oles - shown at pH = © and pH . Li Te electron aff of 0+ 80, meme eae 0; + 21,0 + de = 408 S3eV Tk eae, | was mioge-see 7 #20 YY occa) Wi, ee ip iaern eae natural diamond powder, equil~ s ee a | emai ee eee ‘der nas added. For the former, jue s pmaimiene Seal ricerenee SNe late, (a) < ean), and ace eee EZ nae ae SE Sor ned rcnas and N1aHS0,, Now the expanded vetial sale. 30 NOVEMBER 2097 VOL318 SCIENCE Equation 6 describes the siuation when the acidity is generated by atmospheric COs. In this cave, the counterions inthe agucous passe are dominantly HCO; . The type of counter- ion will depend on the ambient, For example, in the presence of HCI vapors, the counterions will be predominantly CT We note that the electron affinity of the ‘agucous oxygen redox couple is much greater than that of molecular oxygen and other gas phase onidaass in the absence of water. For ‘example, the electron affinity of O3 is EA 0.481 eV (26), which places the energy at E=-BA~ 0451 e¥, farabene the enciy of the oxygen redox couple at any pl (Fig. 1). The celciwn alflnities of other highly oxidizing sgasphass species, eg, NOs, Os, and Fate, respectively, 2.273, 2.103, aad 34 eV 25) and thus are also not suifcient wo directly ‘oxicize diamond. Therefore, direct electron transfer fforn diamond w gacphase species in the absence of water is thermodynamically improbable. Titation of diamond ponder. Natural dia- mona powder wih a nominal sie range of 0.5 to 10 jum was purchased ftom Advanced Abrasives Corp. The aspurchased powder rod and was theretore hydrogenated piers ence eeppotog «byron placa at 750 W, at 2 gas flow rate of 196 standard ‘cubic centimeters per minute and pressure of 35 tor. The powder was cooled in hydrogen {Bs W room temperature, stirred, and the process then repsated two times to ensure good. hydimgen coverage. X-ray photoelectron spe troscopy (XPS) analysis indicated that the oxygen concentration was lowered St 14% by this process. Bosause the samples were transfered in ar, dhe butk of the residual oxygen likely arose from physically adserbesl species Diamond powder ‘yoriate ina plana, inductively coupled 13 56-MHz plasma teeter at M90 W witha de bias of -307 V. A source gas of SF, a125.scem wasused tomlin apresureof (0035 tr for 10 min, The substate temperature was maintained at 20°C. Subsequent XPS. analysis of the powder showed both fluorine and exygen on the surface in approximately a 511 atomic rai, In all of the tivation experiments with diamond peneder, I of powder was added 19 20 ml of solution. Ths solution was stirred continuously before and afler addition of 0 diamond powder. The pH and dissolved oxygen concentration measurements. wer made with an Accumet XL60 meter Analytical sede chemicals (HC, NaOH, and NallSOs) were used in all experiments, and soluions swore fered through aD, lsum fer before u Airsturated water was used for mest ex periments. In some cases the dissolved oxygen concentration was redueed by bubbling nitro igen through the solution for 4 hours and then ua scioncemag.orgadding sium bisulfite up toa concentration of 103 M. The ceiginal dissolved oxygen concentration of sirsatunted water was 7 mg, the concentration aftr the nitrogen purse and bsulfite ucament was less than the sensitivity ‘ofthe disohed oxyast probe (0.01 malt For experiments with sodium bisulfite, the ‘chamber was closed 1 sola the soktien from the open air, and nitrogen was cortinuously purged through the chamber Figure 2 shows the changes in plY when |g of hydrogenated diamond. ponder added separately 10 20 ml of ai-saturated aqueous solutions, one at pll = 5.2 and the ‘ther at pH = 8.5. The pH inereases upon addition of the hydrogenated diamond w the Acidic solution and deereases upon addition of the diamond powder to the basic solution, These changes are in the expected diretions deserbed above. The inset in Fig. 2 shows the change in pHU when hydrogenated diamond povsder is added t0 a solution that has been deoxygenated with Nz and NallSO}, The change in pil is very small (note the magnified vertical seale), This resuh indicates that dissolved oxygen is necessary for charge transfer 10 tke place Figure 3A shows a titration curve obtained by addition of 1 g of diamond powder equili= brated with room air to 20. solutions of known pl The ordinate is the number of celectons, AN, transferred ine the water and is given by Ea. 7, Ne = Nar ANE = Navlino % {10-(108% — yor") —.o-m—10-#)) a Whore Nay is Avogadeo's number, ANy-and ‘ANonr~ are the changes cr of Wand OID ions respectively, Figo isthe volume of ‘water in es ard pi an pHpare the inal and final pll of the solution, At high pl, the Gist term dominates: at Sow pl, the second tem dominates In the Ma portion of the tivation {uv bath protons nd hydroxy ons re present in low concentations, and the tarsfer of a small ‘numberof electrons Kea wo Tage changes in pH “The crowover point in Fig. 3, A and B, is where the eleetmehemical potential of the sobation is equal to the Fermi level of the orig- al diamond epilibrated with ait, The ekcito- chemical potential ofthe solution at this pont is a irext measure of the Fermi level of diamond ‘guiliteatad with room ait. The exossener pot is most easily scen in Fig. 3B and is esimatad 10 over at pl which from Eg. 460 respordls lo an election chemical paxcntial of Re = 8.3 eV. This value is in close agreement ‘with independent Mot-Sebotky and Kelvin probe measurements that show jj, = 82 eV (22, 27) for diamond in equilbsium with aque- ‘ous solutions in at We estimaid the change in the concentration of disalvat oxygen upon addon of diamond powder by using an electrochemical biochem: ical exyyen demana (BOD) probe corsisting of 4 FEP Teflon membrane covering @ god and sier clectde, When hydecgensted diamond powder, previously equated wath oom sig is added to an acidic solution, (dia) > (09): clectons transfer eto the solution and the concentration of disohed oxygen decreases {duc to the wexsion ip Fg. 1, which proceeds in the forward direction. When hydrogenated diamond powder is added ta basic solution, (Cia) < py(aq), and the concentration of isoied oxygen increases. In this iter case clecions leave the solution, driving the wacton in Fg. 2 in the reverse dretion, thus increasing the concentration of dissolved oxygen, The 1 sus are sunumarizad in Table 1 In bo cases, RESEARCH ARTICLES I the change in dsselved exygcn concentration is in the expocted direction. Funhomnore, the os tinstod change in numberof moles of issalved ‘oxygen agrees with the change in number of molgcules of protons and iydroxsl ions cal ‘ued fern the pH change within the expected ‘or of the oxygen measures, Zeta potential measurements. Zia poten tals ae obtained by measursmnt ofthe elt: hort velocity of dipersed solids in aig ‘The magnitude and sign of the 2etx portal depen! on the net amount and sign ofthe charge within the shear boundary separating the mobile liquid fo the stagnant layer near the soli sur- fee. A postive eta potential indicates net post live hae within the shar bounduy: a neve 29a pote inate a net rayative charge (28) He we use measureasnts of the zeta potenti as confirmation of the direction of leetron transfer between diamond powder and water We performed 22 potemial measurements with a ZETAMASTERS asing I-um nominal size naval diamond powder purchasod from SIKS, Kay Industial Diamond Comp. Hydro- ‘genation and fuorination of tne powder were performed as described cathir. The dimond powsr was equiloatal with rem air before the zeta pont! measurements were made, From Fig. 4 i i evident that hydrogen teominatod dhamend powder shows postive -xta potsatal at all pl] ales fess than 7. This result consistent wih electron trans" fom, the diamond, whieh increascs with decreasing plas proictsd by the transfer doping model The sigh negative zot2 porntils for the hydroger-serminatd diamond at pH > S are consisieat with a low coneenimtion of resi ‘oxygen-contining sifice funcional groups on the surfice compared 10 the aoniy drogerated smples. The zeta potentials for both oxidized and uorinaed diamond powder differ makedlly 3E+19 a 3 1EH19 2 s1E+19 4 Zoot es aEH9 4 a \ west a4 * ° 3 6 . RB 9 4 8 2 Fig. 3. (A) Number of electrons transerred from the diamond upon addition of 1 9 of hydiogen-terminated natural ‘brated in alr to 20-ml_ aqueous solutions of va ‘rosiover point is where the Fermi level of the diamond equals the wunuscioncemag.org pH wand powder equi initial pH. The initial pl. SCIENCE chemical potential of electrons in equi ‘couple, (B) Change of pit upon addition of 1 9 of hydrogen-terminated ‘natural diamond ponder equilorated in ait 10 20-ml solutions of varying pH im with the electrechemical YOL318 30 NOVEMBER 2007 14271428 ESEARCH ARTICLES fiom those of the hydrwgentemninated powder The potentials for these sarmphs. are simiar in magrtuxe and negative at all pH yalucs greater than 1. The negative charge ean anse either fiom tans of decuas 1 tie diamond ar ‘ovization of residual oxygen-cornaining fic- ional groups on the surface, e, carboxylic acids and hydroxyl radicals “The similarity of the ress forthe oxidized and Muoriaaed! sam Dies indicates thatthe charging process fs similar spite the diffrent surface chemistry of the wo pes of samples, Contact angle measurements. Adssncing ccontt angles on hydwger-iemminged, 3m diamond aiacles i solutions of dilerent ptt ‘wore determined, Macks are natu, tanguba, unpolished, twinned single crystals of diamond, We measured the contact angles on the (111) facets of these ena by the Willchay plate technique (29) using a KRUSS KICO Processor TTensiometer. The diamond samples were fist hydrogenated under conditions describe above for diamond pondars fora period of 2 hours. Afr livalmgenation. they were cleaned uta sally in Mill-Q water for 10 min ad dried flowing No The mocles were suspemed by a comer fiom the arm of the electrobalance and ime Table 1. Summary of results showing changes in the concentiaton of dissolved oxygen (DO) and PH with the addition of 1 g of hycrogenated natural diamond powder equlibrated vith air 20 mt ‘of solution. The change in number of fons of H and OH is trom Eq. 7. Steady-state value Steady-state value Novof © before adition after action aransforted D0 ingle 78 73 19 10" of 0 47.6 x 107 pH a 53 8.9% 10" of HY and OH” +89 x 10" 0 ‘matte 68 73 23 x 10" of Op -92 x 10” oi 13 a4 4.8% 10" of H ane OH” ~48 x 10" 4. tee poential asa «$0 {uncon of pH for Fycrogen- . (Hydrogenated diamond] ated, ore, and fornated 39 f 8 + Oxidized diamond natural diamond powder fg Thosinated diamond Hyegenteminated do = 1 mond shows a positive zeta Potential (postive surface Zeta potential (mV) mersed in the solution at a constant rate of 3 rmmimin while we measured the force an the ssiple de 1o wetting The contact angle was calculated flom the measured free, wetted enw of dhe sample, and the surfsce tension fof the solution, yyy 29). Fxtrpoltion of the results 9 zero inmenion depth eliminated the ‘effet of variation of the length of the wend pevimxter, The surface tension of the solution was measured with a eughene platinum pl, Which is wetted with a sontoct angle of vie tually 0°. By measuring the fowee acting on the plate when imaversed in the selation, we eal. culated yy. The advancing contot angles ofa hydrogen. teminated diamond macle in contact with aqueous solutions of varied pH and dissolved ‘oxygen content are shown in Fig. SA. We tribute the deemease of the contact angle at loys plT1o the elecaostatie atiaction between the positive space change layer in the diamond and the compensating anions in th agueous phase (8), The solvated anions provide a mechanism for binding a water film 19 the hydrogen-terminated, hydrophobic diamond surface, Reduction in the concentration of eke chemical acceptors ie, the dssolved oxygen, should decane the cletron tasfer from the ‘dimond and edce the effet of pH on comct angk, Fist, we lowered the disolved oxygen ‘oncentation in the water by bubbling nitrogen through the seluion. Further removal of dissolved oxygen was achieved by reduction with SA, itis char charge) at pH < 7, Fluort 10 sodium bisulfite (30). From Fig nated ad ced damond Te eee ge cess ee rei esngeile =O 9 entation of dsohed oxygen daseaa. Fur Lester ‘ thea, the depen cone ang on 50 i 4 ptt esentally dsppared arth aiton i ff exces sodium bisulfite. whic reduces the a ‘ disolved oxyzen concentain to very how bevels o 2 4 6 = s » BM The work of adhesion, Wig which is the P “energy required to separate the liquid and solid A 6 B 3 a @ 2 = : ‘ 2 32 . zg ® 3 in : ‘ 3 3 *aii¢ < 2 a 7 5 74 . oN, + bisulfite ¥ i : = N:purge 2 a + Equi witha = wf 70 1 o 1 2 3 4 8 6 7 o 1 2 3 4 5 6 7 pH pH 5. (A) Adwercing exit angles on a (LL) axrlace of a yciooere terminated dlamond mace asa function of pH. Experiment were perfrmed with ater in equibium with at, water purged with No, and water purged with Ne 30 NOVEMBER 2007 VOL 318 SCIENCE and teed with NallS0,(B) Changes i the work of adhesion calculated fram the contac angles. periment were performed with water in equitrium wth water purged wih Np, and vter purge wth Ny and treated with NolSO3, ua scioncemag.orgphases is reae io the contact ‘the Young-Dupre equation (6, (Eg. 8). ) Was = Yuv(l Figure 3B shows the work of adlsion as a function of pH of the solution. Higher work of adhesion is four t lower pH for airsaturated solutions: the work of adhesion for deoxy gen- ate solutions is read and is indpendem oF pH. expected Role of exygen. Our ress support the sur face transfer doping mechanism (10, 11), but are consist with charge transfer betwen diarnond And an electrochemical rodox couple involving (On inthe adjacent water phase as suggested by Font et al (/7) and Chakrapani er al 08), aber than the Hy” couple a8 or Proposed! Fixing the cleetrochemieal potential and pinning the Femi level inthe diamond require facile eketeon transport between the diamond and the adjacent agueoas phase, The redox n= action (Es. | and 2) must be sulicinty rapid vo pemit measurable time of the measuremens, The relatively kage tio of O3 (Compara Hs) in a= god water facilites the reaction ki netics of Eqs. | and 2. The relucion of Or in the aqueous redox couple may require trace metalic impuritiss on the surface t proceed at ppresle res. Furthennoce, an adsorbed ot= ‘ganic fim can displace the aqueous phase and suppress the ef (78). The use of epasy resins to insulate electrical contacts may provide such a barier ‘We emphasize that under certain contitions, oiher processes not involving electron iransr may take place. For example, surface fc effcet deviees have been construct with the use of induced charge (7) Also, on an oxidized di mond surface, function groups sich as caibox- dle eid ana hydroxyl ean ionize (Egg. 9, 10), ia)-COOH <= (dia) COO +1) (ia)-OF = (ik HF (Dy “The tctal nunber of sites on a (111) diarnond surfice is 10" enr?, Therefore, even a 1% Fig. 6. Changes in pl upon addi- 9 tion of 20 of hycrooerterinated natural damord powder and 1gof ‘oxideed natural ciamend powder, each equated vith air, to sep" 7 atate 20 soiutions with pH = 5. = ‘The ans indicate the point where = the diamond ponder wes added. 4 af wunuscioncemag.org surface coverage with ionizable sites can mask the eflees of transfer doping, vshich gives ise to sheet charge densitis of =10" to 10 em ® eo Because the wacions in Es, 9 and 10 do rot insole escirons their eculibia det depend on the chemical posetial of electrons, i. However, these reaetions do involve rons, 30 their equillvia depend on the chemical poixtal of prowns, ie. on pH, The reaction in Eq. 9 will proceed tothe right i the pl of the agucous phase meets the condition pH > py 44 similarly, the reaction in Eg, 10 will proceed if pll > pk = 10. Here pRy is the acid dis- sociation constant of the functional greups. I these conditions axe met, protons tans ito the solution, lonering the pl. This of issrated in Fig. 6. The pl of a solution, ally at pit = S, decreases upon addsien of ‘oxidized diamond powder. In contest, when Inydrogeraned diamond & adds 1 a solution wit pll = 5, surfoce tansfer doping causes the pH increase All ofthe experiments have boen performed swith nara type LA diamond, which contains ppresiable quantities of nitrogen, However, the nitrogen in nauwral diamond is pres aggregates and, as such, dogs not provide active donor eeruers (31, 32) In contrast, in type Tb i diamond, single substitutional nitrogen ‘which have a donor keel 1.6 ‘eV olow the conduction band minimum (35). If present these donor enter can ionize and compensate holes generated by eecton teas: fer to the aquoous phase (2). In this situation, postvely changed donor sites and. holes shargobalanced by excess anions in the aque fous plese, Because all of our experiments were conducted with type IA diamond, which has negighle subsiutional nitrogen, compensation by ittogen donors des not oeeur. Indeed, we found that the surface conductance ofthe type A dimond is four orders of magnitude greater than the conductance of type Ih, 2 = 2 man (100) synthetic diamond crystals (Sumitomo, HTP) Electrochemically mediated charge transfer fn other material systems. Flecimchomically mediated transfer doping was fist votod in diamond (/) because te effect on conductivity isso striking, However, the phenomenon should Added 77 eiidized ponder “S Added hydrogenated powder 10 20 ‘Time (min) 30 40 SCIENCE VOL 318 RESEARCH ARTICLES I ‘occur in many other systems if the bund Hin-up 'S appropring. Furthermore, the clectrstatic attraction between the resiling space charge layer in the solid and the compensating ions in the adsorbed fin will enhance the formation of adsorbed weer films The electron affinity of semiconducting Singlesvalled nanoiubes (SWNTS) has. been ported 9 be 4.8 eV (35), Alo, SWNTs have fund gap of 0410 06 eV and a work function ‘of 48 to $1 eV (26, 37). These values ploe the Feu level above the eleatoehial poral of the aguacus redox couple (Ex, 1) for astra water, which is ~ 5.3 eV, Hence, when SWNTS are exposed fo humid aig, electrons can transter ‘out of the SWNT into an axhorbod ater fi. Several stalies have obseried_an brut ange in sign of the Secbeck coeticent i negative to pesitive when SWNTS that wore previously ancaled in yacuurn were exposed to air at room temperature (34, 39), These welts show a change in the domint charge caioe fiom dlectons to holes coasisent with the lac trockemical transfer doping mechanism, Fi ihermore, small concentrations of gases such as NOg, Nils, Oy, and HO change the conduct iy of SWNIS (40-43) in the directions pre- dised by our model “The election affinity ofa ckan GaN surface fs reported o be 30+ 03 eV (44, 45). There fore. with a band gap of 34 eV, the conduction ‘hand minimum is st £= ~ 3 6V'and the valence ‘band maximum is at E =~ 64 eV. The ck twoshemical potentials of the oxygen redox coupks ( 443 eV to $466 eV) are therefore in the mighgap region of GiaN rather th the valence hand exige as inthe ease with diamond, Changes in the chemical pone of gue cus redox couples in an aorbed wats film can therefore change the occupancy of the mid- gap surface states tht are believed te play a role in the ubiquitous yellow-band emission from GaN. This electrochemical mecianism may be responsible for the eninaeement of yellowsand emission in the presence of HCL ‘Yepors and ciminishment in the presence of ily vapors (46, 47) Change transfer between polymers and metals (conmzetelecrificaton) isa wellknown, but not filly undersood, phenomenon that depends on the diference in work functions of the contacting amsteals, presence of suc sles, relative humidy, atmospheric adsorbate, humidity, and pF of the ambient envionment (4S, 49) Sliding friction is: another complex phenomenon de in some situations on ihe ambient. We suggest that charge transfer 10 celeirochemicalaceoptons in adscebsd water films may mediate these proxesses and should be consierad when intepyeting esl. Futher- more, charge tansfe eletroxhemieal accepios in adored fms may influence the propertics ‘of nanopartck’s and other nanceeierscale Structures, This point is of particular intrest ‘Because the electron energies in small nanopar- 30 NOVEMBER 2097 1429ticks will depend on size due to quart References and Notes AME tones KY Fa Ape Py dae 88, 975, (ase. 2S. Giet lon. Ap Py. Par 130, 3 (999. 3. AC Ha amano, H Oba «Hj, Apt ys let. 68, 376 0980. 4H state a, Diamond Relat Mae 7, 80 0998 5S. chet an jp. | ppl Phy a2 36, 2057 (957. 6A rics, A Alea E Ken Diamond Rot ot 19,47 2000. 7. towards, An. tk 1. Og, Urea, ys. Sous Sl 88,79 (2000, 8. Map e ol. Ape. ys 8, 748 9RD. 9 TM ot Jp App hy 3, 34s a, 10. F Mai Me ed Marl. Ri, Ley, Pp. B Lt 85,3172 200). tin, Mind L ey, Becton See. 18, fas B00, 12. Larson, tin | Phy Chom 6109, 1304 (a 2, D Ft en yp Om a, 1 | stein, Seane 323, 1057 206, 45 0.Qiot | 2m. Com Sac 128, C84 0067, ALM Adm, Pysct Chom of foes (Wley tne Yor 1982, | Sourde ol, Deond Ro Mtr 1, 856 (oa, 1B. V. Chapa 5. Eten fern aby, Cage Heochom Sous lt 8, 642008) 9, A Bod Memo, 8. ore A Chem. 43, 59 com, 20. Va arse, Yo VF Mas, Betrabin 28, nTime) C2.C Handbck of Chmity end ss Ex CRC eve aca ater 84, 002 22. WN. Peon, AY. satin, MO. Hate CU bawton 8 Spt, Bereonl Chm 228, wasen, 23. G Pianta et lj Aap Ps 89, #259, (Goon. Fie iti Ly, Pgs eB 88H (ean. 25. TAL, 9A Tok Minas, Fjshina |: lecrorem See 146, 80 0099) LA Den Lange Hondo of Chany McG il, New Yoo 5,199), 27,8 femh ota op Pye. let 82, 2366 0002. 24. 9.C Rimura 8 Ragan, Pcp of Calli nd Suze Chit (eth, Non Yt 3, 1997. 29. AN Nourar, Gnd Tega ef ering Cote Suc end Clad Se, va I, ‘simon Matas.) God, RR Svonberg Es ‘eum Mo 1979 30 Nita, YMataga, Kl, T orami, Dwatnen 54,16 (84. G5 Whe, Pgs ae Guth of Diamond, 6 Dawe, Ms ree Se 9, sono Ghia epee, Lec, 198) pp B88 22. fee he Bape of Motel ond Sythe Demand £ Fit fear: P, Loc, 192,» 238, BATT. Caine € © tgttmlo, Papi: of Obnosd, FC fell Eh eae free tender, 1979), 98 at. fit, AL Rel AL Sti Fe ey, Demand Br Mor 1,380 (2002) a 2 a 3, 5, Kazu Nina Ad, HK, Yh, Ap et. 78, 30 200 3h De Moy Pgs er 9, 19300. (ey 37D Look, Murs E Gaur, RP. Ades A Raentegy, Py 86, S683 2000, 3B Bay et, Pps oy. ee 85, #61 (2000 37.6 U.suanaaer, CRW. Ady fog, Slag, Php Rr Lt 85, 1096 2000 10,96 Cli Kae, Migs A. 2a Since 2, s0p1 ue), 44 | Hong et ol, Sence 267,622 E00. 42, 5. ee a We. et eco A, 1046 2008, {A 2a L Spin Peer Mtr Ph Boe 6, sccbo ceo, {AAC Trg Pe Hay Da Har 7. Wena 2 App My. 94 3939 200) 45 VR Bema 1 op Ps #8, 13901960, 46. Chaar, Coe Wester Reeve Uris ‘evan OH Boo, 7. Wethapn,© Ragas, A dere, 6 Sumanairs ste es Se Sp Pr. 956 pe 0736335 8 2007) 48, A Wis, Mls MR dow 5. ML Whey BA. Gapewl SPs Chem 4, 20236 (6 9S, Cb, U. Yor Shr, We Manus, IE Fans Id fl 3,79 20D, 50. We thr J All Mon and) ts ae a Sass Haman posed raucle ‘Spaimentl spp Fare app ts HS get ‘ha 051298) Cae Rate Rae Urey satel ariedye 46 Aogst 207; apd 6 Comber 2007 toiiiereece e882 PeepopTs 1430 Coherent Control of a Single Electron Spin with Electric Fields K. G Mowackt FH. L. Koppens,t Yu. V. Mazaroy, L. MK. Vandersypen” ‘Manipulation of single spins f essential for spin-based quantum information processing. Electrical ‘control instead of magnetic control is particularly appealing for this purpose, because electric fields are easy to generate locally on-chip. We experimentally relized coherent contol of a single ‘elecen spin in 2 quantum dot using an oscillating electric field generates by a local gate The electric field induced coherent tznsitions (Rabi oscilations) between spin-up and Spin-down with 99° rotations as fast as ~55 nanoseconds. Qur analysis ilcated that the electrically induced spin transitions were mediated by the spin-orbit interaction. Taken together with the recently demonstrated coherent exchange of two neighbor fully electrical manipulation of spin qubits. mation processing provide the possibility af adding new Ranson 0 to tuonie devices by using the elston spin in ade S pintroniss and spin-based quantum infor Xa este of anode, Olt Unvesty of Texto, Fos ofe Box S46, 2600 GA Dal the aera “To whom conesponderce should be adiessed, mal .cooved@udeftal Lnkvandepenusetat ‘ese outers erred equal 85 ver 30 NOVEMBER 2097 VOL318 SCIENCE ‘spins, our results establish the foastity of ition tothe electric charge (1) In this context, a key element is the ability to induce tations Ihenween the spin-p and spinon states of & localiza eleciton spin and to prepare arbitary superpositions of these two basis stakes. This is commonly accomplished by magnstic resonane ‘whereby uestof'a rosa osciluing meat field arc applied (2) However, producing strong cosciling magnetic fields in semiconductor devise requis specially designed microwave cavities (3) oF microfabricated stiptines (2, and this has proven to be challenging. In comparison, electric feds ean be generated much more easily, simply by exciting a local c electrode. In addition, this allows for rater spatial selectivity, which is amportaat for local addressing of ingividual spins. It would thus be highly desirable 9 contro the sin by means of clttc fick Although oletic flds do not couple die realy to the cletron spin, indict coupling can sill be realized by placing the spin in a ms nic ld gradiont (5) or in a stuctare with a tilly varying ¢ teasor, or simply Ueugh spine ‘obit interaction, present in raost semiconductor sructures (6, 7). Several of these mectumismns have boon used to ekectcally manipulate eke tron spins in two-dimensional electron systems (8-11), but proposals for coherent eletscal con tol atthe level of a single pin (5, 12-15) have so fat remained unrealized. We demonstrate coherent single spin rota tions induced bya oscillating elect Field, The secon is confined in a gaiedefined quantum dot (Fig. 1A), and we use an adjacent quantum dot, contains one electron as wel, or readout ‘The ac cleinic fol fs generated trough excite tion of one ofthe ges hat form the det, thereby ua scioncemag.orgpeiodically displacing the elec wave fnction ium position (Fig. 1B). nt consists of four stages (Fi 10), The device is initalaed in a spineblockade regime whore two exces electrons, one in each dot, aw held fixed with parallel spins (pin ‘mple), either pointing along or opposed to the extemal magnetic field [the system never bcked inthe tripkt sate with antiparallel spins, because ofthe effet of the rca fells REPORTS I in he vo dots combined withthe sal inert tunnel coupling: see (16) for details}. Next, the to spins are iolated by a gate vollage pulse, such that electron tunnsling heticen the dts o¢ to the reservoin & forbidden, Then, one of the ‘ins i rotted by an 2 voltage burs applied to that depends on the Fig. 1. (A) Scaming ole A, ly the spin tron micrograph of 2 device sith the same gate stouctre a5 the one used in this experiment Metallic Tia gates ate deposited ‘on top of a GaAs hetero- stoucte that hosts a to- dimensional election gas vo he songs). Filly, th readout the kf electron co tunnel tothe right dot if and ‘only if a spins are apr, Subsequent tane neling of one easton w the ight eservoe gives a contibation to the cumeat, This eyeh peste consinaousls, rx the erent ow tough 29. bow te te Fr, Posten edo sts meni we pay Not shown & a colenar S of having riper) sp afer exchtion svipline on top of te eo "o comma ta deel exciton can metallic gates, separated by a delete [not used in this experiment see also (4). In addition 'to a de voltage, we can apply fast pulses and microwaves to indeed induce single-lecton Spin lips, we ap jrowaye bust of constant kagth the wenge cure uch the quantum dots as a function of extemal magnetic fied Boy (Fig. 2A). A finite ‘eurent flow i observed around the single- Isolate Manipulate the right side gate (sind care fos e cared rand Os Sl ‘ated trough a homemade a ston spin rexonance condition, i, when binetee. The oration of ‘ Bas Baal Mim, With Planck's Constants fc the in-plane external magnetic fl is as shown. (B) The electric Field generated upon excitation of the He excitation frequency, and jy the Br magneton, From the position of the resonant ‘gate displaces the center ofthe elecon nave function along the electric fit dtecion and changes peaks measured over a wide 1 eld the potential cepth. Here, is the orbital energy splitin, {yy = Wy A. the sie of the dot, rv the ‘fective electron mass, the reduced Planck constant, and Et) the clectic Fld. (©) Schematic ofthe se (Fi. 28), we daternine ag tor of Spin manipulation and detection scheme, contlled bya combination ofa vltage pole ard burst, Ve), 0.39 = 001, which i in ageemem with ots applied tothe right side gate. The diagrams show the double dot, with te tick Dack tne indicating por values for eletrons in GaAs quanta the eneray cost for adéina an extra electron tothe let or right dot starting from (0.0, where (nm) dos (18). ‘denwres te charge state with and m elections inthe left and right dot. The energy cst for reaching In adtionw the exkral monic ik the (2,) is (carl independent of te spin configuration. Homover, fr (0,2), the energy cost for forming 2 cleciron spin Recs an elftve nuclear Fick By singlet tate linicated by 5162 is much loner than that for forming a trpet state (not shown). Tis arising. trom the hyperfine interaction wit Ailerence is exlited fr inialation and detection, as explained further in the main ea. tecker opin in the host aaseral end actus im time (79, 20), This nuclear field moxifes the secon spin resonance condition and i gener Fig. 2. (A) The current A poem] OC eral ov 0 ra ae Sy cto nh Te an itd 080 netic field sweeps is 7 Ae ‘The peaks shown in Fig. 2A are averaged over sven for aie dee Kou | 2 ee mgt eld pecs ie ie ‘ent erciation frequen- © 9 s ‘of about 10 40 25 mil. This is much larace than Gauvanicome Eyl eee |g thseapstel now, which eay 02 bustnghotisom, sue : bs avon bythe satel tans of By The aces are offset 2 gy 2 (2l, 22) Looking at individual field swoops for clarity. The mic & —_ a ‘measured at constant excitation fiequency, we sive ample Yoo aes] uarone fo tas be pea ue tds Be a eae was in the ronge 09 ioe (Fig, 20), but thatthe peck postions change in 022 64, depadag gpvese _ ae lime over a range of ~20 mT. Judging from the is onan Beet) Bath dependence of the positon and shape of the medion tceved 5 avemgad pals on snesp divetion, th origin of pom a Desi ths pe vrai in te moka eld rat ecard te Hey dpa nou’ polzaton (23-20) i aa F donmanne canter enmiot tees See 2 we ved te kaght of fe mizonne bas seitching creat used ~ gf ‘and monitored the eurent kvel. In Fig. 3A we ‘acon irene pot the rcinam curet por mpi ld bursa. (2 Peston oth esp ut oe wane bn cet aoe deals chet weroermd areqe mies cover wider frequency Fan more sensitive method than averaging the traces: Sst and then taking she maximum) 7). The mavimnura current exhiits czar oscillations asa function of burst kngth. Fiting with a eosine fiction reveals a lincae scaling of the oscil ‘and Feld ranges. Error bars ae smaller than the sia ofthe crs. (C)Indidual magnetic fd sneeps al = 15.2 Gil measured by sweeping from high to low magnetic fell wth arte of $0 mU/nin. The traces are offic by 0.1 pA each for cary, The red trace i an average over 40 sweeps, ncuding the ones show and scaled up by 2 factor of 5. SCIENCE VOL318 30NOVEMBER 2097 1431 wunuscioncemag.org1432 ORTS tion fequeney withthe driving ampliude (Fi 3B), characteristic feature of Rabi oscillations and pmo of cohcrent contol of the election spin via eketie fed, “The highest Rabi iequeney we achioved is 47 MIL (measured at = 152 GHP, core responaling to a 90° ration in~55 as, which is ‘only a factor of 2 Sewer than these ealized with magnstic driving (). Stonger electrical driving was not possible because of photon-assises tune neling. This is a process whereby the elect fied provides encwy for one of the folowing transitions: tunneling of an election to a reser voir or to the ples with bods electrons in the right dot Ths its spin bloekade, imespactive of whether the spin resonance condition is met Small Rabi frequencies could be observed as vel. The bottom toce of Fig. 3A shows a Rabi ‘oscillation with period exceoding 1S ps measured at fe ~ 26 GH), conesponding to an effective driving field of only about 0.2 aT, ‘onc-tenth the amplitude of the statisial Must tions of the miclar field. The oseilatons ae neverthokss visible because the dynamics of the snuckar bath are show compared 10 the Rabi period, resulting in a show powerlaw docay of th escilaion amplitude on driving fick! (27). ‘We next tum to the mechanism responsible ‘magnitude too smal to produce the observed Rabi fseiluions wit perio up t0 220 ns, which roouinss a ceiving fil! of about 2 anT(J7). Second, we have soon that there ae in principle 4 number of ways in wih on ac elastic fed ean cause single-spin transitions. What is ‘tcouired is thatthe osilating eben fold give rise to an effective magnetic field, By acting fon the spin, oscillating in the plane perpon- dicular 0 Bw at frequency fe ~ gh Bol ‘The gtensor anisotropy is very small in GaAs, so g-ensor modulation can be ruled ‘owas the driving mechanism. Furthermore, in ‘our experiment there is no extemal magnetic field gradient applied, which could otherwise lead to spin resonance (5). We are aware of Fig. 3. (A) Rabi ose tions at 15.2 GH blue, average ovr five see) and 2.5 Gir (ack, 2% ‘erage over si snes. The two osillations at 152 Gh ae mesurec atdferentampbtudes of the microwaves Vow > 3 5 Doteurront fA) $ g | leating to ditleran RIB frequen. (8) liner TBM og 868 dependence ofthe Rabi “eel gece frequency on apps mi Sean crovare amp mea OO TSS ma Suredtfee 14 Ge. Bure time (08) Vn Fig. 4. (A) Rabi troquency resealed with the applied electric fel ordre echt frequen hs. Theertor bare are given by <4, Snot “(06/6 + fein)? j exe where if and sEaretheerorhn theRabitequency ancl eld gay amplitude, respectively, The grey y 4 ihesatete SeconMemetouncs 2 oot ay for 3 near ft through the éata ‘weighting the data poins by the = ghey inenweror quared) (8) Esinated ous] 8% let fil amps at hich the Z Rabi ovations of wee mee= 4 sured at the respacive excitation feequences 17). (€) Constution > a athe decion of wresiting fom & the Rashoa and Dreselnaus spin 3] © Gatccineuccnd © along [310] foloning Fa, 1 The ° + feordinate sytem ts et to the 4 «eystallographic ais [100] and (010). 30 NOVEMBER 2097 VOL318 SCIENCE fe (GM) ‘only two remsining possible coupling mete ankms: spin-orbit interaction and the spatial vaiation of the nuclear fed tn principle, moving the wave fianction in a rclear field graent (5,28), as was recently observed the measurement of each Rabi escillaton lasted mere than Four, much longer than the time during which the nuckar Fld gradien fonsuant (-100 js wo afew s), Because this field gradient an, thvefore, the coresponding clfctive driving fee, slowy fluctuates in time around 22m, the osilations would be sronaly dampal reganlles of the driving ampliude (20), Peosibly, a (neatly) sate gradient in the nuclear spa polaizaon could develop as a result of olectron-maclear feedback. However, such polarization wouk! be parallel to By and thus came be responsible for the observed coherent oscillations Tn contrast, spin exbit mediated driving can induce coherent transitions (/2), which can be undersea flkwss. The spin-orbit imeraction ina GaAs heterostructure is given by Hoo ~ pas, Py) * Pa * Py). WheteU a are the Rashi and Dresselias spin-orbit co ceffciea, respectively, and pay and dy are the momentum and spin operons in the andy diretions (long the (100] and 010] ery ivee- tions, respootively. Assugaested in 3), the spin cotbit interaction canbe con enenlly accounted for upto the ist order ina, by applying 2 (gee) transrmation, reuling ina posiiordependent scemnal magnetic fed. Thisef- I acting on the spi is pro- Br) w ‘An electie field EL) will pevioically and adiabatically displace the electron way tion (Fiz. 1B) by x(7) = (elas? AVEC, 30 the celoiton spin will feel an oscillating effective fick! Bor) + Boa through the dependence of Buon the pesition The diection of can be comsrucied fiom the direction of the elt field as shown in Fi ycther with the direction of Buy determines how effec: tively the electri Hel couples to the spin ‘The Rashba coatribution always gives ALE, while for the Dresselhaus corsribution his depends on the oration of the electric ld with respect w the crystal axis. Given the wate mnt, we expect the dominant eave ld bre along the double det axis (Fig. 1A). which hone i ir the [110] or (170) crstallogrp lection, For these orcatatiens, the Dresses ‘contribution is ako orthogonal fo the electric eld Fig, 4C). This is why’ both contributions will {the Bip? 0 and lead to coherent oscillations in the present experimental gcomety, where E | Bo Jn (26), 2 very Simi 8 used, Dut the orientation of Bey as diferent, and it ua scioncemag.orgcan be expocied that E- Bey. In that experi ment, no coherent oscillations were observed, ‘which is consistent with the considerations hoz ‘Aa important chirctenstic of spin obit mediated driving iste Hinear epoadcnce of the cffoetive driving field on the extemal magnetic fiekl, which follows from Eo, 1 and is predicted in 2, 13,29) Wesim Wo verify this perience bby measuring the Rabi frequency as a function of the revnant excitation frequency (Fig 49), which ‘proportional to de extemal magnetic il. Each Point is rescaled by the estimated applied cht fied (Fig, 4B). Even at fixed output power of the microwave sour, the electric fick atthe dot d= pends on the microwave frequency due to various resonances in the Hine bowen te micowave me (caused by woflections atthe bonding wires nl micronaye crmponcat). How ‘ker, Wo ase the photonssisal unneing Re sponse as a probe for the ac voltage dp acres rc tune! bate, convert in amplitude by assuming atypical 1 oF 100 nim. This allows us 10 roughly estimate the electric fie a the dot for tn exquency (17). Despite the large error, ‘which predominantly result from the emer made inesimauing the eet ek, an overall upward trend is visible in Fig. 4A, Foca quaniative comparison with thoy. we ‘extract tho span ort sre in GaAs vi Lo es. pression othe elective field Bur perperlicular Yo Bu for the geometry of this experiment 12) bax ho 8 [Bee(t)| =2 [Boa @ With ko the spin-obie lonath (for the olber defindins, se Fig. IB). Here gg = ht Bb forthe case with the gate symnmety avis along [1TO} or (110), rspactvely. Via fas ~ Bar Y 2h, the contidence intent of the lope in Fi 4A gives spivorbit ength of 8 o 37 jam evi 4 love spliting A i the right dot of 0.9 meV ‘extactod fiom high bits tarspon measurement) is due to the este mate of the inierdot distance and te assumption ‘ofa homogeneous electric fk, deformation et : mgnitude as ‘other ror! vals foe GaAs quantum dots (8). ‘Both the observed tend of Bag ith fu and the extracted range for fgg are consistent with ‘our supposition (by elimination of other meet anisms) that spin tansitions are medited. by spin ot intraetion, We note that ako for ela ation of single eine spits in which cleric Tied Mictsaons fen phon couplet te sp, itis by now well established that the spin-oxbit interaction is dominant at elds higher dan Fes 100 ma (22,18, 28, 29), can thas be expectad vo be dominant for coherent diving as well ‘The clvtieally driven singk-spin sons repored hoe, combined with the so-called SWAP gate based on the exchange interaction wunuscioncemag.org between two neighboring spits (20), brings all clerical universal contol of losin spins within rach, Wheweas the VSIVAP we already ‘operates on subanosccond time scales, single spin rotations sil take about 100 1 (He min Timiation is phoon-asistal tunneling). Faster ‘operations could be achieved by suppressing phoonassised timeline (eg. by increasing the turnel barriers or operating deeper into Coulomb bkekade), by working at stl higher manic Fela, by using materials with stronger spin craton, or through eptimize gate d= ns. Furhennore, the eletcal contol offers the powntal for spaaly selective addressing of individual spins in a quantuen ot array, becasse the electric fel is produced by a local ge Fall, the spin rxations were realized at mag. neti fields high enouel 6 allow for singlosbot reat of 2 single spin (3/), $0 that both ele- ‘mcs ean be integra in single experiment Referances and Notes 1. Aucalo, Dos, M Samar, Semicon: Spinronis and Guonton Canpuiion Gringo, 2 C Pde, Eben Sin Resnere (ley, New Yn, 42 390), 2. 8 Sra o, Re Seen 77, 44702 2006, {FHL Koppens eet, Aone 842,16 2000) 5. tla, Wn dr Wa, 108, 5 Teh, ye fa Let 96, 017202 966. 6A Behe, ERs | Phys C27, 6939 (2988, 2. G Drees Phy Be 100, $0 0955). 1. at eC My A Gas, DAs, Noare 427, 80 2009. 9 Yate, Scone 299, 1201 2003) DG Ste eo, Hetue438, 639 2001 ALM Stat 1165 ot 6 Deninger, M.D, Py Be et 98, 133601 (008. 2M alc, Brhn et, Py Re. 874 652192006, REPO! 1B. testo, tab, Pps: Ps 8 67, 135328 2003, 1,5. abs C tang, oy Be Lt. 8, 26803 GODS. 1, | Wal, Condes Maney, Mio obs T5231 (aon. 6 FH, Lips Al Pps. 10 091705 a7 1. Supporting mater aaa on Sence One 18, Roa, UP. Keweeove, eB Feta, 5. Tas LEM aadenpen, Ro Mod. ye 29, 1217 GOD. 7, Ave ast Bay Geman ys Re Lt 1602 0003. 20 LALMedulo, tho, Moen, Pye Br 865, 205209 2000. 21, ACh ay Matar 435, 925 2008 BE PALL Hoppe ely Soeee 309, 46 QD05) 23 | Oagh¥tamrs, KOs, Tach, ys Re et 9, oheb0 (207, 24 MS ture LS ter Phys Roc Ln 9, 0364020007 25. 0 as ACA Ceih Ls, Pps en B73, 208302 (eo, 2h, CA Lede, Conde Mater, Mplninergaby oon.0887 a0. 27 FW Kappa a, PR at 9, 104803 (07. 2A SLE Many ye Ro B66 155127 202. 29, AN. ae VA Pp Ri BA, S216 00D. 3D RP tl cree 30,2380 2009). BL A tenancy uve 430431 (200. B2 ethan Ptoowrboven Barbe Ea (Msn, 1. aed. Mee er dcesos ord Fe Seheten, on de Eade aed Bole fee tech icone and). Rarer and PC Groat for hp zwar arene Supa by {be DaehOrgsraon er Furman Rach ‘Maer GORD a he Methane Ogoton fr ‘Soe Rescrch (0) “Suppeting Onine Materat wnncieagegeerertfll148092001 Iwate date Sc ioe figs i ane 82 Renee 20 py 2007; etd 6 Oem 2007 on12dece 148092 tree hs inoatin von cig ft pope Flow of Mantle Fluids Through the Ductile Lower Crust: Helium Isotope Trends B. Mack Kennedy? and Matthijs C. van Soest® Meat and mass ere injected into the shallow crust when mantle fluids are abet flow through the ductile ower cust. Minimum *He/ He ratios in surface fis for the northern Basin and Range Province, western North Ar Increase systematically from low crustal values in the east to hhigh mantle values in the west, 2 regional trend that correlates with the rates of active crustal deformation. The highest ratios eccur where the extension and shear strain rates are greatest. The correspondence of helium isotope rat and active transtensional deformation indieates a elormation-enhanced permeability and that mantie fluids can penetrate the ductile lithesphere, even in regions where there fs no substantial magmatism. Superimposed on the regional trend are local, high #He/*He anomaiies indicating hidden magmatic activity andir deep f production with focally enhanced permeability, identifying zones with high resource potential, particularly for geothermal energy development. ane vols, princily water and M: Ds, lay an pare ef > sph aeobogy and he groection fof buoyarn Maids that can be injected ini the shallow cnss. Regional and local tends in the ‘russl oveunenoe of mantle volaties provide insight into the coupling between mante-crist tectonics (2, heat and mass exchange be rnveen the mantle and erst (3-3), and the oe ‘currence and distbution of cconomic sources SCIENCE VOL318 30NOVEMBER 2097 RTS. I 1433such 33 ore minsals a oil gis, and oor thermal lus (0). Man-deved volaties in the cast are taccale tou le Sotope come poskions oF hydrolie us (7). Once inte into a cnasaltlud syn, mande He will be continuously dikted with adogeie heb (ie) aegaied For the U-Th rch eras, and therefre surfaced He snap compositions ako provide a measure of the mane He hs and the integraed perneabiity-tuitpresre seodivat (Hl te) though the east (1) To emer the hydrologic stem, mane He rast pass though a ductile lower emit, which #8 Believed wo be an impermeable boundary Decae of a inability 10 mainin open Tae tures en long tite sales (8-10). genera a sumrion fs thatthe passage of ids Lough this boundary mast occur either by dice av tris ara dansing of mareeived me anus (6) 0x by diison th bona Byer (7), However two reert adic. iv areas von of recent yoksnis (2,12) have found evidence for fi flow of mantle vids theoush 1 boar. How and why this oc undentood. ductile not well Fig. 1. Sample location map of the BER and surrounding areas. The expressed as Rea tthe ait~ symbol colors delineate "Heri 1 Corrected sample ratio normalized to 30 NOVEMBER 2007 VOL 318 We coniucied a regional study of He isotopic compositions of thermal fhids collected fiom surface atures and wells troughout the northem Basin and Range Province (BRR), west em Nonth America (Fig. 1 and table $1) (13). As a sul the Tectonic influence exert by the relative motion of the Pacific and Noh American Plas (74,15), the BR. a Vast eX tended region of ancmalous thermal gradion, Farge heat fl, high regional clevaion, thin (nec) ena and lhespherc- and asthenospherc ack: mehing. Presently extension is accom: ‘modated by high-angle nommal faults, and the locus of myer extension and is. associated ‘magmatism oovurs at the margins of the province (16), primarily win the Walker Lane, aon 10040-200-km-wide wanstmsinal Yolanic zone along the eastern side of the ‘Sica Nevada that extends nom into a tans: tional zone between the Sima Nevada and the subtion-elatal volamiec are of the Casas. East of the Walker Lang, BAR extension occurs ata slowing rai, over a much wider an and in the abscrce of ative oF recently active vole ism, The highest extension es in this area feete vse of the central Nevada seismic belt fe-cors esse catenia Peon TGRSIR CNSB; green, and BER (NSB), Hblocene seismic activ ‘Across the northem BSR, three general He ‘Socope trend are apparent. Fist, ratios 23.0 Ra (7) occur only at the western margin of th BAR, reflecting ative o recely active sallow. ‘russ yoleanisn within the Walker Lane Cose, Long Valle, ant Steamboat) and the Cascade son, Mount Shasta, Medi cine Lake, Crater Lake, and Newberry Crater) Second, the prependerance of raios >0.6 Ra ‘occur inthe norivest BER in an area ranging fom the CNSB west to the transition zone ics. Thin, cas of He ratios dosing, ranging fro 0.1 10 ‘8 nonh-norticas- trending belt of ¢ Cascade vole fiom west to eas, as illastatal by the shaded ‘come er top Geechenisy, far Sdevees Onsen, lana Bey Nar baa, Behe, CA 98720, USA "Note Gs Geter ad Geoconlogy aba ton, Scho of Eat ond Spe Erato Azo Ste Unrest, Tene AZ 85267-1408, US “To wher cavespondnce shoud be adesed. Emait tntemaiy@bign @> sor evewconp eee northern B&R; yellon, the Walker Lane ranstensional zone (WD and the the Sierra Nevada batholth (SN); Cascades volcanic zone. TZ, transition zone between the Cascades, Wl, nd light blue, the SCIENCE wunvscioncomag.org2. Aircorrected He isotopic ‘composition of geothermal fluids above 38°N latitude in the BBR, TZ, and Gscades plotted es a function ‘of longitude. The shaded curve depicts an east-o-nest baseline teend, defined by minima in the local 'He*He ratios a ma ae 240 1609 1509 1409 1309 1209 11.00 1009 9.00 800 100 600 ro REPOR pepe eer eee eer eae) Longitude ("E) 2a Increasing total strain and NW Shear Component 500 400 300 200 100 0.00 Tota! GPS Velocity (mmr) me 230 tat 2a 243 24 2527 Longitude 'E) © Bennet 2008) 38-47 N Bennett (2003) Average © Hammond (2008) S85 -40N Hammond (2004) Average Fig. 3. Compilation of present-day GPS strain rates actoss the northern BER relative to the North America reterence frame. The data are from GPS networks located in a bard from 38°M to 41° N latiude (25, 16). West of 242°E to 2425° E longitude, the data show 2 combined increase in total magnitude of stain and an increase in lateral dextal shear stain super wunuscioncemag.org SCIENCE imposed on te east-nest ecension. Tiss most evident in the Walker Lan, but it impacts mest of the BER in and to the northwest of the CNSB, 35, iastrates by the Fig. 3 insert ten from (19. The broad colored band isthe ‘regional baseline He isotope tend from Fig. 2, shown superimposed on the teends in sian rate. CP, Colorado Plateau; GV, Great Valley. 1435 YOL318 30 NOVEMBER 2007i REPORTS 1436 band in Fig. 2. East of the Water Lane and ‘Caseades, the oseurence of mantle He as indicated by baseline Hee ratios (02 2.0 Ra) thit are much greater than those of average erst He (002 Ra)—is not supporto by magma inmsion, as this region has no ev idence for curent or recent yokznic activity. Insead, the baseline seis strongly comeated with a change in the destin and magnitude of strain detected by presentay Global Positioning Systom (GPS) voces (Fig. 3) (17, 18). West ‘oF the CNSB, a nearly pure east-west exten sion rate of -3 mnvear shits to NAOW and increases 10 [2 to 13 ra/year, The accelerating dextal shear component is dven by a cg force duc w the reluve movement of te Pacific and Noth American Plates (J9). We hypothesize thatthe increase mn otal train an, spitically, the nomhscstocigntted dental shear component greatly eahance average Nidlow mates, allow ing fora more pic how of mantle Mids trough the crust and preserving the high "Hee ratios “observ at the surface. The enhances los rate must penist hough the britle-ductle transition, through the ductile lower emus, axl into the mantle lihosphere, I 36 expected Huds pass ing theugh the ductile ens enter the base of the brite zone at, oF nes; lithostaic prasure, then the easto-vessinciease in the flow rate is primarily governed by an easttoswest inreass im average pemacabiliy “The high-angle normal faults a presen accomarodate B&R extension and that re probably fluidsNlow pathways are not expec 10 penetrate the duce lower crus In extensional regimes, because of grivistional leading, the maximum principle stress is pomenicub tothe ‘geod, With inerasing dpi, thetrinfesuetle hil in shoology reacts the maximum sts i, suling in pearly horizontal shear zones of detachment hus at the bound: ary bericen the highviscosity upper ens and low-viscosity lower crusts (20, 27), Strain Ioeale ization induced by an inerasing dowel shear ‘compenent superimposed on the extensional te fel must mechanically couple the brite and dette enssal zones, generating veically or ‘ented downward fault splays that extsnd though the ductile crust and tothe mantle. These spe would act 8s conduits for fui flv, The less ccomspondence between He isctope ratios and the rte of desta shear stain suggests that uid ow rate (and ene. permeability) through the duotile zone isa function of the rate of desta shear strain, Once the flow fs insted, the ‘prsire pore and faciure Maids may help 0 maintain permeability, cohancing flow dyoush the dacile zone (22), ‘Thete are many sampled atures that have levied *Ho"ite ratios with respect 10 the fe sional baseline wend, Akhough a few, such as Roosevelt Hot Spring ant Covefo, a asso ‘ated with crustal magma systems (23), the ma Jesity awe not An exaiple ofthe later the Dixie Valley thermal system (Fig. 2), where a 30 NOVEMBER 2097 VOL318 SCIENCE Alvi! stuly 24) found a range in He isotope ratios (-03 to 08 Ra), withthe highest ratios restited 10 fluids emerging dinclly fom th Slater ranger ul spstom, ‘onnal fal that defines the wes margin of the vale. A recat eases high -esaton dsp aznctotelurie study tha crossed trough Dine ‘Valley reveae a zane of low resistance a pase sible indation of ids at a depth of -25 km within the mane fihesphew C5, suzzesting the the Disc Vale He anomaly celles son bination of loca deep prexketion of rmaile fluids (one of low resistance) anda ange-feet fault systam with enlannced penneabily’dhigh ie rats ane, bens, high He“ H ais). ‘The rangeiomt ulti the primary condi sup phying -20 ~ 10? kg of 250°C water in suppon ofa (2-megawattektic goodhemal power plant {G01 fll hat thor “toca He anomaio say be indiatve of simibr systems and rep rescat goshermal tages wi high pete. Our regional He isotope sy of his across the noniem B&R clearly demonstrat a strong comeation batwsen an east-west increase in the magpitae of dex shear stan and an east toswes increase in baseline He isotope rates. In the dence of ative or erly aetve sation, te elevated He isotope ratios reguire amagmai flow of manile fais though the dace Tower cn, suggesting at the ere dextl she stain mas erates and moinisins permeable path ‘ways trough the dette zane, Eleva He 0- tope ratios surfice Mids along amos sections of the San Andreas fault (J) anda recently observed series of nomeeanic tomers Aeep (20 0-40 kan) fase the same section of te Soult provide atonal suppor. forthe ei tence of degp-mante his, he peter impor ‘noe in faut mechanisms (27), and nonmagnetic uid Hor through the dette 2 Thehigh He“He anomalies superimposed on te regional nen indicate enhanc eros pt _meability coupled with local zones of dexp tid ‘rodbxtin alr hikcn magzaticactivi. These local anomalies may’be indicate ofa beter cous dsrbution of mantle voaties that promote melt praction. Assuming a COy"Me rato of 2 10” M Charette of michoean ridge bor salts) (6) 282 proxy for mantesterWved votes, the caleulatod fhi-tlow rate through the Dinis ‘alley geothoma syst (24) rashes ino a mantle CO; hx of -2 10 10 20 = 10 ® mob env year Asof yet no deinthe geochemical corisotopic evidence forthe presence of mantle (COs hasbeen found, other da ethon Borie compositions (8°C = 68 25 per mi) @2) not dissimilar fom those of mantk: COs (6) An estimated COy/ eration Dive Valley tu of 401% WP (13) in -20 times the mid-owean ridge ‘vals suggesting that most (95%) ofthe COs sot marteervad or alfsmatvey hat the sib- continent mantle CO "He rao frase ureter than tht observed at midkoccan ris Earth's erst sores an enoancus resnee of thermal enengy pros pint fom t dioactive decay of U, Th, and K that is ispersal ‘roushout Eat Ith been estimate that, with inne United Sit (excuing Hawaiian Alka), there are ~9 « 10" kilowattshours (RWWh) of ae- ‘easible geothermal eas. This ina sizable se- source compared 1 the ts energy consumption in the United Staes of 3» 10! kWh anally, In ‘onder fr geothermal systems to develop and mine the heat seurce naturals, adequate uid sources and deep permeable pathways are a necessity ‘The deep pathways provide aoe 1 high tem peratures that can drive uid convection cll. He ‘Sovopes provide a quantitative, at ast, qual ative estimate of dep perneabiliy fom surface measurements, anc anomlisssuperingesca on “ is can demi potemial resources. References and Notes 1. BIN ere ly Sece 278, 1278 947. 2 DL Neel a SA Todey 35 8 200 3. eh Sper Cons in Pel 0, 217 3938 4. 6 Hatand .Btene,nowee #4, 186 240, 5. C Being 8. Moy, 65. tai, Hai, Mere 193,23 205. 6 C Btn, Bugs, 8. May, Re Meal Creche 47,599 (202). 7. Nac reefer, sl ais redo ‘ie rote onthe wool ani ef sed Th sed tarred by yon He" tar of 002 Re Ietee Ro 14% 10 he atin a Monte ie thet otal ctl iuence se Sengy ened Ire wih yal tor egg am £105 Bs, tepetig one re ere eg, pre exis, ‘ere mi cnn rege hak era dare een 8, 1 Bjetee Gees es Ltt 47, 2306 970) 9. | 0 yen elegy 23, 303 (993. NW Sep MCL Hepler 359, 487992 UL tigen, J Geophys Res 98, 16257 (2). 2 Tonge Do it | ih, Geen, noch. Ata 69,387 (2008 1, Wess 3nd meh fe mob apart steal en Scene Dane LC nese, fey 203, $7 0982 5, MLL Zabock A Thorsen, elegy 6 311 (978, 1, T-Rosrs Dewlopnenh in aeons 25 Pat 244 of enteral vote rayon, KOS, Es, New Y 1995), p. 27-326 {VLA Bnet Pri, MA Mr, ML eh, [om tenes 2, 3008 (003. 8, WC Hamman, Tate, Gop: Re 309, Boao Good. 19, LL eth WE Ht, ies, 8. She Tu Sone 27, #34 Goto. A bade m0. 2b, Gelgy 36, 271 (988 21H Med, Mote 33, 333199) 22S. F Cn Eoth lt Space $4, 121 2002. 24.0 LM, Wee, Gvthom Rear Cae Ha 3,503 076, 24, 8 med, M10 Sos etm 38, 26008, Soc Ans Roe og 7, 495 2909. 24,00. Sle & Golan, 0 eet, Geter Rese Coane ars 24, 223 230), 27, RMLs, nee See 307,39 268 paced bene 9 Erbe 24 1031201073) 2% Tet na pata yt U.S paren Evy. fee of ae Eney Scenes fe of Gather Teele unde tri UE AC?OSCHIIDS- Suppeting Ontne Materat tnmeclenemag ogre 1858591033003 Matera ad mec ‘ase st felererces 4 hdy 2007; acy 17 acer 2007 ua scioncemag.orgIn Situ Determination of the Nanoscale Chemistry and Behavior of Solid-Liquid Systems Santhana K. Eswaramoorthy,? James M. Howe,” G¢ ‘Muralidharan? ‘Many fundamental questions in oysta-rowth behavior remain uransnered because of te dlficlies ‘enccuntered in simttancously observing phases and determining elemental concentrations and recistibutions white cystals nucleate and grow atthe nanoscale. We show tat these obstacles can be ‘overcome by performing enetgy-dispetsive xray spectoscopy on partially molten Al-SLCu-9 alloy pails daring in stu heating in a transmission election microscope, Using this technique, we were able to) cetermine thatthe alumirum ard sian concentrations change In 2 complemeataty and symmetric manner about the soli-iuid interface as @ function of temperature; i directly measure the solié- and Uguid-phase compositions at equlitrium and in highly undercooled conultions for ‘quantitative comparison with thermodyramic calculations ofthe Uquidus and solidus phase boundaries; ‘and Gi) provide divet evidence for homogeneous ruxieatian of the aluminurrich soli Fystal growth isan important technolo ( ical process alceting feds from metal lay to soldat physics and applications ranging from the presi of mesakallay ingots buildings and aderatt to single crys se co 1 oxi for electronic and optical devioes (7,2 sas involv in everyday nat ted phere such asthe oz of water, ad biominealztion of living onanism (3), and in volosical processes such as the formetion of stalactites on stalgites in canes), Understand ing fundamental phenome such asthe structure and composion of the soidiguidinrface, the Pastioning of elements besween the solid and Tiguit phases durng crystal growth, and etitcal factors involved in the auceaton of phases is ‘esl soliicuion science an the develop- ert of no matrl srucares under ogi and. nonequilrium conditions (7, 5-7) The challrges of diwcily datnining these features have boar hrgely beyond the cap perimentalists (8), although recent progress has been made in deermining the woric stuctae of based alloys have wcived atenton as model 33+ ters haxas is posible wo proche ae uner- coolings in atomized liquid droplets less than 20 am inciameter (7). In these tues, theaone- mentioned difcules have precluded dest ob servation ofthe nucleation and grow behavior Rk A ies of ex the solgigad iri (@-1/) This pron ©) we ¢ partial sverin norgmicenginwing mae = Takis myc stapes ape RE cally eater thin 800 K) and are optically opaque. % . “Tumba an Cech (showed tatteenges Sy ‘cous nucleation on impurities could be eliminated i ae rng te tee wre cembomal by a using quid drpetsa for ten ofrmicomctensin fq” fh dianeer Sin iztorshave banditea ° P A8 seliiieaion of alloy deplets uncer conditions far fro equiva isthe potersial to prctoce mse stable phases with novel propertios and tron the study of solidification processes 7.13). 1. (A) Bight-eld TEM image of a partially molten ALSi-CuMe powder part REPO! ‘of phases, oF measurements ofthe lol come sitions present during soliitication (er meting The closest eypcriments of this type have i volvain sity heating of submicrometer diameter binary ALSi alloy pticks 1 obtain quiltaive clemental profiles ross soidtiqu imeriaces (14, 15) n the taasmission electron microscope CTEM) In his Sud, we used a nowy desiened thermal shield on a eonventional TEM heating holder (76910 obtain quanitative chemical come postions aeross a sol-iguid inertave ai tndercooled liquid phase as a funetion of sermperatune, The sample chosen for this investigation w 4 multicomponent commercial, ALSi base alloy {AA 390) powder obtained fom Vaimet (17) Thesalloy is typically used in azwspace and ato noble applications because of its high sue tonoight ratio and good Wear and corso resisance. The alloy i mstinly Al, with imine tional additions of 17.87 Si, 183 Cu, ane 06 Mg (here and henceforth, all compositions are atomic percent), Aitional minor impurities in the ally such as Fe and Ma ae gencraly present at lower amounts and are no relevant 10 the preseat study. The powder particles were ss pended in ethanol and dispense on a ula B 8k ° Compontion ot. “epatnent of Mates Scence and Enginerng Unvesty of vigil, 10 Che ity Dave Charts YA 2e904—a74s, USA. “ates Sena Tedicoy Dison Oak Ride Natorat Uberti, 2 Bethe Yaley Fad, Oat Rie TH 37831-6332, USA “To whem covespantence sould be adessed. Ema Wxeveahacds wunuscioncemag.org temperature of 828 XK. The faint, cashed tine parallel to the interface indicates the approximate appearance of the column of mate‘al sampled by te electron beam normal tothe plane ofthe figure. Circles a though f indicate where xray spectra nese acquired. [Regrined from (2) courtesy of the Microscopy Socoty of Ameria and Cambridge University Pes) (B)Bright-field TEM image of the same powder particle after further heating to 872 K. Cices L through 4 indicate where way spectra were acquited. (C) Cncentration profiles of Aland Si obtained from xray spectra taken at 828 K sod ines), and at 878 K (dashed lines). (D)Concentation profiles of Cu and Ng obtained from x-ray spectra taken at 1828 K (cli ins) and at 878 K (dashed lines). SCIENCE VOL318 30NOVEMBER 2097 14371438 caubon film supported on a coprerimesh TEM. which are generally better than 0.2%, i, The in sit heating experiments were per- provided inthe Supporting Online Materia foe at 200 kV a JEOL 2000F XI TEM with Figure 1A shows brihifild TEM image of 42 Gatan doublesik heating Holds. A thermo- an ALSH-Cu-Mg powder patici about 350 rm in coupk: in the holder maintained the specimen temper 1S K. Chemical comport tions were determina by ery specimscony (EDXS) with a Gesharn high ray detector and sn incident clectron bes diameter (probe sizs) of about 25 nm. Petals regarding ian the accuracy ofthe composition me swnete, ken ata tenperature of 828 K. The pomige is pari 8 Primary Si particle on the lower left and an oval shaped solid Abrich paniele that is surrounded by Abrich liguid phase in the upper right An snes alamium oxide fhm several nano meters hick forms naturally on the patok sue ication ofthe ray spect and wrements, face and thisoxie sel preventstheBiguid phase Fig. 2. (A) Bright eld TEM image ofa second particle at 833 K. fn arto points to the position ofthe eecton probe i the iuidin the analysis anc the bright spot in the lones-Let comer isan image ofthe cual probe used. () Image at 753 K wth an inset electron cifraction pattem showing the diffuse ring Pattern corresponding tothe liquid phase, as well as Si reflections (C) Image at 743 K with an inset electron diflraction patie showing additional spots due tothe formation of crystalline Ach phase Fant rings in the itfracton pater correspond toa small fraction of remaining liquid. () image at 743 K taken with 2 loner electron beam brightness to eliminate beam heating, Arrow points to the Al,Cu phase identified by EDXS. The election beam i condensed to just lurnnate the particle inal ofthe images, so that the remaining background appears dar, The contrast visible i the Sherysal due to detects, which ‘change ther orientation and contrast slightly with temperature, Fig. 3. 00 Si and (8) cu A Bw Concetotors in he under om = Cooled Gaui pas plated as on ae net of temperture in tn gua pucucbinay phase cingamt wn ise ong wi tiecsctedewur 2 | >. ibrum tigidas solidus and" |* the thin aluminum oxide shell ‘contains the molkn rtal in the high vacuum of the TEM, Nonponider forms can he examina by using spec! TEM spocimen-prpration metas (20), Low vapor pressure alloys comtining ee- ments such as in and Sa, or the roscrly suid Aue alloys (27, 28), can also be observed high vacuum For other metals and materials that have a high yapor pressure and do not form a sve stble osde ayer, some way oF cane taining the fiquid phase has wo be aie. Special Figuit-cell holders have been developed for the ‘TEM (2%, 30) and in combination with a heating and cooling cpabily may be she w accom date these materials in the future. For materials higher msking temperaunes (eg, grea than about 900 K), futher imgrovemens inthe SCIENCE VOL318 30 NOVEMBER 2097 1439terperaure cyubility of the heaing-bolders-ray detscice system are nosed. The use of sina, brighter clea beams in sticottheat TEMS (Gd) can provide chemical rsoltions approaching atomic dimensions, Hing such dicet meqphor logical informaon, cornbined wih hig-sti resolution chemical analysis illustrates the porsn- tial for solving many important questions in ‘enwllzation science, Examples inckale whether the fimdamenal assumption of local equilibrium at solid iguid interfices in themedsnamic and Kinsic moxkls is valid whether soltfiguid iner- face compositions change with erysial orient tion, and if segregation occurs at solidlguid interfices. Cniestion of the formation mccha. niss shown i recent sts of nanedoplets (27) and naanwire growih (28) may abso be possible Releronces and Notes 1 Me Rape C Beka, KT, Eb, Sbfiction Recs ond erstnenr A Sposa i one of ef re he Mines, Mel and Meals Soc, arene, PA, 20041. 2. Bynpp Oa, Cyst Gra echecleay (Wilander, New Yok 2903 alae at wr. KewlcontnneoipitonD-7S28Qer “0 3. Msi, € Tamer, ey HS, Banepa Mate. 2.57 0003) 4 W.Orebd, Boer 28,347 199) 5. MC Menigs, Sian Pocesing Uo Hl, 6. WA Tile, he ence of Clan: Miele reac! Phramero Cambie Uni, Fey, imbrdge, UE, 1990, 7.5. as fH. Pepeca, ty 6 Wide, More Si fag, 4304306, 159 (2009 0. osm, See 296,47 (202. 9. Rehr ety Hate 400, 39 (200. 11.5, C Oomay @ ol, Soon 296, £07 2002, ALS. 0h ¥.Rwfmin,C She, WD op, Mle, Scece 320, £61 2005. 2D Trbul Cech J. pl Ps 28, 808 (950) DLR Piy OM Hee Me vndouse, AL Ge, Meat tte Fem 384, D7 200 14 SAS: Tainos Moa Saf. ecror iro. ely) 48, 317 (955. 35. GA Stk, CT Mob, LAL Hone, Pi. ag, A 0, 239 2009. 1b. SC maramaey, EM. Hon, FP Meese goers 33, 2712007, 1. Valet, (31 fat pry Rea, Sto (495206 USA, 2. GA Sosa JM Hoe, Mate 5 tng. A368, 183 Geo 17. C lpi in ical emcee er Src, Mo el 703 p98 TITS, A752 20. LTeWandll in liom Aly Screed Peperis Dstt Lerdo, 2976) po. 366376 21 FM. this, Pore Dagon in Neelge hr Devcon ard Apictn enw Me Yok 1950. pp 5-4 229-20, 22. Tot Howe, AL Maram, Py Rex Let $4, 245504 003. Fluctuation Superconductivity in Mesoscopic Aluminum Rings Nicholas C. Koshnick, Hendrik Bluhm, Martin E. Huber,* Kathryn A. Molter Fluctuations are important near phase transitions, where they can be dificult to describe quantitatively. Superconductvty in mesoscopic rings is particulary intriguing because the critical temperature fs an ‘oscilatory function of magnetic fel. There is an exact theory fr thermal fluctuations in one- dimensional superconducting rings, which are therefore expected to be an excellent model system. We measured the susceptibiity of many rings, one ring ata time, by using a scanning superconducting ‘quantum interference device that can isolate magnetic signals that are seven orders of magitude timaler than applied ux. We find thatthe Ructutiontheery describes the results and thst a single parameter characterizes the nays in which the flucuations are especially imporent at magnetic Fields were the critical temperature is suppressed, rperordbciniy ues bo decten SEs hen Cmtpeorie qeenre eae oh ew rmge pinches silly deb ss fh we Roc In eid pemer ther fl en alma cotibons fom male ‘eve eons cantly charge bes froth) Exe kx fat cantons tous denon (1D) Tage etd fn ean esas) ch sue cect cons ela cay “epainet of Apteg Psi, Sunt Une, Sar fo C9505, BA “Depuanert of Psi, Univesy ot ‘rae Dee, Das, C0 80208, USA “To whom corespndenee should be adlesed, Eat rler@itnlocedu 30 NOVEMBER 2097 VOL318 SCIENCE appli cunt We used. cormetes tohiga sty fuctstion effin soled guts 1D rings inthe temperature ences comparable to he temperature dependent Ginzburg-Landau (G-L) coherenve length, $0) With use ofa seanning miero-superconducting ‘quantum interference device (SQUID), we detected the curent n many individ guesi-1D alumioum sings, ying punicuar ateation t Shall currents near cach rng's supercondacting transition temperature, Z, Such measurements havemany advantages fy 1D, the cunent about a ting, 4 is related to the ve enensy, Evia 1 = -€F thy, wine ty fhe flax tough th ‘ing from an applic magnhotic field. Measuring 7 as a finction of @, thus teas a indamental thermodynamic variable ans our undersanding 23. WE. ing et LJ. pe Ps 97, 131208 2009, 2A CA. ode, iu eal ne Slidiceton ames Sect fo Wel, Crear WS, 25, A Indi Iroc Se. 1, 159 2300, 26, hang Ska ih Pi oy. 85, 207 (2008) 27, BW Sater CA Str, Mee te 6,38) Qa? 38 Sedans, teeta. Rew Ro, Seance 316, 129 2007 28, EL Dawon Lie, K tw, pec eae, |. Micebiot Metod 50, 36 2032, 90, W Milner, ML Tom, Po. Yee, 8 Hall, FM Ras ha ete 2 53212008, BLS.) Peck Mae, C0. Hetero, 1 Lica SB 3, 36 2000, 32 hs sar wes supped by the HS urd gon DUR SORES ard OALOSSA92 Th pt of i war fone 2 Ost Rae Nia aon sprog by the estar Seta te tery tery ane Feces repusion ae Progam, US. Onna of ry, ue contac OFAC 0082725 mth benny of oat UC Uso trenton eB Rana Nar (arate ay 2 UV fr i sya strode ‘Supperting Online Materat vwonsenemog orgie 1OSE551007002 IMs wed bac 50 Tet Mees 1 un 207; pied 1 eb 2007 1odaeieer 04811 ‘of the sing’ state, W' there are superconducting pas that are coherent about the fing’ circum. ference 2, the ring’s cument near zero applied field is proportional to the density of pairs Deviations fm this mean field sobation provide infomation about amplitude and phase Mactan tions in the ring. “The mean fed G-L solution predic that the ‘urrent near ro fgkd should decrease linsaty to revo as te temperature, T, approaches Te. For small ings, we find 9 measurable curent above Taclear signee of ications, The qussi-ID _ssometry allows fll numeric solution of ther: tal lactations in a G-L famework tha ine lads non-Gaussian effects (3, 6). Provious resis on a single ring at zro applic field (7) disagreed sonaly with thar thoory. We stcied Auctutons in 15 rings and found that 13 rings agree quantitatively wih a fall numeric sosion, which was numerically inactable for the other2 Fings [Supporting Online Mtrial (SOM) tox), “The results in an applied fick! are partic: ularly imeresting, Lite and Parks () showed ‘experimentally that 7 varies asa periodic fun tion of Wy. TU). At haltineger mubiles of tho superconducting fax quarsumn, thy the enc: xtc cost of maintaining the Minded super- furrent can be larger than the condensation ‘nergy, destoying supewondtivis. Previous results (2-1) indicate qutatively that ucts tions are especially important inthis oxime. We Sind.an enhanced respense tb, = Why? ta can be quantitatively exphined by G-L thenmal lac tuations and demonstrate tha a single parameter ‘an chamcterize the Gaussian and non-Gaussian ua scioncemag.orgregimes and detsrmines where the Litt-Patks cflet is entirely washed out by tetuations Unlike stationary sensors a seanning sensor (Fig. 1) can measure many samples dusing a sinwke evolown. We report measurements on rings with red (R) of O38, 03, 1, and 2 sme annuhis widths (3) fiom 65 to 180-nm and thickness (/) = 6 rm, The scanning SQUID ako allows excellent background cancellation 112). Aer background subxraction, the sia (Fig. 2) is proportional to curent inthe ring Many of the atures in Fig, 2 cam be ex- pinod with the mean fek! esporse obtained by the mininiavion ofa 1D G-L functional, By assuming a hemogenods line wid of ‘wd this proves gives the sing eure (7, thy Ina oun —P aaFg ol") A Pamas(1 2 fay /ay — ny 8) where VACTP represen the superfans and nis the phase winding number imposed by the singlevacnessof the macrscepic wake funeton. The dillowet sss resuh in ponds i, curves. The Oetiesr tom isthe London reson, whet 1/3(T)*=7e(0) 7 bobs 120) inte emperatur range of intrest and 0 when > 140). P describes paicbreaking dus to an Abaroaoy-Bolim phase azound the ring, which leads to a downtim ofthe response at nite field when (7) = RInsinall ings. sfc ‘occurs ll botow 7.40) (Fig 2, AC). The Li-Picks effet occurs i the temperature range whercE(7) 2 28, bringing Pto zero for 4 range of applicd Mas. The dashed green ine in Fig. 2, Cand D, shows the best match to Eg. Vat T~ 122 K. The cuss lrge omnent response in the epi i whi the mean Feld ‘ure vanishss isa eso demons tha fh tate eee ae fnporant in this regine. In large sings (Fig. 2D) Hstations doriate the reapumne hele the elle of the pecking tor apparert Tne theoretical farcwork of) the ent isgiven byl = “0/20, — Kyl sb Za, the sapereonducting parton frtcns Ze. i the path tcp overall possible wave ces and hy isthe Boltzmann constant As shown in (5, 9) COM exp, Zon he writ a Z Yin OPC 2x10) V5.9 96-7"? Ease where Fz are the eigenvalues of the 2D Je-pavtisle Harniltonian, A= $V? isthe angular momenturn index and ris afstitiows position coordinate, This {ormalation allows 3 numerical calculation of F that fs exact yp to truneaton eroes. We ‘emphasize the parameter = 250) fo 087 : Marks Mar laby wunuscioncemag.org whew Ee = hiyle/M2 is the correlation or TThoaless enor. ¢ isthe Riomnn zeta function, vis te Feri velocity, isthe mean five pa, and Sys the Pippar oro temperature coberencs Feng and d enter into the effective number fof channels, Mog = (/e/L) kya Az), where kp is the Fermi waveknath, This ccmbinatin of parameters dharkctrizs the sizeof the ring ‘Temperature only appears in the sccond term of the Hamitonian, which can be revrten using Seby\T ~ T(0))/Ee = 12/5(T)? 19 illustrate therdation othe ten of Fg, and to indicate there T4092) in Fee. 3C, This, once the comeet Fara! 70) are ‘known fora given rng, the curents function ‘of by and 7's entirely detemnined by The data poins in Fig, § were derived from baby curves (ex, Fiz. 2) by fiting koe onde polynomials near, = 0 and ‘by = =bj2. We hhave compared these suscepiliics as @ funetion of temperature to the Uacory by using the measured goomety factors, the Ap and yp of bulk aluminum, and tee parameters chosen by hand: ey TAO) and Mag, the muta indic- tance between the SQUID and tbe ring. fe is identified by the shape of the curve aa function of temperature, Mga is dktermined by the ‘magnitude of the response, and Z(0) is chosen A Field Coils Mod, Coils, CSE SQUID Body SCIENCE VOL 318 REPOR to allow the thor ature dependence of the bebo 7201 The 720) ofthe sings varie fiw 1.237 6 1268 K, with up 10.7 mK difference fo nownially identical Tin-widh ngs, Tae fied Mg ie within 15% of the indactan calculated with a model based on a 0.75-am Fing-senso-loop sopation, The fits (sare between 14 and 25 am with an increasing dependence on fine width. We atribute tis de= pendence to exygen absorbed during the fabri. cation process and, 10 a lesser ext ‘observed 20%» variations inv» (2). The foe nominally ietica fine-wiedh rings shown Fig. 3 havefe = 19.5 £0.53 nm. The auoomet with the ID models we have discussed demon strates dha the finite lne-wiedh effets are not ‘esental to our physical result and that small ‘atiations in w do not quiatvely change the response above 7) ‘Nea TA) yoharaeteries the non-Gaussian luctations that interpolate between the mean field behavior far below T, Auctatins that dominate Non-Gasin flocuations ‘quagtic expansions of the five exergy cio Aeserbe the physical resalt. This is particulary appent a 74,), whore any Gaussian approx 1. (A) Diagram of the diet current SQUID susceptoreter. One field coil applies up to 50 Gauss of field to the sample, whose response couples 2 magnetic uc ‘ino the um pickup loop. A second counter wound (CW) loop cancels the SQUID response to the applied felt witin fone part in 10%, Addi tional modulation col mmainain the optimum working point ®) The SQUID's pickup loop (white) and field coit ‘ble ate postioned over single mkzoneterscle ‘luminum ring. In stu badkgound masses allow the megretic uc induced by currents in the ng to be unambign ously distinguished from the applied fed, nich up to seven orders of magne Lrg. 30 NOVEMBER 2097 ns 1441i REP ORTS Fig. 2. SOUID signal (left axis) and ring curtent (sight aig) 35.2 function of applied fax cb fortwo rings, both with d = 60 am and w= 110 nm. The fluctuation theory (Gashed ree) was fit to the data (blue through the tem> perature analysis shown i Fig. 3. (A to ©) R = 035 pm, fitted 7,(y=0) = 1.247 K, and y= 0.075. The green line isthe theoretical mean field response for T= 1.22 K and ‘shows the characteristic Little-Parks tne shape, in which the ring i not superconducting near +, = ‘/2. The ‘excess persistent curtent in this vegion indicates the large fluctuations in the Litle-Parks regime. (D) R= 2m, fited T.(j=0) = 1.252 K, and y = 13. The periodic response (ight inset) shows 10 tveatment is appropriate and can be approximated by a thermal average over mean field G-L fluid states (Eq, 1 and SOM tect) until additonal fluctuations contribute near Te Fig. 3. Susceptibility data (symbols) and fits (lined a1 @, = 0 (Positive values) and ay/2 (negative values) for 110-nm-wide 60-nm-thick rings with various values of R. (A) Smaller rings have a larger temperature region where the Lite Parks criterion £(7) > 29 is satisfied and thus have @ larger region with a reduced &, = ‘042 response. (B) ib, = O susceptibilty scaled with the cross section and radius to show the effective mean fied superfluid density eround J, Smaller rings have an enhanced fluctuation response. (C) When the temperature is scaled by the correlation energy, E, the susceptibility is uniquely determined by the size parameter. ‘The gray and green shaded regions indicate the temperature above 7b) hen D = O and by2, respectively. The fluctuation response above Teg) is enhanced for by = ty/2. The dotted tine shows a Gaussian prediction SOM text that ‘is valié at some y-dependent temperature stove TA), When = 1, the response at , = 0 and the response at , = e/2 are comparable in the Litte-Parks regime, which corresponds to 2 fluctuation dominated sinusoidal! ~¢, response. did, (WA) 30 NOVEMBER 2097 VOL318 SCIENCE 05 -025 0 025 Applied Flux (®,) ing (A) -04 ow t & k & 18 1.2 1.22 1.24 1.26 TiK) 0.5 Bk (T-T,(O))/nE, = + @ART))® ua scioncemag.org 0“O5-025 0 025 05 ‘Applied Fux (>) Fig. 4. Mean field theory (green), fluctiation ‘theory (dashed rec, and data (blue) for tree rings with different 7 paremeters. The mean field response & derived fom the fluctuation theory Parameters foreach nga the given temperature. (A)7=1.20 K.In smal rings. the Litl-Pars tine shape is clearly observable. ()T™= 1.25 K.When += 1, the reduction ofthe response eue to the Line-Parks effects significantly suppressed. (©) T= 1.25 K Inlarge'y rags, the Litle-Parks effect is completely washed out by fluctuations, which affect the responses at al lux valves. limaion would predict a divers sascepibiity (Fig. 3A), By using Eq, 1 to dei superfhid density fiorn the zero field response, ie the suscepi- bility deviate from the mean fel response bolo 0), graally smoothing the wansiion, Our pe ramotezation ofthe theory shows that isthe only sanplockpennden parameter at 7 ~ TA) The temperature range wh actutions are important is typically param ‘terized through the Ginzburg parameter as 7, TATA.) < Gi where T- Tel y)|/Ex 2 Gin 753) side this ara, y determines the magnitude of the response. Far cove this Gaussian Mucweeions dominate, and the susosptilty isa finton of [7 ~ Te(0) Ee non-Gaussian The theory's dependance on 7 allows us to state the criterion for the visibility ofthe Linle- Parks efloct in tho contest of fhctuatons. The region that is shaded in_groon in Fig. 3C is above Tig?) became 37) > 2K. The sus- cepiibilty ould be ze10 in this exime cwation effets were not considered. When 1 € Lathe distnet Littk-Pasks shape is visible in shat the suscep is smaller at, = Wo than at ®, ~ 0, However, when y> 1. the Litle-Parks shape is entirely waste out by ‘actuation (Fig 4). Foe sullicinty large wunuscioncemag.org susceptbiltes at @ ~ 0 and by ave equal and ‘opposite even beiow Te(Dy = e/2) 80 Uhe we sponse appears sinusoidal This depundnce on y. rater than £ and £7) alone, isthe reason ‘why the Litle-Parks live shape does noe occur inthe ring shown in Figs, 20 and 4C. stanially smaller than the well-preserved skulls (oF Probus from Swankrans (such as SK 12, ‘SK 46, SK 48, and SK $3) and Kroméraai (TM. 1317) alle SI). Diflerenaes between DNH 7 and lager sdividuls are consistent with those cexpoctod enveen females ang! males. For ex ample, the larger individuals have sagittal crests ‘eacing ont the frontal bone, as well as deeply projecting masioid processes, whereas DN 7 Tacks a sagital crest and has reduced mated regions. The degre of size ellerence botwoen DNII7 and laruct, presumably male specimens can be exphhined by a peilike level of sex dimorphism but not the lower vel of dimor- ‘phism soon in chimpanzees an mera humans (supporting online materi. Funhermore, i is any ofthe other iatively complete skulls ae female. In comparison © extant hom invids, the dtfercnce betwexn even the smallest of the previously known crana (TM 1517) and DNIL7 is statistically grater than would be es pected within the range of variation of fms {supporting online text and ible S2). The size of DNIL 7, in combination with an understexting of male life hisery, hops resclve the sex of spe inns such as SK 48 and TM 1517 (25,27). The wo Tater spociners ae best regard young ‘adult males, A bias toward milesamong the wellqneserve shal: ialboreflestd in the sample of masilasas ashok (2), Specimens such as SK 21, SK $21, ‘and SKWW 8 re similar in ste and prpertions DNIL7 ang! are probably females (hese at size reaks | and 2 in Fig, 1), wbereas all ther spec mens show mide size andor mmrpbology (sa poring online matial). We conclude that the masilofcial spec | Smalesenera, and 3 fomaes nd 4 males the Snartkrans sample enly. This distribution do- vias significantly from random samping of an unbiased population (asing a two-tled bincrnial ‘stand assuming 0,50 ratio, P= 0.0192 forthe ‘overall sample and P= 0.0127 for Swart) Aan abundance of males is perhaps nt sr Prising in @ fossil sample that resulted largely fiom predation, Dirt exidence of carnivore a= livay is present on several hominin specimens Sarthrans and member | of ths ite fs among the most dotinitve examples of a predate: ‘of hoeniins (27, 29) hic primal, nadia males spend more time alone, on ihe pernbery of a social cup, oF in smal lle bands (30) Solitary behavior plces males at risk (32, 22), For &x ample, when male haboonsispors, they suller ‘mortality rage at Hast three times as gevat as ‘hat of groupsiving make or males (32). This, Table 1, Ranking of size and age (displayed in Fig. 2). Swartirans member 1 (NM) includes all specimers from deposits refered to as the lower Kank and the hanging remnant [see (12) for explanation), maxis Specimen Provenience Age Size Sewil swartkane mS 1W41517— KomdasiB 24 ska Swentanc m1 sky Swerthane ML 1 SkA21 Swarms M2 Sk 6s Sworthane ML 2 SK 57 Srorthans M27 SKW29 Swarms ML 27 DwH7 Drimoten 31 sea Swanton ML 4S. SK 826877 Sworhrans ML 48 SKW12 Swarms ML 49 SkW 8 Seerthas M52 Sk a6 Swanton ML 5S. SK79 SwrerthansM = «5 Sk1512 Swarms 5B sk a3 Swarthons ML 67 ska Swenians M6 ska Sworthane ML 7D Mandibles imen_Provenience Age ‘Size Twas? Kemdaai®@ 1 SKWS — Snartkans M2 DWH & ——Drimolen 1 4 SKESO13 SnarthansM2 2 SK23SnartkansM2— 2 Sk3 — Swartkane M22 SK 856 SnartkansM2— 2S. DWH? ——Drimolen 2 3 DNH 21 Drimelen a2 Sk 84 SnartkansM2 SK 74 Snarthans M253. DNH 19 Drimeten 5 6 SK 1566 Snarthans M15 skal SnartiansM1 5 DNH6 ——Drimolen 6s S12 SnartansM OT ua scioncemag.orgegw of difference in mortality matches th male bis at Swarthrans, F males were apparent ly move shiek! fom predation. Putin observation together with the conclusions about bbimatrism and sexual dimorphism, we infer that 9 Maxilas * 8 e? - : a6 . af 78 Male Halas ds. 2 . + Fomaie 1 . : : ee g: ta ae 1234567 Rank by age Fig. 2. Comparison of size rans to age rans for adult masiladal and mandibular specimens (Swarthrans, diamonds; Drimatea, squares; Krom rai, tangle. Table 1 lists the specimens used hete.‘The Lbnest ge rank contains individuals, with MS recent erupted. The Larges individuals are mare advanced in age, showing a least some dentine exposure on MZ. When a randomization test of correlation coeidents is used, age and site are significantly corelated among the male manilofacial specimens = 052; P = 0.027, one-tailed test, 5000 permarations), eri ky Fig. 2. Size and morphological emparsons ameng DNH 7, SK 48, SK 12, and SKW 12 (rom left to right. They ere aligned epproximately on the Cementum-enamel junction ofthe molars, indicated by te Lower tine. Upper line indicate the level ofthe inferior nasal margin as a general sze com- ynwuscioncemag.org SCIENCE the distribution of fond sours allowed sable soups of 72 robustus females o form in sponse to pasion presse, ana males i tur su monopolize productive access to these eroup (23). If fomaks ciated from their natal ‘troup then ts key teat they pene Tite time ‘alone and transfered dinslly to a male or an «established group [as oocurs in gorlls (30,34) The P robusius pater contrasts with that of some ater asiralepthecine collections. For ex Ne the sample of ntrlopithocas acne is cither biased toward femal cr stars no bits (2 The dl. ajcannsaccunsltion foe Ste toain member 4 muy also be the product of predate ‘bekuvioe, but this copelusion is ss cman than for the Swarkrans sample 27, 35) 1F both Swarkrans member I and Sterkfontein member 4 hominin collections are largely atuibuuile to ccamivore etn, the ference ia Sex Bis ses the posibilty hat P.robustus and A. aficmes difersd in social srt 1 lea, Pan Seah ar al 32,345 fase, 2M Meh, Har, fat 27,7 (298. 3. fly PC oe, Snce 283, 901 (959, 1 Cond, Am. Py Ahrapl 306,97 (909 5. PL Ream RS. Meindl M.A Metin C0. Ly, Pe. et Acad So) USA 200, OH 03), 6 Werk cifewce nl ets maphdlogy ‘ote mes and es wi Baebes ‘arte wit vel Sepia a ty ras. 27M can, Vt. Ds Athol. 12628 86D, 2. Seateram sefrod af ex difrne in tineg of satin ol oth U7 9. RN Roth 5 He. 5, 96,199 C00, 2. AW. apr, 6.Merie | Mog Ce Pies, Ci egu, 5 Af se 96, 193 2000. 11 ES. Wa noni ren: ot sent nd te Nain hig, Yr, 985) 195-20 22. CE bain Snore A Con's Chie of Ely Hin, CX Brin Gb enoyaph 8 Wont Mean, Ptr, Seth Air, 1999) pp. 23-38. 2. 5 tla eth (Camber re, Mew Yo, 2, 2005, 25, Inspcinns th HO cet eal (ot 3698, "NL 3S17, snd ONM 1) nl den nde prac Praboe WE raped 33 yor Mond {the mandible and cr ra au wv. a 2, 2 2% 2 2 2, 2% YOL318 30 NOVEMBER 2007 cron f ise 9 ems OM Ah np st ots er Toye ope dieser ye Raced ne boy bees rd. 1 Some enya smarts ef vn es ivan ogre opt ete on 12 we at a9 yeas ole tho oe age rk. ‘ta eAaralgtoctne foe Oakes Pet, Sly Am j Pps Atop 7, 23998) Sel a, Se Sn Pate PMU aptis Cn see ag Oe thes Cage, 200, 15-198 Tham, Bet to 28 16 090) Cimon Cast sk, nde ites Ue fetoy ootapnan, on Bev Re, aa vo Us Gn, 390, Ca 8A Wee Fla intel. Bot 2611 tsoi, Jomagot 7.7 0, 0 Pats A hay nel ioe Uf oy Scapnet and Bo hk Pca, Fal ord Us Ps Orr 172, miei Pa Sechl Wingy, eo iat, od 75, ‘atom IES Wings, LA Rog An 1 Pye Johopa 134, 68 200 IK ln Rtonoploy May on erg 1 pea Cota elites ob Meek con San Car lponny foo eg es Sis, 169, echt an ses tomate, bc thy a8 greuhy oon geetay oe eave por ore Bint been madd sete voy dow TC cn, Ben.) Him el ‘Sty € Pai nin Sci, 8.8 Sats 8 Sing 8 soya Nghia, T pe oie 2 cotikon Bos 133, 292 00. Cher) Aton hot 48.298 955 ENA Ser OF Wate © Pn Sh, Boor Ko Seat 201 ct, eect 329 TS erg.) ne} Hopi cect A Py Jno 15, 88 1A Ran tom ft 2,599 O38. 1c ean ia Mo Rb etree, Fe, On Nw vt 90, yp. 2-50 pation. DNH 7 ie the most well preserved female specimen of P. robustus al vault are not shown heel. SK 48 is one of the largest young adult males. Sk 12 and SKW 12 are olde adult males. ravings for SK 12 and SKW 12 are for schematic purposes ony. Scale bar, 3 cm. 14451446 pure ene an Tey nS A, Fete fanc appot ws poli CAL by Be Fl Scty UK AMR 9d CA. th a 1 Sn on woe papery Den tie feméson cco te cosinor fore. FC rece ppt to te ha ry of aon ‘Mats rezoe Geren per Prana ‘Supporting Online Materat Copecsone Cale fi V, coed, cod the wan xen reeeterISRBSOSSANYOC. elun tntasy an aon Cal iam Pete. eS ex ‘ta thak 9 shina sd, ear orth caved Feb fahley St and 52 ‘wth be Dindenexavaten 8. apd 5. Pee fr Hrs usa wh seine ard 2 Aenoee, 8 Bade, {CDeon Kn teh Pavan} Ste Sat, nd Sport Season sd coms Boron-Toxicity Tolerance in Barley Arising from Efflux Transporter Amplification Tim Sutton,* Ute Baumann, Julie Hayes, Nicholas C. Collins, Bun Shi, Thorsten Schnurbusch, Alison Hay, Guenda Mayo, Margaret Palltta, Mark Tester, Peter Langridge oth Uniting and tox sol concentrations of te esental mlrorutient boron represent major limitations to crop production worldvde. We identified BotZ, a BOR ortholog, as the gene responsible forthe superior boron-toxct tolerance ofthe Algerian barlay landrace Sahara 3774 Sahota). Botd nas located atthe tolerance locus by high-resolution mapping. Compaced to intlerent genotypes, Sahara contains about four ties as many Bold gene copies, produces substantially more Bd wanscript, and encodes a Botd protein witha higher capacity to provide tolerance in yeast Bott transcrot levels ‘identified in harley tssues are consistent with a role in limiting the ret entry of boren into the root andin the disposal of boron from leaves va hydathode guttation, {apes nascar asthe On mp’ bees tc a fon soll ston corre sod oti bondetcecy on ony seventy eprodasbavorinteG 9) ccbty ime diteitiomonge enor end xed wir ty ng Fonsi vce. Gn ‘alton fr berraiy loans ow ume ep ple Tene Sonmtnly amped wih he alty © main (ow ten eoncenaons in th sho (0) tn bay oem no) he naga hihi bros 277 (Saas elf a apt aoe oftakeace he vay mpnrcnen (7) Great brennan and bo bo tlt Austin neiagvocy Chpse sen! amr tae rahe (QT) cotg wan ee idol (The myer eas on choo vas kat Spon exposon eA bore econo ig) nt hgh roma ond wes pode vs bore tone coniions (3. The aly of Satara to mainin awer sa orn cunt ica pitalydistoamschanin fact baren cio tr NX lowed mapa pres o ole the bommlsace gee Uy a eon peering O20 ces nd gue leery iti ayneteragon onde chemmsone 3 -usralian Centr Pian: Funconal Geom, Soa of ‘ofc Ft a Wie, Univesity of Aleuice Wate ‘ergs, Pivate Ma B40 1. Gi Ostend, South Aastala Sees, Aaa “To whem covespandence sould be abessed, Era sinsator@aqigcon.ay 30 NOVEMBER 2097 VOL318 SCIENCE crore muskers, we delimited the terme Ibeustoa(. Sccaimonsan interval tween make crs XBNMI7S xl xBV1162 (Fig. 24 and fg. SI) U0), The conesponding 12-Kb itera in ioe contains two intact copies and one 3sruncated se showing similarity toa Saily esine monephesphae (AMP) dependent symihetass and ligases and no other prodicted zene, Barley exprosed sequence lags (ESTS) ‘mest closely matching one of the inset copies swore usaf 10 derive the mirker 1860, which cosegrogata wit the tolerance locus 14 fount 2007; sete 24 Octobe 2007 edaadcence BASEL Ina pall appodh, we ako mappal several candidate genes in barley, These sere barley ‘genes showing similarity to the Arabidopsis NIPS.1 major intrinsie protein (1/) and the Arabidopsis BORY ells transporir that is relat to bkarbonate transporters i an (22,13) Bah Arabidopsis pees are nuit foe hslity growts under conditions of ow baron plans the genes involved supply, Howe horon-tonieity ole show to fart patisons of barley FSTS revealed four ORY (AQe4716@0.1) related genss. Mapping lcalizad ‘one ofthe barley genes (Bor) the wagon of the horon-elerance QTL on 48} Subsequenlly. a marker devekped fiom the ¥ ent of Barf w fund 1 cosegrewaie perfectly with the toleran locus in our high-resolution mapping population 24), siromaly suaaestng that Bor! encodes the boron tolerance fom the $11 locus, Abou huatleytice gene colincariy was found tobe igh in te region Fig. 2A). the cerresponding iner- sal on ize chromesome 3 hes a BOR/ ortokg and the ice ame most closely resembling Bor {0s01g0804) (Fig 2B) esides onchromoseme Southern hybridization with the we of a Clipper drive Bort probe gave a sioner sia in Solara than in Clipper and other boron- invokzant genotypes, ingiatiny the occumence of ‘additonal Bot! copies in Saker (fi. $2). Anum her of rsrction enzyme digests revealed hybrid ‘eng Sahara ragment of mosly a single sie (6, A { 8 \ \) #)=aeeme I be i: boy ry 7 1S 2 11808 pow sun it) Fig. 2. Genetic variation for boron teerance in barley. Coe _ sare, loge Sane 3s oo (A) Borortoxiity symptoms in leaf blades of boron Intolerane (poe) and borer-elerant (Sara) tavley Plants. Approomatelycightof the oldest eat Blades from singe plats are shown 14 days ater boron treatment.) Sahara. accumulates less teron in leat aces afer ‘groning for 14 days ina range of solution concentrations. Data are mears = SEM (n = 3 plans) ua scioncemag.orgAbu D (ig 2), sugueing that the Bos! copics in Sahara are highly silac With Dr 1, which dis- linguikies the Bot! Clipper copy fro dhe Sabra copies all Bot! guns could be mapped and wre four te cosesexate wi boron tolerance Clippersy.SaharaFy derive pulatien, iia ‘huser. Quaniarive realtime orescence poly= merase chain waction (QPCR) analysis using ‘genomic DNA as the templae indicated that Sahai contains about four times (3.8 = 0.17) mone copies of the gene than Clipper QPCR performed using 008. This tranlaes int at Teast 1% of homozygous Nean- dental individuals that may have had reduced pimentation kvels, possibly even simiby to the pale skin color andor red hair observed in modem humans, These results once more raise the question of whether redueed pigmentation may have been advantageous in Europe, for ex anple vin ukravioletlight-mediated vitamin D spithess oF whether it just reflects a Hoss of constant foe the wel gene in regions of w= ‘duced solar imadiation (25). Our data donot support the hypothesis that phenotypic similis ‘etveen these wo human gweups are expnad ‘by gene flow (26) and do suppot the hypothesis fof converges evolution of reduece-unction MCIR alleks, as suggested between modem European and Asian populations (27, 28) References and Notes 1 Lees Aru Re Genet 37, 67 30). 2 Res. Pert el es 33,35 (2000. 3. CFikson el Rone 43,250 2008 6 ke caeene ly Mase 44,3990. 5. Lf Moos ea, Sco 334, 113 2008. 5. 0 Caramel ea, cur eb 26 R30 (2006, 7A Res eal. Po et ced. USA 409, 15266 (ao. 1. Sere tof, 0 Bi. 2,57 (2008, 9. C leur ot, Cr BL 26, 629 C0, 1, 5. Pb, ens Be S24, 13 99) AL Nea and etd ae ovale a apart rate or Scere Dale 2 THC ng. Rees Pees 26 195 (2005). BMC. Gentebtit, A Ne oxen MC Fas, Fess Lind, Mors, eat 28, #95 207. 14 Wlotthe, stice, Bee, Av Hoesen, 5. riba ice Aches 29,4793 2000. 15, Mile eof, Pa Ma Ac Sc USA 103. 13578 (208) 6 HM iat, Scene 333, 392 (2000) 1. HM Gane ay See 296, 2615 2002. Ora. Yolen, Hat Mian, Sina, 4 Bt hem 280, 32089 Gt. 19, HD. Somoy LA Goce, 5. 5. abou, ce hom. 28, 12836 200. 20M Teka 5886 Kl, MO, Mayen Endcnlgy 14537322000 21 8 toe ol, Cotes 15, 1547 (99H. BE PA Nancy a Concern. Biaarers Pe 13, 8 2000 28. KA Bent el Han. Ml Gere 1,285, (ons 24, Sunder aren | Sane Ma MC Tei, LC Gane tron, C ene anes Call ik 52 39 Goo 25. ding et al Am Ham, Ge 66,381 (zo 26 MEHL Wolpe 1 Phys Atop 1, 27 @000. 27 Stiles Sm Tar | Keo, String, am Gee, 120,613 07, 24 HL meton eo, Ml Bil Erol 24, 729 GED, 29, WethankC Geno mary sprsnes and ata tng sf pe msneagt 5. Po rep th he i Ses an eit) a Cuore cece Sam ard Be Generate Gslae (EL dB) ‘eae Mac Soc (OH, ME, RS, on MH he Date oncarggeneinal Bnet ua scioncemag.orgig wed Frc, he I ei A a 1S and he uti ce Deter Woke (SD ee fwd ppt Te exo at Sern ppc ty he Atonnous Gormnt of Aura The ty a fhe Ment Les eset tamed 9 sch fj en hae fs eis ae Yeon it! Wey seo The Mendel mc eee tas been deste at Grr, sees suber wane Sepperting Online Materat figs Ste 56 REPO! Tobe 5 4 Manes 5 uy 2007 cepted 2 Oc 2007 ited one 5 Oat 2007; ade he nloratin ven cing ths pape. 5’-Triphosphate—Dependent Activation of PKR by RNAs with Short Stem-Loops ‘Subba Rao Nallagatla,™ Jungwook Hwang,?* Rebecca Toroney,* Kiaofeng Zheng,” Craig E. Cameson?*t Philip C. Bevilacque"+ ‘Molecular pater in pathogenic RNAS cn be recognized by the innate immune system, and a component ‘ofthis responses the ntrferon-incucederzyne RNA-actvatedprtein kinase (PKR). The major activators ‘of PRR have been proposed tobe long double-stranded RNAS. We report that RNAS with very limited secondary structures activate PKR in a 5'-riphosphate-dependent fathon in vito and in vivo ‘Atvation of PAR by 5 triphosphate RNA i independent of RIG and is enhanced by treatment with type _Limereron (IFN), Surveiance of molecular features atthe Send of transcripts by PKR presents 2 means ‘of allowing pathogenic RNA tobe distinguished from sel-RNA, The evidence presented here suggests that this frm of RVA-based cierminetion may be acta step in mounting an erly imirane vesponse. jinn immune resporsc offers the best | y protection fom foreign onganisms nd viruses (/). As part of tis response. the double-stranded RNA. (RNA) setvated prtein kinase (PKR) becomes activated ty auiopbospboylation inthe presence of vir RNA (2), Subsequently, PKR phosphorylateseu aye inition fit 2a (elF2a), which ini dation, dhusspreventing pathogen replication ¢ PRR can be both sotivaed and inhibited through fis imerzcton with RNA which és me- dlisted by de RNA-binding motifs ((RBMs) (Fig abo exist ober divene proteins, in- RNA-specifie adenosine “deaminases {ADARS), Diger, and ribonuclease HIE), This interaction with dsRNA is soquonoe-independent (4,5) and alhough at last 16 base pais (bp) of ASSRNA are requied for inhibin of PKR, 33 bp are needed for activation (, 6). We have pre- iewsy shown that shor GRAS with singh stranded tls (s-6SRNAs) activate PKR, with the rica determinant hasan imperfect tem of 16 bp and is fanked by single-srande tail (Fig, 1B), and because it was prepared by transcription, it com tains a5 tuiphospate (7), This raises the ques- “oepatnwt of Chemisty, Fennyaa Se Univesty Unters Pa, PA 16802, USK. egratne Bexcerces. Ferran Se Unis. Unity Pak, P6802. 5 Sochemsey and Woecla Boy. Ue wash Lives Pat, PA ABBE USA. “The autos conve equaly to this wok flo when covegendece shoud be adivese. Era peigdhem pueda CBI; et@pa.edu (CLC) wunuscioncemag.org tion of what features ofthe al might be import in acuvating PRR. nous inital expstiments, we observed tha a 79.4p pefetly dsRNA (USRNA-79) led 0 poset activation of PKR, with an RNA-dependency factor of ~35 (Fig. IC) (8) PRI was ako activated by sedsRNAs and gave the expected =O.nucleoide (nt) wil-ngth dependence (6) (Fig, IC), Maximal activation by si-chRNA was as intense a8 th by dsRNA, albeit nuiing 10s more RNA, The sdsRNAI9L) (having A 67 % 169 280 581 REM) “GEREM2 “KINASE DBirain LF BRED Ben ocornerrocceo S=-46RNA (811) eeSSAINA pees nd tals of) apd II nt, spoctnely) transept (Fig, 1B) vas next eated with eal? inestal phosphatase (CIP) to remove te S"siphosphate lig. 1), leading t abrogation of activation ev at higher concentrations (Fig. 1C). Further stiernicallysymbesizetsdsRNA(9) having & 5’ hydrenyl (fig. 2) ako filed to activate PRR. ‘The presence of the Ssiphosphate kal 40 100 ‘Old highor PR activation than occured init absence (fig. $2) (8). A mintue of C1P-wated ‘and untreated ransrips showed fall activation ‘of PKR (fi, $3) indicating thatthe reason why CrP-reatad RNAS do not activate PKR is 10 bocause CIP tsniment anders PRR incapable of axctivaion Totes whether the presence of triphosphate alsoatfets the ability of fang &SRNAS i ativate PER, top and botion strands of dsRNA-79 were C1P-reated and annealed (ig. $4). Unlike sedRNA, CIPareated as wall as untreated ASRNA could acivate PKR, with a sandded bull shaped dependence on RNA concentration (Fi S4D) (6). Thus, long dsRNA docs not require Stirphosphate, suggesting that the contibuton ‘of this motif to PKR activation is dependent on RNA structure iven that ss-dSRNAs have functionally ienportam on-base elements (6), we next tesied activation by the single stands of dsRNA. 79, which also have secondary trucue ig. $4, N and B) C1PAwated SRNA-7STS (TS, top c opss-dsRNA 031) aen0i.70(03 m9 ray — PKR PAR AGL (:20 100 126 40 16 20.38 Fig. 1. Activation of PKR by s-¢5RNA is Sctiphosphate-dependent. (A) PKR has two GRBMs (the dsROD) and a Kinase domain, (8) f:perimental structures of s5- sRNAG (6. (C) Activation assays using ABRNAS [10% SD5~polyacrylamide get electrophoresis (PAGE). RNAS were tran serbed and untreated (5-999) or CP- treated (5-OW). A. no-RNA lane (-) is Provided. Phosphorylation activities are normalized to 0.1 pM dsRNA-79 (no CP) 63) SCIENCE VOL318 30NOVEMBER 2097 asi 14551456 PORTS strand) and sRNA-T9BS (BS, bottom stand) ‘ranserpts (8) wore poor activators, whereas un treated transcripts activated PRR at levessimilar to those prouccd by cSNA-79 (fig. St, Band F), with @ measurable S'tiphosphate dependence “The experiments thus fi sugges that triphosphate makes the greatest contrition when the $end is unsructred, To examine this further, 47= and 1HO-nt transcripts with minionl secondary structure were prepared (ig. 85), and despite having ‘only a small number of sheet serdoops, beth transcript activated PKR at eves clese totes produced by dsRNA tig. $4, G and 1), CIP treatment gatly duced activation, confirming dependenee on 3righosphate ‘Many viral and bacterial RNAS possess 5 triphosphates (9, £0}, whereas most ella tean- scrips have a 7-methylauanonine mG) cap 0 4 Ssmonophospbene. Thus, i is possible ta PKR uses the S'end of mRNA apart of aquality ‘contol mechanism (77) S-monophosphate or = hydroxyl termini, prepared by chemical syethesis OF RNA, wore a as. SO-ld Hes effetive thana S-phesphate terminus, yckling no detct- able activation (8) [Fig. 2nd supporting online material (SOND text. Transerpt primed with 3 mG cap o€ yuanesie diphosphate GDP) ako fui o ince measure PRR activin B to), Parallel experiments on sRNA-10 ave similar results (igs. S4G and SO), indicating that longer transcripts alse havea dependence on 'axiphespbate for activation of PKR, Show S'sriphosplate doublesranded small imerfering RNAS, which induce an interferon response (12), a8 well 38 related short SRNAS, failed to activate PRR (fg. S7 and SOM text). This suggested a minimal kngth of sSRNA equine lor activation, which we detemined 0 bbe ~47 nt dig. $8). In axition, a short (5p) stem-loop enhanced the mogritude of the 1 sponses wellas he dependence on tiphosphate igs. 89 and S10) We also cbsorved at the ‘optimal pestoning ofthestem-loop in otherwise ‘unstructured ss{NA. was 2110 46 t from the 5° end (igs. $9 and $10). One passible season for this is that short stemvloops asst PKR binding, dca that is supported by data on a dsRNA- Dining domsin (dR) hinkng 0 20-bp dsRNA that is consistent witha site size of 610 7 bp per AUSRBD (13). As foroaher support, RNA/PRR. Dining ass recaled aconltion between RNA binng and kinase activation (fg. S8, Band C). One ofthe biological substaues of PKR is lP2c, the fianetion of which in tanslaion inition is inkl upon phosphorlaion of Ser" (2) Upon activation by S'riphosphste SRNA, PKR cllicienly phosphorylatad elF20 (gs. SLI and S12 and SOM text, with cach activated PKR molecule phosphorylating more than 109 fF molocukes wih a PKR P20 Stoichiometry of I: Those results ae consistent with a recent eqsal srcure, whieh showed coch monomer in a PRR dimer interacting with an cfF2a protein (4), “To explore the biological relevance of our Sindings dhs fr, dro el ines were sect to test components of the ate immune response Hhal-7, Mu? 3, and Vero (Fig. 3). All ae responsive to interferon (IFX-a) but prod diferent levels of IFN in response to RNA Views (Fig. 3A). Traasoction of dSRINA-79 into Huly7 cells induced activation of PKR and increase keels of eIF20 phosphorylation (Fig 3B, lane 2). Phosphorylatad PKR was not ob served in mocketransfected cols, although some lF2ecp was detweted (Fig. 3B, lane 1). For the SSRNA, 2 110-r¢ oligomer was sslcted, which has the same Send requirements as the 47-nt oligomer (Fig. 2 and fig. $6), becuse longer RNAs possess superior tanslection propertics Although |10-nt oligomer pppssRNA (7pp. ‘ a ae sss ig ee ea a a ee OE a et) a ee al of 5-OH and 5p on PKR orecibed —tansorbod E wanscibod ersctoed c PRRtERNAAT — TmGaaANAAA7 (61) TmG-eAINA4T (84) TrOSSRNAAT (121) ie Ne ray PKR ““THPT’--rpeet Se (@ashad tne, white boxes. Phesphoyation actives ae normalized 1 0. jt dsRNA-79 (70 CIP). (aN a 30 NOVEMBER 2097 VOL318 SCIENCE adistion &RNAT was yn these wth 5-OH or 5-9, and ‘concervations were 0.1, 05, 2, 2, 3,5, and 10 uM (0 ult is mited for 5p). (B) Efets of SS-pp and Sppp on PKR activation. ssRN-47 as tvenscibed with guanosine triphosphate (GTP oly with 2 GDPGTP ratio of 12:1 ard tested for PER action. RNA concentrations nere 036, 031, 043, 125,25, an 5 ul (0 fect of 2 7G cap on PAR activation. sRNB-A7 was transcribed wth 3 TmGppEG: GIP ratio of 4:1, 8:2, o 121. RNA concentrations were 0.46, 0.31, 0.63, 1.25, 25, and 5 ull (D) PAR phosphorylation data rom (0. The ati of TmGpppG: GP was 0 Gol ine, lad ces), 4 dashed tine, white cicles), Gold lire, Hack boxes) and 12 ua scioncemag.orgStariphosphate} filed to activate PRR to a d= testable Keel, a significant increase in elF2a-p was nevertheless observed (Fig. 3B, le 3) lative to mock-trnsfcted cell. The reponse rexuited the uiphowphate, because the C1Pareated SSRNA transcript showed only baekgroand lev els of elF2a-p (Fig, 3B, lane 4). These results suggest that SRNA requires a S'tiphosphate for PKR-medisted phosphorylation of clF2a in celular “The helicase and innate immune sensor RIG-L has also recently been shown to be activated by ssRNA with a S"tephosphate (15,16) To eter: mine whether actvaticn of RIGeI conebutes to PKR activation by S*trinhosphate ssRNA, we used he Hab-7.5 subline of Hub-7, which acks a functional RIG signaling pathway (7). Acti- vated PKR was not detsi in this ell Hine in response Ruud G. H. van Leeuwen,?{ Matthew Tyreman,2* Danuta Tomkiewicz,* Nico Nouwen,z Harald L. Tepper,2s Arnold J. M. Driessen,” Sander J. Tans*] How chaperone intecactons affect protein folding pathaaysis a central problem in biology, With the use of optical tweerers and all-atom melecular dyramics simulations, we studied the effect of ‘chaperone SecB on the folding and unfolding pathways of maltese binding protein (MBF) atthe singlermolect level. In the absence of SecB, we find that tne MBP polypeptide fst collapses into a molten globulelike compacted state and then fold into a stable core structure orto which several aelies are finally wrapped. Interactions with SecB completely prevent stable tertiary contacts in the «ore structure but have no detectable efecton the lading ofthe enteral helices. It appears that SecB only binds tothe extended or molten globulelike structure and retains MBP inthis later state. Thus during MBP translocation, ao energy s required to disrupt stable tertiary imeractions ll pays are radial sed Pissise peter tah te actions with other mokeules during the folding process. For instance, interactions with chap frones aw often crucial 10 guide folding, to prevent aggregation, and to facilitate protcin translocation across memranes {/, 2) However, how folding pathways arcaflected by chaperones remains pooely understood. For instance, it is uunckar which sops of the fokling process are ‘parent of Nedecle Wibiley, Going Bor amaeclat Scenes ad Ssecnctog este an the Tere hte fr Advanced tel, Unversty ot ‘Glonngen, Kean 36, 9751 AN Mate, Nether. "Fo ite ler tom and alee Ryses(AUOLF, Arslan 407, 1098 5 Asterdan, ethers “These authors cobiuedequaly ths wor Pest aes: Phils Reseach, High Tech Campus 36, 5456 AC Endvoven Netbernds, reset adds: Labeatae de Symbiosis Teiales et Médieraniemnes 1A 4821, Campus International de Bailogue 38988 Montpelier cde, Franc, ‘reser adds: Metinsy and Company, Ansel 34, 4017 AS Anseraan, Nethetands. Tio whom correspondence shoud be addressed. Erma tan@anat ol 30 NOVEMBER 2097 VOL318 SCIENCE fiver by chapewnes, whether the protein- chaperone structure is stable and well defined, and wheiber native or akeratve tetany ine actions are formed inthe presence of chaperones. We adresed these questions by using p> tical ecczo to induoe the mechanical unfoking and refolding ofa single protein molecule in th absence and the pressce of molecular chape ‘ones Single-mokeule techniques can reveal fk ing tansitions, has been shown fe isolated molecules (7), although they cannot direlly concte the trinstéons with moleclarlevel changes in protein sicture, To oblain structural insights inv the chaperene-fne folding pata, ‘wecombined all stom molecule dyemeni simukions with optical wsexzes We studiad the unfolding and fokling of Exchericha coli maton binding protin (MBP) and its dopenddonce onthe chaperone Sec (8, 9). ‘The imeraction bexwoen SecB and MBP has be extensively sued by bulk biochemical assays, smukiny ican ideal sysiem for exploration atthe mokcule evel, The known physiological lets of SecB are to reduce agesegation (J0) promote tratslocation across membranes (1), and delay frmation ofthe native stte(2 13) (74,15) ana does not require 2 Sara sequence fx intenetion (76). Although the structure of MBP in complex with SecB.s unknown, i boon suggested that MBP forms tertiary sructre ements in the presse of SeeB (17). Such cloments would need to be disapied during translocation because ony an extended cain can pss through tho narrow SeeVEG channel (8). Tnnivigual MBP melocukes were tethered hotween two polystyrene spheres 2 yum in diamcier by using @ 2853-base pair DNA molecu spaer © prevent undesta beaded interactions (Fig. 1A). Conacetions to the N- and ‘Cminal ends of i.aype MBP were obainal viaan engineered bictin group and aquadnyple cecnye tag, repocively. Purified cans, bere refered to as MBP, reined thie ability to bind to an amylose rei, indicating proper foking. The DNA protein tier was stetchal by diplacement fof the pipet heal, and the resting fre on the sccond bead tapped in the ls focus as m0- sured. These eyrriments yiekled free-extesion ‘curves (Fig: 13)showinga skin hang in exten sion atan average appa! nce of 5 «8 SD) pN. When MBP was replaced with a plain bio. tinylted crnye tag, there were no sexiden exe tension changes (ig. Si). showing that they comespond to MBP unfolding events. Alerthe unfolded MBP was relaxed by moving the beads together, it Folded Ick tots native tae, ed by a second stretching expe ment that yiood a similar eure, This unfold refold eycle was reproducible several times on single molecule until linksge inthe ether broke: Seeching cures ofa sand consiruct com posed of four tandem MBP repeats (SMBP) produced the unfolding at an average force of 234 pN (Fig. 2, A and B) As previously ‘epowted for atomic-Fore microscopy expesonts ‘on proeins with repeatal domains (3,6), 2 saw tooth patien was observed, coresponding 16 the scparte unioing events othe wpeats, Inerest- inal, however aller the orce adboen loweredto allow refoing ia tne second Sretching curve the pots ofen fied to unfold atthe normal 25 pN ua scioncemag.orgREPORTS I (Fig 2B, 02, inserting over 40 pN ‘ais the igh fal eoncontaton ily promcted garni 9 of te MBP repeats © prods 8 = ., Steir more sable dan the rte ste z ‘Once proper fll boven was ett inhesingl MBP corset. the chapeneSecB 2 Iweeaer. The fist stretching curve (Fig. 1C) was = simi o Uose without chaperone present, +Sec8, omavable erage unfidng frce FOE Oe PN, So thus doe no promote fling Extension (um) of native MBP, even when itis hell rn tension, Subsquent sttching caves dil ot ‘vel with Ue fist sci curve De rahe floes the ebaton curve ad Heke ees typical of Iking era icra. Sec ths only binds filly unfolded MIBP and hen ain of any stable ere say tenctees in MBP aon, too MBP. SecB eopen appearsto fom more rap than the finest foking event (9), Simi, the azgrozaton sition te second steihing cure Oe eR) eee AMBP verona wah the pron rina tae cane eee, son cure Fig 20), showing that despite tei heres atthe N temins itis attached via srestviin-bitinHnkoges to a DNA ether, nich in turn is_ Sly the aren erations cao rm sued to the bead safe va an antbodyigaigerin cnnecion (8) Foceesteson cares te Pecan inertrence by See absence ofS showing unin at igh ore (ed) refling at rcs green) andagainunfeldingat The frce-etesion curves show move de high fore (bel. Foweetesin carves inthe presence of Sx (01 MD The Secor seeing cuve su! etre, andthe challenge so cate those (red cs te ypialuneiing features, sang that stable teary interatarsare absent. (0) Comparson 0 situ changes. For example a gral betcenteitation cures and VLC model Indkated the average extension Irom several carves ihn 9 exlesn incre involving several sl farce ntdow,nornalzedto the exesion expected om the WLC model. 5) Athighe foes, data and scps is observed bslow 15 p before full made oretap, neres a ower valve pods o adios compaction ofthe pobpepte. The ever bars unbling at ~25 pX (Figs. | and 2), The low ‘nate te stonarddeitons ofthe etenson fom varus rellding cues. and hgh-fewe teanstons cannot rellet a sey fre unfolding of sevcural dante: the 80 bbs of MBP (20) cannot unfold independent Ieease the popeyriie ne back and fh ‘tse than “To deni stuctral changes asc with tannin the reeexkion ves, weed mlecuar dynamics (MD) a situs dh une fokiny of mative MBP. Whena forse was applied co the MBP tsi (Fg. 34 ata movie SD. fivoresidve segment athe N termins unflded fir, fllowal by the Catena a bel Cone snug such unfolding simulations is poems fg, because the increasing protcin extension rakes the siulation vokune computationally inrcabl, To cveumvent this, we dled fide segmens and then corfu the sim bons onthe new canst This method lis ee o7 08 09 a Extension (um) Fig. 1. Experimental setup and Sect dependence of MEP force-extension cuves. (®) MBP is wether between two beads One & held on 2 positon cor- troled micrpipete; the other is held by an optical trapalloning force detection tthe Cterrnus, MBP Extension (fraction of WLC) A 7 ‘onthe asumpiinn that unibldatsegrents emia +8008 } — eutendod and do not fold back, which is wa 08 1 12 O8 1 42 sable given the force applied on the kin Extension (ym) ‘Extonsion (um) “The subsoquent simltions sowed tht another Fig. 2. (A) Schematic iustiation of the AMBP consti. (B)Foxceextension curves in the absence ofr C-terminal a tices local on the exterioe Set. bated nr repent the theoretod WLC complane oft DNAprean conse rue OVE HVCMME Wee agurtialy un stages of unfolding, as precicted by IMD simulations: curve 1, DNA tether alone, representing the native or #1401 91 residues had unfolded and assablecore aggregated MBP stae: curve 2, DNA tether plis compliant palypeptide of four times 91 residues, Sfucture remained (Fig. 3B), Note tha the sim tcpresceting ou folded MBP cores and the uneded external «hls and curve 3 pojpeptidelengh ¢wltions invohe ling at much faser time increased by 279 residues, representing the unfolding of one core structure. For each subsequent curve (4,5, les (27) and therefore act as a hypothesis that a6) te kngih i ened by ene additonal unde core fer hil ld () and sauce wll st ognins the expetimers tekaatin too fc, te stele at ed tac) flow WLC ave I, rang AMBP hse mifoled ni a The sty of the simulans inermedite core Uphtagpeptte (forever curves inte presence ofSecB (0. MD Aggregation reve ding srt implcs tht might be observed as @ telat a erences ytelon forces requte er exercingte polypeptide ed vac) The SEP esx din stein th Nicer mesons. The ae conaten withthe sgl HBP rest data, thos fere-extesion relation for ib ste wunuscioncemag.org SCIENCE VOL 318 30 NOVEMBER 2007 1459based on the wom-like chain (WLC) model(22) measured gh changes. Also th indoad overtps withthe data after the lows-foree ofthe low-fove extersion increases is consent unfolding below 15 pN (Fig. 2 and fz. $2), ine withthe simuled sequential union The AMBP dking goodagreament betwen sulted and data show an increase ay kth that four times lua nature 3. Results frmall-atom MD simulations. (A) MBP native state. Alter applying a force to the Cand th terminas, the red segments sequentially detached until a stable core structure remained (B) Unielded ‘segments were deleted from te strucure to keep the simulation vlume computationally tractable * sa 3 as o ~SecB Q| +SecB_ a Cn a a Extension (jum) Extension (ym) = & fe a) 2 couprereD : E. — - wine he Bos \4 2 ‘EXTENDED: 2 ol seca} a -F SF Se AGGREGATE é Seoreasod sabilty 4. Loweforce unfolding and refolding and translocation assay ests. (A) Force-ertension cares of AMBP in absence of SecB. The construct was fist stretched, resulting inthe predicted gradual unfolcing of the external helices Fg. 3), and ten relaxed before the core structures would unfold. Duting relaxation the neat-equtibrium refling ofthe same a helices was observed decly asa shortening of the tether. The dotted inesindkate the WLCbehavior The first denotesthe DNA atone whereas in the second a 8x92 residue-compliant polypeptide was added, representing the unfolded external helices (ls fig. 52). (B) Force-estension curves of AMBP in presence of SecB (0.1 ND. The SecB interactions do not alter ths tefoting aration. (© ATPase actiny nn ancocation of BP tabity mutans, Flding sain elimol is, respectively: 11.2 [Th?™*—e™? (345)], 10.5 Wild type), 9.4 (Val*GyP (BGI, 9 lata?*ciy™ (42760), and 7.3 [ly?--asp™ 2830)]. All mutants exhibited a similar energy Feourement forSec8-mediated tanstocaton dark blue). The preMBP translocation kinetics wassimilarin the presence of SecB. Contols without SedB ight blue) or NP showed low ATPase activity and translocation as expectec Te eror bars indicate the standard deviation. (D)Force-induced MBP folding ‘and unfolding vansiions observed in the experiments and their dependence on SecB. 1460 30 NOVEMBER 2097 VOL318 SCIENCE Larger a low force compared wih that of naive (MBP nicating that al extemal a helices unfold before the ft core unfolding. The lengthelnass ‘ofthe high-foree unfolding events agree wit the unfolding ofa single 279-eside ere, showing that MBP folly untois, WWe note tha reported stabilty-dccrssing MBP mutations (23) arc all Positional near tbe N temninas of the presctad nt of the extemal heices suggests that the reverse folding proses, in which they sould ip hack cnto the core surface, might be dirsstly observable in the foree- ‘extension experiments We texted this idea by stopping the strech experiment alter the low: ‘erce unfolding and thea bringing the beads wo gether agin, A gradu refolding of the extemal ‘chelices was indcod detected dretly as a nsat= ‘quilibekim proces, as evidenced by the short ‘ning ofthe tbe at nonzero Toads (Fig. 4A). “The native state was recovers afr relation, seen by the characteristic unlling atures in the suibsoqion siesching eurve. Now that ater fill unfolding (Figs. and 2) ane does not dictly ‘observe elding of the core nor ofthe exiemal hstioss during relation, whic is consist with the opened fokding pathway in which the lowly folding core strvctre must form fis ‘The extemal a helices refilled wih similar dynamics in the prosence of SooB (Fig, 4B) SeeB apparcaly docs not aflet this transition, despie the existence ofa pusaive SeeB binding site on the unfolded external « helices, a iden Tfed in peptide binding assays J), These we sults may indicete dha this refolding triton ‘occurs ister than Seok binding. Alteratvely SeeB may only bind sits buried within the core ssructure(5)and not effckly bind the putative binding sie on the extemal & helices, possibly hecause ofthe neatby bulky cow. Inthe experiments withoue SecB, when the polypepide i rd alter being fly anfokdod (Fig, IB), the measured extension increasing! sev inks fom the WLC bebavior, showing lengis tae ver tv times smaller a the est Free lig, $2 and Fig. ID). This compaction of the Polypeptide ie consistent with previous bulk studs incating an MBP molt globule state (24.20, a compacted fon thatisheld together by umstale teary and socondaty intramolecular in tercions: When Sicha ration experiment was rpidly followod by a second sttching exper: mem, networesing curves oxertappa (ig. $3), sic indicate dat the transition is nor equi thatthe key erations an unstable The cuve shape forrelesation to low forces it similar in de presence of SB (Fig. L, Cand D). showing thar protelt- Seek interactions dy not result in a axonal compocticn. The MBP- SeeB complex has beem showen to hae a tit [21 stoichiometry (25) and likely involves mu tiple contacts 20), Ono might therefice anticipate that their npturing during stetching would also show up as siden extension changes, The lek fof such features suggests that MBP-SceB con ua scioncemag.orgtacts arent stable or alternatively that bing ‘occu effectively at single MBP sits, Next we followed the trnstien from the ‘compacted molten globuleike sate the native ly folded siae, Considering this tansition as a Singlehamer proces the probaly to fod within 4 waking time ris PLO 1 &*, where kis the felding rate. This probability can he ested by perfomning muliple sirtcescaxation eyes in ‘which the poiypentise Bowed to rf at a0 leadin the wit tne swoon the raxation and the sxtching curve, Whether fling had occured was dtemmined from the unfolding features daring the subsexpontsaching. We obtained a fling rate of 0.760.195" in the absence of So (is SW. This vahe & sintky to MBP folding rates feud n bulk 23,20), which iscorsisont wit te iat itis tbe lowest Solin step. Weeould ‘quasi the revolting rao inthe presence of Sos hocause this egies waiting times inthe onder of mines Ir i clear however tt SeeB-MIBP interactions maintain MBP ia the molten globule compact staicby petri te fonreion of stable stir interactions plication for MBP tanslosation acwoss the cellular membre. Whoiber sublet intrations re actively disepted by the tans i a mutter of debate (/7, 27). Our data suggest, that Soc eflicintly prevents any stable tentary interactions in MBP. To testis prediction in translocation reaction, we meas the adenosine trpbesplanase (ATPase) activity daring te tanslocation of MBP mutes with alert fling stablty(77, 23). Weindoed found hat all mutants displayed ATPase atvity similar tot sional sequence) (Fig 4C), Comal experiments lacking MBP or Soci showed low ATPase activity as expecta. These rests supper our y= pothesis ta for SeeB-mnedted translocation eely limited energy is required to divupt SecB-MBP interactions o Well a6 trtiny intermolecular n= teractions in MBP. The single-molecule dat provide an estimate for this energy requirement of 2SknT (ig. $2, which roughly comesponds to the hydrolysis of one ATP moc “Taken together, the data indicate a folding pathaay with a lae vary of tanstions and modes of folling (Fig. 4D), which an each affected diferendy by Sec: () The euenitat peptide is compacted toa molt globule satin ‘ther the presence or the absence of See. Gi In the absence of SecB, folding proceed rom the molian globule w a core intermedia, but SecB. prevents the foewation of sable tertiary ints: tions thereby maintaining the moter wbulelike slue. This efect may well be moe general and apply also to other chaperones such as GroEL, for ‘which there is support Kom orher ude (28,29), (Gi Once the cow intermedi has formed, SexB ‘cannot bind, ands Uherefize has no effet on th foklng ofthe external «helices onto the surface of the core smacture. Folding fee the core tothe fully foldod state exhibits sindbis with a wunuscioncemag.org anucleation-growth made of faking (30, 1) and ‘contrasts wt the comple conformational search ine molkn ghbulelibe phase that characetizes the folding ofthe cre. iv) See aso prevents the sable azerezation interactions tha occur at high local MBP concentrations “The results provide an important fist step in ‘undersanding how s protein fokting landscape is altered by contacts with asevondary protein, Th approach is general an can be applied to inter= rogate other proteins and chaperone syste, Roteronces and Notoe 1 FU. Mar Marka Sceee 295, 282 2002. 2 Cm ation, Rene 426, 884 2003. 2m Ras) ovat Soc, Hf ah J Mo Bel 246, 82 0995. FE tet 8 Star, VBS, Cat, Bopiys | 8, 2364 200, 1 Canes fngns | fern J al ist 319, ‘2 cnn), 6 ir 9 Mei) Fm, Mt Sac, ol 7,719 200) 27 C cee A Stnk Btn, 5 eee, Seer 309,057 0005) 1. Pifeltes 1M. Doese, Miso Mal ie Ae 63, L999, 9, L310. Yosie Wat Sout il 7.1172 (aca, WS ter, 8.1 Onan Wie 80 9, 2108 1930, 110. M, Sa Sel, Hen 12D Cle ¥- A nts | Wes.) Bast ca 53,273 0980, 18S Hay, UL Rand cence 251.49 (0. 24 LTA ees a) Ba Ot 24,323 5.1 Taping LL Randall Pet St 3, 750 4990, REPO! 6 LL ard. B epi 5 Hardy, Se 248, 060 liso, 1. He Ceo Randal Mabie 27, 469909 1, 8 yan en Brg el, Nore 427, 36 0008. A Fobehae, PE Most, MC Vat, A ean, Met. Seat Bo 6, 1025 (999 23, Xuan} AGH Moi FA ui Mol Bik 306, 715 0G, 21 Mel and ead aval on Scene Oat 22 LF € 0 igo, Urol 28, £759 (595. 2), 5. hay Sie P.O L Hanh J. Bol (her, 268, 2855 09) 24 Seo} B, Upon, Venn, Biche” 18,3408 G00, 25 fle, Car ders A LM Dries 0 1.26, fos 97, 24 LM. Ge taj Ma Bak 362, € 2000. 27. GF bu TO Topping WH Cv LL onl) Bik ‘hem, 263, 1090 O88) 24 Rs dal Pox Nal Ad Sd USA 192 27900051, 22. G'S Cle Lomer, Dama 3.8 Beds, roe Ni eo Se USA. 264 8303 209? 30. AR Fea, W gen, Cl 108,573 0. BL A A fom, Cor Op Stuer Bil 7, 3 (299 BR We Bark Fen Wale, Tet and MDa lercival eadng 3 te eauners & Tee teas ler dren: aed Dae eal sane Ths necks fred bythe Crspese Cemeanty Donat progam; Nantes, snarl eos egam ‘coceate bythe Oth Missy of tonic ib ed Orgsraten oFuecst Rese Se water GOW aed te Funan lr Le Sere Ulu, tic ae bth ancl spore bythe NeheardsOrgenzaton fr Set Reach (IMO, ET ma sapere yo VIM gant om VD ‘Supprting Ontine materat seoncaneg ogre fl ALBSHSSASEOC, Ite rd mote figs it Renee 1 iy 207: ace 17 Gober 2007 oiaaince 160972 Carbon Dioxide Activation at the Ni,Fe-Cluster of Anaerobic Carbon Monoxide Dehydrogenase Jae-Hun Jeoung and Holger Dobbek* [Anaerobic CO dehydrogenases catalyze the reversible oxidation of CO to CO2 ata complex Ni, Fe, and S-containing metal center called cluster C. We report crystal structures f CO dehydrogenase tom Carboxydothernus hydregenoformars in thiee different states. In 2 reduced state, exogenous COs supplied in solution fs bound and reductirely activated by cluster C. n the intermediate structure, CO, acts asa bridging ligan botween Ni and the asymmetrically coordinated Fe, whore it completes the square-planar coordination ofthe n. I replaces a water/nydroxo ligand bound to the Fe ion in the other two states. The structures define the machanism of CO oxidation and Op reduction at the Nie site of luster C. he biological redox transformations of COs, I ‘No-ana Ip are event processes in lol bgaochernial eyes an are caalyed by «enzymes conn complex metal users hase fm ion and sur whose dtd fonction i sl pootly understocd (/, 2), Carbon monoxide hydrogenase (CODHS) axe the ological ‘stalys or the reversible oration of CO 16 COs, ‘wih wororas the soe of oxygen: CO + HO 0p) 2 I" (Eq. 1). Two peicipal types of ‘CODIas have boon dascriba that fein thie ‘oficcr composition, struct, aa stability in the presence of dioxygen: Anaerobic bacteria and archaea use oxy gemscusive Ni-and Fevontaiing COINS, whereas aerobic, caberydarophic bocteia use a Cur, Mor, and. Fe-containing Avery (3) The NJB-CODH bes ae mon fineticnal ce bitirtional enzymes asccat with NiFecentining aceyl-coenzymne A (CoA) sym fses (ACS) (4, 5) [oe review, 2°, SCIENCE VOL318 30NOVEMBER 2097 RTS. I 14611462 ORTS C0 oxidation and COp reduction atthe active site, cluster C, of NiFe-CODHases are proposed toraire dec differentiation sists dileving by ene eosin (Crt, Cia 34 Ca) (0) this rnodel, the Crt state of cluster C converts CO 1 CO, and i formed at redox potentials below 200mV (7). Atpll~ 7.0, the migpeint potential far the conversion of Cysy 10 Cas is 530 mV (8, 9), similar wo the noemal potential of the COm-CO couple of $58 mV, Consequently Gas is generated by a two-slectron reduction of the Cg Nate Via Coy (2). The structure of chus- terC tas reveaked by exystallographic analysis of CODIases isolated fiom Carhoxvdotermas yidrogenoformans. (CODMey) aad Rlxto spiriuan rubrum (CODY) at 1.6 and 28 A, resolution, respectaely U2, 13), Clas C in ‘CODIIIlcy has been described as an asymnmiciic [NiegSs] comer which coenpises an inal Ni on coordinatedy fou sulfa ligands with square- planar geomeny (J2) An asymmetrically coor- inated Fe ion (Fel) is found elase tothe Ni ie. In COsreated CODHp, cluster C fas a similar structure wit acubsne [NiFe\S,] enter linked to a mononuclear Fe sie (13). Comespemcing src= tures of cluster C wen alo identified i the ery structures of bifunctional ACSCODH isolated fiom Moorea thermoaceica (CODHya) (4,5) Mechanisms propesed for the reversible oxila- tion of COtoC Os posit the action of HO and CO-as well s the stabilization of @ meta-bound ‘COM intermediate Howeeer, tae struct oF these states remained chusve. We doscibe hovr HAO and CO, are bound and sctivted and propese a stucture-ased model for CO exidtion and CO reductions the Ni-Fel ste of chstr ‘Aa expression system for CODIIlley esab- lished in Feherichta colt onal. a one-step tion of active enzyme. Crystals of recom rk CODM Ik iia up © 1.40 A resoation ‘onarotating anode xy generator (able SU). The foverll sruciu of recombinant CODE is entical othe structure of native CODH Hy (12). CODillleg eystas were held ata defined redox potential of 600 mV for 3 hours with use of Ti(ll citrate, These crystals wer citer diexy Stoven in figuid nitrogen, generating te 660 mV ‘sate oxidized via incubation with methyliclogen (MN) and dithiotctol (DTT) and they incue batad with DTT fo give the 320 mV state: or incubated in the -600 mV’ soko with Nal COs asthe C03 source, generating the 600 mVvsCOz ste. The -600 mV’ sate (quivalen in its redox pote to the Cpe tate) ann the 320 mV’ state {ecjuvalot to the Gaye Sate) cisplay practically ‘dntical stctares for cluster C (Fig. 1, A and), In buh structures, the Ni ion is coordinated by Ueoe sulfir ligands with distorted T-shaped ‘ooninaton geometry (Fig. 1, A and C, and table Latootorum Proténktalogasie and Foschunecer ‘Tm ir vaca, Vesta Bae 95440 Borah, German, “To wham coresponiene soul be adtesed, Ema Andgedobbe@an tape 30 NOVEMBER 2097 VOL318 SCIENCE 83), Fel is cooniinate by Hiv"! M1261), Cy (C295), 2 jysultido Ligand, and a monoatomic Tigand (Fig 1, A wn C. and figs. $2 and S.A. weakly occupied altomative postin for Fel (FeIB) is observed in both states (Fig. | and table $2} The monoatomic ligand is distance of 2.7 A from the Ni ion and occupies the posion that would complete the square-ptanar ‘cooniinaion of the Ni The electron density of ‘the ligand canbe modeled 22 light stom (C. N, ‘or 0) with occupancies of about 60 to 70% ora sulfur aon with 30% occupancy. The observed Fellgand bond lengths of 1.93 to 1.95 A are atypical for Fe-S bonus, whereas a HaO/O1T Tigandis consistent with the refined bond length, the elative occupancies ofigand and Fel (table 52), ant spectoscopic investigations of the ‘Cay state, The Tong distance between Ni and the ligand suwgess a weak Ni-Olly interaction AHO O1F ligand has been detested bound in Fe" ion calla foneus capone I AFCTI in fe Gy Sate (U4, 15,90 the erystal struct is consistent with Fel in the 320 mV slate being FCT Inthe presence of appropristereducingasens, NiFe-CODHas en cay’ the rxbuetion of (C0, (J6), The stracuae of CODHIly sith COs 00 mV COs Sate) reveals a triakewc Higand bridge Ni and Fel, which repooos the water! hydro Higand at Fel (Fig. 1B), Modeling the ligand as COs ally sasos the observed eleckon dnsity maps, whereas modeling wih one oF two Fig. 4. The~€00 mV (A). ~600 mile (B) and 320 i (© states of uses C. Fae ~Feae maps in ble a¥econtouted at 10, and Fay — Fae MADSn green ae contoured at 4.5. For the calculation of te Fn — Feqc ap, the OH tigané (4) ard (Cand the CO, ligand (8) have been removed fiom the mods An aternative positon found for Fel, termed Fe1B, is depicted trspaent light ery. The occupies fo the alteratve position have been estimated to 10 tb 30%, Selected cistances are shoun in A. For more etalsonthe geometry ofthe thee states, ee figs. Sto 4. Al pictures ere prepared by using Py (23). ua scioncemag.orgREPORTS I ato does no. COp bound to cluster C acsas a A! OCO ligand at NP* with « NEC distance of 1.96.4 and completes the squan-plasarcoondins- tion geometry typally found for NP" sons, COs acts ava ni" OCO ligand at Fel with ay Fel-O1 ddisance of 2.08 A, reuing in a tf binding mods of CO; bridging the N-Fel site (Fiz 1B). Like the HsO/OH tigand inthe 600 mVand 320 mV sates, O1 of COs i in hyd bonding disance to Lys" (K563) (Fig. 1B). 02 is in hydrogen bonding distance to His. CO, binging w cluster eases only miner the geometry ofthe cluster (Fig. 2). The change of the disorted Tslupe to the squa-planar atthe crbon atom resultsin act econ tranter fiom the meta ine the aie Nh ate Miia) ‘he -320 ni state hasbeen sed a8 a med Tor ya, he sae of | luster C competent of CO oxen.) The proposed transibon state ofthe eaton fn which CO nds wo the W?* on and eas withthe Fet- made forthe naa tate, wich suposed to contain twoadeitional elections bound OH group. dH) The ~600 m/sCO, state fused as a model for the compared Mh Cay at, Genoted a3 formal change ofthe oxidation stabilation ofthe metal earbeylate tat. (N) The 400 mnVtateis used asa state ofthe Ni" fon. wunuscioncemag.org SCIENCE VOL 318 30 NOVEMBER 2007 14631464 bonding lowest unoccupied molceular xbital of (CO, This activation of COs increases the nx tive puta changcs atthe oxygen ators, which are wabilized by binding to electem-defisient ceentas Tike transition etals oF by fos hydrogen bonds (17) In the ehsier C-COs complex, Ni acts as the Lewis base, and Fel is the Lewis acid that together with K303 sabi the negative partial charge on OL. The deviation fiom linearity along. the O-C-0 axis [0-C-0 133° (able 83) s in agrecment withthe ati vation of CO. by binding to cluster C bry previous structural dugeterizatons of tive reparations of CODHHly, we identifi a sulfido hgand (S2) brging Ni and Fel (12,18). The enayme wsed for erystlization as wall as discohed eqstaks had high spite stvtes of 144.000 unis mg" and en the basis ofa postive correlation ete the presence ofthe S2 ligand and rzyme activity it was postubed that $2 is ‘eset forthe caalyic CO oxidetin (18, 79). However, the nsossity ofthe $? Higa for cata ysis wa chad when sulfide vas shown 10 reversibly inactivate CODI gad CODI yy Kad ing o Shon ay planes (20), and no S2 Tigray were identifiod in the enstal structures of CODA nat CODIIgy (5, 13). Hee, we dete the sinuciures of [NiFe(S(OHL,)(CO})] clusters without 32 gan in crysis with high specie CO ‘oxi ett (11.000 9 13.500 us mg", slowing that the presence of $2 is not necessary Forcatabsis Funhermore, the SP igand oseupis the binding site of two substrates of Nie CODHases, water, and COs, The HOON Tigand identified requires the same coowtination eat Fel as theboridging S2 ligand (fig. SS) and (COq binds to the two open coordination sts of Niand Fel. Thus, we suggest thatthe $2 ligand Ipetween Ni and Fel is absent in caalytisaly ‘competent ezyme species annca be redutively ‘or chemically replaced, aetivating the nay, “The thee presented structures offer diwct insight io the reaction meshanist (Fig. 3). The [Nife,S,OHL | clusterdctemnined inthe 320 mV stae isthe Rinetional sate tat activates CO and contains the H,O°OH ligand. The N#* ion is Positioned. the endo to subsite channel, and its the sulfido Higands act as x donors to the metal, enabling CO to bind tothe NP™ fon (27, ‘The Ni ion has two open coordination sites, allowing ctheran apeal binding of CO worm a diverted choad geometry oF CO binging ‘equatrially compkse the squue-panar coor ization gcomety. Binding of CO in the apical Position has been propessd for COteakal exys- ta of CODHg and COD ag (3, 13). Modl ‘070 in the apial position places the CO-carbon stom more than 3.5 A apart fom the HLOOH ligand and makes further rearrangements neves- sary forthe eaiion to proceed. In ontast, CO a distored square-planar binging 10 complete ‘oonination ofthe Ni OCH, diance of les than 2 A (Fig. 3, step ID. The binding of CO wy a weakly hackdonating met like NP resis in an eletrophilc erbon ater 30 NOVEMBER 2097 VOL318 SCIENCE and ficiliaes i action with the Fel-hound HOI lignd w a meta-cabenstie species as observed int -60 mV (COs sate Figs fd 3 sp il), Product tease may be assed bythe reverie Huan exchange of CO> sist NOatFet and isaccompuniedty 2 worecton reduction of luster C,ssnsating the Cnn ta ‘The HaO OH ligand can he replenished ya acighboring nawork of sohent mokeuks (Fg, arson ot the [NiFeyS/OH,] and $(CO>)| tates wea the positions of Ni and Few be lagely nafeted ty the prsense orabvencoof ths COpH gan (Fig 2). The [FeS) site of cluser © presides a sold metd-slfor frame in which Nia Fel aw hod in place and serves as an eectronic bullet cere for the ekeronc hangs st Ni and Fel cing the catalytic evel. The small stactrl changes of haser Cree well with the Ios reognaton tera expected foraeaton wilh unser aes Of 31000 5 anda ratio of Ky Keastie rate onan) / Kg (Michaels const) of 7» 10° Mt stat 0 22, ‘The stritureasod mechanism outined agrees i all central aspects with the bimetl sm proposed on the has of elton i esonanee (EPR), cleo nica Aoubleresonance (ENDOR), and. Mossbauer spectnecony (14 15) References and Notes DC aes, fon, Rs Bote. 72.721 2002» Ces, 1. near, Srace 300 924 (08 Sw Rope, ite tr al al 39 145208. LL Ope. ers, Seal 5. apse CL train, Scene 278 367 0X2), 5. © oxnat a ut So. WoL 0,273 20 6 PA rai ocho, 2077 G1 L Feng A. Lindh Biches 43,1852 2008 2. Pk Undot C mur Se Fogle | il Cer, 265, 2073 G0). 9. PA Unda 5. Raga, E Mund Bil Cher, 245, 3080 G0), 10.0.4 Gotan, £ Dt Boxter 3, 4617 (295). AL D1 fest PA Lind Bechet 3815705 (999. B.A Debek Set Lem tbe OMe, Seance 293, 1281 G00, BCL Drea | He ALO Sok ES, Plate Pec Mot od Sa USA 9618732000, 14, Le ctely I-A. Gen Soc 118, 50 4990, AB, Det, Teber ME Aner PA dh, 2M Hain I Chon. Se 120, 6767 50 36, § A tig, choy 30,5372 C999), 1. W. Len Cord. Cher. Ret 153 257 0996, 1 Debate, ag. Mee) Am Cher See 326, 5382 2008. 1, SW. He to JB Chom, 282, 30639 007, 22 Feng PA kh Am Che. Se 26, 00408. S.A mucgegee Zl 0 ert, RH Cbs, nrg. Cher 33, 3646 (954 22 Stitt CFs Ree 0. Nee, cl 183, 5134 G00. 23 Pel L-Oeinn Diane Sete, Son Cag, 2 Rethrk Wut and @ En ore edig ofthe marusce ne wonledg the Deas Forschner (FG grant 0 70/0 and the fens dr chron ri UT for dig Vette, aramid or roving C eragenpnars 22901 Grin OS £006) fer swname UNA proparton. eatin sd sce fats Pave Ben ceperted ihe Pte ut. Dek pb.) eter 9052 (600 a Ste) 3852 (600 sO ead 3853 (329 ste ‘Supprting Online materat soon caneg agree fl ALBSHSSNRE OC. tte mace Sou ot fie Ste fetieoe 50 ny 207: cated 1 Orme 2007 voltae eb Solvent Tuning of Electrochemical Potentials in the Active Sites of HiPIP Versus Ferredoxin Abhishek Dey,* Francis €. Jenney }1.? Alichael W. W. Adams? Elena Babini Yasuhiro Takahashi Kelichi Fukuyama,* Keith 0. Hodgson,#** Britt Hedman,** Edward I. Solomon” A persistent puzzle in the field of biological electron transfer is the conserved iron-sulfur cluster ‘motif in both high potential iron-sulfur protein (HiPIP) and ferredoxin (Fa) active sites. Despite this, structural similarity, HiPIS reect oxidatively at physiological potentials, whereas Fas are reduced Sulfur K-edge xray absorption spectroscopy uncovers the substantial influence of hydration on this variation in veactiviy. FeS covalency is much loner in natively hydrated Fd active sites than in HiPIPs but increases upon water removal; similarly, HIPIP covalency decreases when unfolding exposes an othernise hydrophobically shielded active site to water. Studies on model compounds and accompanying density functional theory calculations support a cortelation of Fe-S covalency with eaze of oxidation and therefore suggest that hycration accounts for most of the between Fa and HIPIP rection potentias. tins conning Fea ches catas tbigoesinnunas (These lcs hhave evolved into nvo classes that have differences ia their electrochemical potent hil potent iron-sulfur protcns (HIPS) and hocterial fomedoxins (Fds) (5-8). Physiological -ondtions support a rxluction potential winx ng Rom about GOD to +500 mV (9) Spectroscopic and ceetrochemical_ measure ua scioncemag.orgments have shown that the reting state in both clases of protein is [FeySalP and that HiPIPS react by oxidation 10 [Fes nu (vith an asso cafed oxidation poser i rom 150 mV 10 350mnV) wberon Fare redacedo Feu)” ‘ouith an associat redaction potential ranging fom 250 mV «© 400 mV) 10-14. The corresponding Fd oxidation anc) HiPIP retuction potsatals ie ouside the physiological window, and so the associated processes are not hiolog. cally ative, “The goumetic and spectresconic features of the [FeySq]°" resting slates are Mentical in both protein ckises (15-18), an no significa dere ‘ences ant electronic structures erent from ‘quantum mechanies (QM) (9) and molecular mechanics (MM) simulations (20, 2). Thus, the difference in clectchemial bchavior appears arise fon environmental fetes atthe respoctive active sites. Crystallographic data indicate that the Fe,S, cher is buted in a hydrepbobic care ‘oepannet of chan. Starord Une, Stato, Ck ‘9a0s, USA. “Dgurnwet of Bens ad Weeder Blo, Unbesty at erg ens, Gh 30602, USA“ erty of Baga, Camps Uva Sine ee inert ts vente, 079 47023 Cea, ti. Depa ‘matt Boye scenes rauate chock sence, Oka Unies. oat, Osta S600, an rtrd yr teen Yadation bray SAD, Sf ea Re ‘eto, Cones (IAC, Sanford Une, Nero Pak CA “To wham couespanience soul be addesed. Exit hoton@sslsaestmiec edu KOM) hedran@sssle Sanferd eds (890)edverdsolmronasinioraeu E15) — cepa Cina HP) § 10| — ode Compioe 2455 2408 2467 2468 2069 2070 2a Energy (eV) in HiPIP with ~five conserved H-bonding interactions: to the cluster ligands. from the Dpackbonc, whereas Fas are exposed to solvent, ‘with eight protein H-bonding interactions, and have a highly conserved CXXCXXC loop (outer € is Cys and X is any amino ac) (18) Structurally congnient syrthetic inorganic com- exes fawe beer rqprted Fr the three oxidation tales observed in HPIPS and Fas (3, 22, 23). ‘Whereas the oxicaion of yrahetic (Fe,S* com pees has a similar potential to those abserved in HAPIPs, tbe eduction of the [FeaSq)?* mevet ‘complex fats IV more newatve porential than that of the Fas (24). Holm and co-workers reporied significant variations in the [FoyS.)” ‘redaktion potential forthese complexes with yary= ing solvent cietectri, nature of the thiolste, and prexence of charged residue near the cluster: how= ever, he elution potemiiak of these models were sli very negative and mostly below the physiological range (25), Fhetenttic and MM caleue lacioas suggested that lage dipolar interactions from pei resins anc intercalte water me cules in the Fa environment shit the [FoySa>" redusion poteraal by 14 V above that EiPIP ‘while atthe same time distavoring oxetion by shilling tht povential 1.5 V below the core sponding HIP couple (21,26), However, 20 fie there are no experimental data that determine the telative coninbutions of these effects on the eke- ‘tochemical potentials of these proteins, irwet determination of electrochemical potentials in pesturbed (lyophilized, unfolded, zo Ronormalized Absorption 8 pire airs are Fig. 2. (A) § Kece xray absorption specta of (NEL) IFS (SE) (blac, HIPIP from C.vinosun (blue, HIPIP fom E.halopita J ight blue) Fé from Bt (ced) and Fa from Pf (range. (B) Pre-edge region ofthe absorption spect for the model (blak), WT C. vinesum HiPIP (bun), unfolded C. vinosum HiPIP (dashed bronn), WT B Fd (ed), and yopitzed Bt Fd (dashed green). (C) Kay absorption spectra of (NBu)s(FeqS{SPh] asa solid (Bact) and in acetonitrile (geen) N,N-dimethylormsmide lve) and R-methyllrmamide ed selatons. 3 og wunuscioncemag.org REPORTS I etc.) protein active sites using volian techniques is challenging at best Our previous results have shown that, in a scien of model ‘complexes, the elecroehemical potential variss linearly wi Uae extem of Fe-S eovaleney inthe laser, which ean be dreely mera eqperi- mentally with use of sulfur K-edge ray absorp AS). In this siudy. we ly he sohert exposure to the active site of these prteins in an efor to quantify the impact of hydration ofthese active sites on their eketenic structure and ele shemial potenial With use of § Kuk: XAS, we previously ‘obscrved a lange decrease in Fe-S bond &o- valency in Fa relative to TEPIP,alxgh ‘origin of this diffrence was not detennined at that time (27), Sulfa K-edge XAS is a dive, cesperimersal probe of ligandme bond co sakency 29), The primar featur at theS K edge the S Is — dp triton. The lower energy transitions from ligand Is orbitals to unoccupis! metal-hased 3 antibonding obras owed, but they obtain_absomption ifdhese 34 onal mix ith Sp orbitals hecause thesulivrbasad Is — 3p(AL~"T, whore represen the ombial angular mementur) tra sition is electric dipole allowed (24, 39), This pre-alge intersity & diccily properional the Potent of Syp mixing in the Feyy orbitals and hence provides an expecimental measure of Higad-metal bond covaleney (87). This method vsas previously used to analyze the cketcnic 2485. 2466 2467 2458 2469 2470 2471 2472 2473 D474 Energy (eV) Energy (eV) SCIENCE VOL318 30NOVEMBER 2097 14651466 PORTS structure and boning of models and active sites ‘of mononuclear Fe-S (rubredoxis), binuclear FesSp pinch feredoxin and Ricske protein), trinuclear (bactova!feredoxin), and ternuclear (UEPIPSarn reins) proteins 27, 28, 30,22) Jn mont eases, the FesS bors in the proxin active sites were found to be less covakent than those in te stucturally congrdentcomespending model ecmplenes (30,33, 34), “The S K-edge spec of (NEL FesSUSE 4) (Fig, 1A), whore EL ingicates an ethyl grup, serves as an appropriate reference forthe FeSy chusiers in proeins because an aliphatic ethyl thiolate Figand has a bending interaction wth the ‘lusior similar that ofthe esting Hgands in the prosins fy absorption spectrum (Fig. 1A black) shows a pro-cas peak at 2470.2 ot. Previous $ K-edge da on clusters with S substituted by Se and RS" (where R isan alkyl ‘or an aryl substtucat) substiuted by C1” have shown thatthe RUF Sy: the FOU Sei Table 2. Fe covalencyin FesSqand proteins end ‘over 180% total Fey ased eritals, where ach p-adge vansitions are cemeral at 24700 eV and 2471.0 \, respectively (30) Fom fis tothe black specu in Fig. 1A ana its second derive tive, thopeck intensity reflects 168% diols and 480% sulfide 3p characters mined into the wie ‘eeu valence orbs ofthe Fe chsk (28). Inthe following texte otal intensity ofthis peak, ie the otal valency ofthis model comple. i twsed as a reference for the decreases obser the proteins. The aldionalrising-cdge at 2472 w 2473 eV presents the background Si, + CSee trasiions for all eysicine and ‘mathionine residues ip dhe model or rin (hus, these vary between difens The XAS dota for representative HiPIPs fiom Civonuativon vinosum (Fig. 1A, blue) and Evotiontodespire heloplits 1 (Fig. VA, light blue) show pre-edge imensitis very similar to cea other and only slightly lower than tha of the model complex. The fits to the HiPIP data indica tha the tal Fe-S covalencies are 95% amodel from K-edge XAS.Covelences were summed fof the 18 unceeapies ortitals i worth 100%, ovatency (HS, Relative Seana Sie Toul ‘mode! complex WvetalFessuSEt) a0 210 «168 27 648 = 30 200% rnosum (HIP) 46823 15629 62421 96% C rmasum (untldee) = aaa s 16 1a = 8 sais 23 39% -halophila | (HPP) 468 £9 Mg 27 63217 95% ra aor 1622 557 = 25 BEd Qyophiiee) ‘etl 528 id 396218 a8 = 16 A - B * 2. Computational structures simulating (A) solated model comple, (BH-bonded model complex, (© WibtP active sit the NHS distance ae indicated, and (D) Fd active site (the CXXCKXC loop of te backbone is highighted) 30 NOVEMBER 2097 VOL318 SCIENCE 10 96% of that ofthe make complex Table 1), In contrat, the S Keedge data for representa live Fs frcen Bacillus shernoproteostcus (Bd (Fig, 1A, wd) and Prococens frosus (PP) (35) (fig. 1A, orange) have significantly less pre- ‘de intensity than the model complex (Fig. 1 back) and the HIPIPs (Fig. 1A, blue and light bie. These data indicate that the Fe-S bonds in Fs are less covalent than these of the model ‘complex ana ofthe HiPIPS, The fies intensities fr the Fal give otal Fe-S coyalencies of 86% 1 85% of that of dhe model complex. Both the Swine and the Site Covalencios ar sii icanly reduood in the Fe active sits relative 10 those of the HiPIP active sites (Tablo 1). “The weakening of Fe-S bonds in Fds relative to HPIPs could stom tom differences inthe H. bonding ftom the poise backbone (Live PIP conus oight fn Fi) as well as differences in solvent exposure (ILPIP is buried in a hydrophobic cor, and Fd is soluentexposs) (U0), To gage the effect of hydation in FeS ‘covaleney, we subject a lyophilized sample of BL Fd w XAS, The data (Fig. IB, deshed groen) show a marked increase in preadge intensity relative 40 the pr-edge of By Fd in aqueous sclution (ig. 1B, ne, alwough the ssing edge remains relatively unaiested (for details, se i SLA), The reduction ofthe preedge itensiy in hulled aqueous solution is reversble (ig SLA) and indicates that the Fd chaser forms TH bonds directly with waer molecules, thereby reducing its covaleney. Quantiiation indicates ta the tual covakney increases fem 86% in Br Fé (Table 1, wow $) 10 95% of that of the medkl comples upon lyophilizaion (Table 1, 6). Ina complementary experiment, the C vinonen HiPIP ws wated anaerobically sith 4M guanidinium hyroshloide, whieh has been shown to partly and revenibly: unfold this FBPIP, a» expese the elusir 10 water witout furer chemical mosifieaton (36). The XAS. data for unfoided HPP (Fig. 1B, dashed brown) show a deerease inte prea intensity relative to that of foklod C. vinosun HPIP (Fig. 1B, bhi). This process is reversible fig. $1B), and the similar ising edges between these spocia Fig. 1B, blue Hine and brown dashed Tine) indicate thatthe cluster in THPIP remains inet daring this conversion. Fits to the data show that he total Fe$ covaleney decreases from 96% fof the model in wildaype (WT) C vinasum PIP co 89% nthe uatolded protein ane thatthe FegisSaid ad Fe-Sgitae bond covalencies are ‘gully aMlectxd The result of unfolding the HiPIP prokin is cemplementary tthe Iyephil- zation oF the BF pretein,with both indicating that hyckationof the clster isthe major source of redttion of Fe-S band covalency To gauge the relative impacts of solvent ileeic and H honking independently on FoS ‘ovalency, we disohada well-chanctried FeSy rod comple (NBA FesSuiSPhi, here Bu is butyl group and Ph isa phenyl group, in a ua scioncemag.orgvariety of solvents with differnt diclecsie con stans(acotonarile and N.N'dimethyliornamide, DMP) and capacities or 1 bonding (Vnethyl formamide, NMF), The model complex is stable inal tue solvents, The § Keedge data forthe solid (XBughl FeyS(SPh¥] complex (Fig. 1C black are very similar the data forthe complex in aceionitile (Fig. 1C, green) and DME (Fi 1G blue). These ests sugzest tat there is ite impact on covaleney from changes in dielectric ‘constant around the eluscr Hossever, the data for the sane ccnp in NM (Fig. 1C, rex), wich ean act as an H-bond donor, show a lage ecrcase inthe pre-e incrsity. Fist the dat ineate tha the total Fe eovaleney decreases by 15% in NMP ‘fig. $2). This docease in covaleney duc to H bonding from solvent Parallels the roults observed on petubine water access 1 the protein active sis tyophiliad Fdand unfolded C. iaasem HiPIP) ard confirms that H bonding ofthe water to the Sigands isthe diet contribution to this fect “The above resus were compared te density functional theory (DFT) calculations on the model comple and protein active ses The (NBu,):{FeyS\(SPhig] complex. was modeled with an [Fe,Su(SMedy/? where Me indicates 2 methyl group) cluster (Fig. 2A) fo which euht war molecules were added Fig.2B).The HiPIP active site was approximatod With 3 amefecule who econdinats were taker fom a hhigh-esolution (12 A) enystal structure of the C sinasuan protein fom the Protein Data Bank (PDE identifcaion code ICKU) (Fig. 20), A | 40-stom aude of the B/ FU active sie wil and ‘without seven 11,0 molevales was. sinilarly simulated (PDB idenifieation code 11QZ) (Fa 2D)G0. ‘Upon forming H bonds to sight water smoecues, the simulate [FeySy(SMey)? come pound evidenved a total Fe-S covalmey rede tion of 10% Syp tuble SI which reasonable aareement with the experimentally observed decrease of 15% Shp in an H-bonding solvent (vide supra). The ‘caleulations ako indicate dt both the FestSwtide and tbe Fe- Sevetuc bond covalencies are reduced as mi in the experiment (lig. S2B). The on en the BY Fd (uithoat HO moe cues) and PIP active sts show 3% and retin inthe Fe hon covalency (able SI), respectively, mcative to the reference model ‘This results consisent withthe limited $% and 4% dacrease of FeS bond covalency observed in lyophilizad By Fd and PIP (Table 1, rows 6 and 2), These decreases are lange localized in FeSintac mics (abi: SD, which is con sistent with the lager number of H bonds do- nated w the Spite atoms teatve 10 the Syase atoms from the backbone. Whon the amide REPORTS I groups. inthe simulated backbone replaced with ester groups (i.e, the dipolar interaction of the CO is sil presen, but there ‘Sno Hi bonding trom the NAP groupe), no dccrcase in FeS bond covakrcy is observed (able SI, row 4), Including. seven H,0 molecules in the selvea-ewposed side of the FA mock shows a 10% decrease in FeS co table SI. row 3) etative wo the model cempiex, wich is close although less than) the exper: imemully observed 15% decrease in F valenoy in Fés relive to the model complex Table 1, ww 3). Thus, both the experimental and theoretical resals show that Fe-S covakney is a sensitive probe of electrostatic interactions present in poten ative sites. This decrease of covaleney ‘due H bonding would correspond tothe soe polation term in a total enerey’ calculation (37, 38). In both HiPIPS and Fds, 1 bonding fiom the popide backbone eases only a imited rechasion in Fe-S bond covaleney. Rather the hydration causes two-thirds of the lange deewase inthe FeSbond covakney tha is observed inthe sclvent- exposed Fd ative site relativeto an alkyl ‘hole move Rolaive to the model comples. [in DMF scaled 0 the onal hydrogen eleetode (NE). tho reduction potatial of Fal isa from 1100 A a (Pea oxaion ra 0 | oars es 40] Sane es Bes oo 0 = 2 is) 375 no = 0 1025 = “83 et 8 a “9 wo 6 65 87 60 OF % 8m Fig. 4, Schematic representation of [FeaSel? reduction potential tuning by cesotvation. WFesSaP By wunuscioncemag.org 8 oe 7 Go 101 09 Ws 107 13. Plot of total Fe-S covalency as a function of (FegS,P°* (A) reduction potential and (B)ovidation potentat versus NHE for a series of [FeaSe(SRll ‘complexes (sold black) and 8 Fd (solid ed. The open squares represent the @ oo oe To 6S vor vos 106 107 predicted (using covalency) [FeqS4l2* electrochemical potentials. The [FeyS<)?* {eduction -enidation potetil for the reference complex [FecSuSED i indicated by the horizontal dashed gray line SCIENCE VOL318 30NOVEMBER 2097i REPORTS sn 10-300 mV (black and rl pois in Fi 3). Fromour pastsudis, the lorosbuc efit fof waer Fbondsd © $ Tans will doencase the covakat interaction with he Fe and weaken the Fe boas This will aise the redaction poate by destabilizing the exidved ste mare than the reduesd! site 40), An estimate ofthis effect on the rection porenial of the FeaSy chusiers is given in Fig, 3A, in which the eovakeies deve from S Kae XAS imer- sits for a saies of [FeSuSRyl> compleses with varying thats (oli black squares) ate plotted relative to their coresponding [FeyS.P° foncelocton rection potential (eased in DME scaled to NIE). This plot shows a linear conlaton at! the asgative slope indicates hat itis easier w reduce the [Fesal™ cluster athe FeS covaleney decreases G). For the FesSe ‘hier in HPI, the experimen eovalency de- creas by SY retive 1 the model which eo topo AHO GV ah Ue [Re recueton potential (open blue diamond) Ce sisent with this exrapotation, the oneelecron reduetion of the [FeuSsP resting fom of HPP fiom C snaim has been expoimernally esi- mate to be 910 mV, which outside the ps inlogical window G%. For Fd in aquooas solution, the clvtostac fect oF the kal HT bonds on the thiolate and sulfide ligands ofthe FS luster decreases the covaky by [5% relive fo the medel come plex The plot ip Fig. 3A gives an estimated change of the redtion potential of 450 mV associat with this FeS covaency change. The remaining 380 m\ contribution 40 the observed reduction potential of Fa would oak fom the addtional sailiaton ofthe reduced stae by the asymmetric eksiosatc field sound the IHbondad elise: One fis of the cluger fs expose the high dei of wae and th other tothe dipols inthe CXXCXNC peptide kop. which is highly conserved in all th back Fs (Fig 2D, highihted bo and fi. $3) (40). The CXNCXXC four to make the dominant contrition tothe pretcin dipole stabilization tem in the cal lions of Wah and cosvorkers 2 For the IRPID [FexSq)" onidtion coupe, an analogous pl oft measured al Sp ch terforaseriesof [FesSdSR) compleres with Aifizen thiobee Ryards versus abe (FEW ‘oxdtion potential (//, 42) abo shows a Tincar concation (Fi. 3B). The pron data pois on 3A}, The predicted [FoySyP" oxidation pots for {HPP (Fig. 3B. blue open dianwoad A -265 mV) shows & Himited shift bseause of the 5% decrease in covaleney ative to the alky|thiotte mod couple (-200 mV). Aer natively, the [FesSiP* oxidation potetal inthe Fa protein active site is lowered by 700 mV" relative to the model comple, into the nonphysilogical 800 to -900 mV range (Fi 3B, solid ed) (42); 300 mV because of the 30 NOVEMBER 2097 VOL318 SCIENCE clecnosaic effet of load I bors which deorene th covaeney: and 400 mi boxe of nonlocal protein creates Ths, te same tors ht sf the |FeySP seduction potaia in the Fl active se by #600 Inv relive 10 PIP Coen -900 HV to —300 binging i nw piso shi he [Fe,Sa? oxidation potential of Fes by OD nV relative HPI (om 350 mV to 880 mV) and oof physical mage ‘Our eels indice tht H bonding fom ster i a seveacesposl Fd site can tune the [FeySi? reduction potential up by at ast $300 mi This phenomsian may. prove regulation anochanam for esto taney Fe laste ptsing As example, Fs ae fatale the eduction of formyl a a ‘nin methanogens: bacteria (43), an DNA, Binding & proposed w lower the [FecScP™ reduction potential by >300 mV in endo- auckase IH (44). As illatrated in Fig. 4, intration with an elim transfor pari tay kad to apatial devotion ofthe at site tat ean shit the rection oF oxidation potential ofthe Fa [FeySy)* cher Indewthe tinge saris ae of docirg (1000 A) an the lone contact between the active its (10 A to 12-A) in a miclear magnetic nsonance structure ofa posi oe peiencemples ofa Fl wih is redox pater extocome cas (om Desujoutro. aorvegicm) (48) ane conse ‘sth this possibiy. This, solver ting may Play an inpersant oie fm prowinprocin and Drtedr DNA interactions, References and Notes 116.59 Ed to Sule tei, ok. Wf Met ons Un Sgr We, en Yn, 3982, 2.5. Clee RH Holm, en fe. 104, 2085, (aco 5. Psa, RM, chen Re. 208,527 (2008. X Fura i Hercbook 0 tenets A Meese Hb Fon, K Misha, Es ey, ew Yar 2008, p58. 5. One eee hasten rprte havea ptr 50 0 6S. Goshen H.R Fetal FA Asn, 3K buges J Bk Chon. 273, 3514 998. 7. Sh lh, Bs. igh lB 70, 95 ‘ase. 2 ma Pet 8204 9. A pteabalnthreprt re erence ate tthe uta yee ectoce OF) AB. CWE, te ee, Pe ts Ace Se USA. 6, sae agra. LH Sle Ll, E Mind Sere 27,653 taser. 12 L1G Pou, 66. Lane J. Hose, 6 J Am. Che Soc 336,183 1999. 18, Goa Pou, J An Gen. Soe 197, 7513 am. ACL tape, 66. ete, Moses, 6. A he See 189, 9757 99M. 35. G Bikes etd | A Cher So. 13,2055 son, 36,5 Geman KA Mo ven A Gewith 1. Spi, Am Che See. ‘sen. {VAR Oiejoinon ea chin Bigs. Ata 48, essen. 70 1B RP. Sean LC Alen ©. Cr, Bi Chem 256, 5052 We. 19. A Toes Tel LNoeeman D. A Cae, ho. Ser. 423, 183 GOOD 20. ngey G/M ese Ua, F.] Steens, A Woe Bol Chen. 267, 23625 (992. 21 GM even, A. Wane). Steptens echemity 3310911 1990, 22, 1.0 Ml, J A, Chee, Se 107 096 (ies) 23. 8A bel 1 Hestor, RH. Holm 1A. An Gi 95,3523 0979) 24, RE por, Gc Pps. . Hae Ameo 1 An Che, 165, 7250 (551. 25. C Bou. |W forbne, 8 Sea RH el, tr. chin Aca 300-302, 692 C0. 26, Pl Sees, 0. Tale Mase Cem Re 36 busine. 21 se a Am. Cem. Sc 423, 4839 200». 28 EL Slmor, & Hed, KO. Hobson, A.D. FL Seean Coor Chen fe. 29, 97 (O09. 29 Foes 8 Hed, KO. Hoon Sean, Che 38 854 (999 50 Ge, 8 Fema, KO. Pods, Semon, ‘he Chem fe. 33,659 GOOD ‘Onan ard meats arate on Scene nar. 32, AD el Am Che. Soc 128,383 00. 38. M Usama, 1 Yorod, 1 Ouenra 5. Rew A Malan are Chen 35,73 (1936 34 A Dey al An Chea, So: 327,126 O08) 38, Me fest From Fe coca ty on exentae Usp wes tone hes an Ab' "Cr at, rich is bee Curacenzd faefu seis ‘tsiaatg thet eas he Case, has been ed ot 34 D beta eat, ches 38,4669 99) 3. | Rlran Weel Zs rem 8 20,553 laser. 3 lor, A Wane Cone Cem 13,199 ro 391A Mees 8. Eds, Mapes TE. Mee, fr ec. 252614555. 40. CW Cae Bid Cher 252, 7402497, SL FA Amster, HA. O. PIL 1. Nr EBS et Ao, 28 GPK. 42, HL Bark eo, fom Chem. 3032939 ‘3 U Dapper, Pg. Miele ds Mal 7, 2 02), 44. ER Boar, AL ington, Gil 5.5 Dai, 1K Bata fc. Mo Aca Sc, USA 300, 22563 (axon. 45 X Mei et, Bt 39,2530 2000. 46 Ths eeath ws spp by HSE us (HE 044¢308 15), 91209 KON, ord (E0527 GAATLA. AD. rece an Even Meta Feit tam Sarfrd Unive) SSL operation are ‘apport bythe US. Deprtneet of Energy, Ofc ot hc tneay Scene The SS Sct! Maker Balagy Pera supe by the Mi, Natal Center br Resench Rees Bien eordeay gam ad te US. Depatnet ef Ee Dee of Belogial and rwroertal Resear Te proetcocrbd ma npoted gros unter PAL RRO02209 Fen the sel Cert for Beer Resouces WCRE,a compen lhe Nad tarterts resol the espensbliy a he baths and Sonat csr epithe afi en of ERR NK] oven aclowledgedo sages nthe Supporting Ontne Materat swoncinaog ogee fll9205 655146403 Tt dec Mis 5.1058 Table 51 Felerences 12 by 2007; acted 18 October 2007 ua scioncemag.orgREPO! . A Fed and 200 Nady sc 7, an its 3 The Obesity-Associated FTO Gene peek lagiy camcte! os peaern nie fo mecoliopaneiscey led tat Encodes a 2-Oxoglutarate—Dependent 2 ms--%sisin sesermom 4 = iehdne DNA roa: iy wel pool a Nucleic Acid Demethylase postrinslaional mocitications, for example, pro fe lpdeotind i sats tert ‘Thomas Gerken,? Christophe A. Girard. Yi-Chun Loraine Tung,> Celia J. Webby." iron [Fe(I)] asa coficter, we oxygen and, alae Vladimir Saudek,*+ Kirsty S. Hewitson,™*f Giles S. H. Yeo,?+ Michael A. McDonough, t+ always, 206 as cosubstates, a ‘Sharon Cunliffe,*+ Luke A. McNeill ™*+ Juris Galvanovskis, *t Patrik Rorsman,? Peter Robins? Cis and carbon dioxide as by-products xavier Prieur,? Anthony P. Coll? Marceila Ma,” Zorica Jovanovic,? |. Sadat Farooq)? To detennine whether FTO 4 Barbara Sedgwick,* Inés Barroso,’ Tomas Lindabl,® Chris P. Ponting," t5i) ‘genase, we expressed the murine Fio gene Frances M. Ashcroft,”381| Stephen O'Rahilly,*5ll Christopher J. Schofield"+81, Escherichia coli arx\ purified N-terminally hexa- His tagged Fo (70), Some 20G axyuenases cat ‘asta a the 70 tat mas and best asl gen are sated wth neeased by masse HBB! Pe (0), Sone 20% aaa feds i una ews we show hy Kchaoennts milpas tat 0 acs merce meas Onaga Tee eine Fol: aul erence apenoe mygrome Rehaiaa jemminseiae nace: foe esters atest the Fil and 206 depenertdenetyoten of emchybnyrine i shfestinaed Oka, wn oemoctwrcupll tuo, 206 wna Couonhaapadeilr # actors brutes oeeaeniks Casunewane rrr aeabe Were meme nara puatiielllnauckicadd deceit Relecaicem Gesecuswiartecna cas oli cfaou ime Chesbon Sees 1, ces of oh-you sic locas Wl Fe esr MAA GORWO Wat cine he GR, Soest ceatin 200 eae, heey Ripon ia eanace ers a keorenca aie Seca eee cree rod sk Ux Tod by ody sed AS Sue cn be iene eae oe ina a FAS ientaytn roel mean: Sakianan mena cmmeneesaiag mo sumrereenGe! Fined to Purened ft mas Uieccoliee! ae Hert af Sek FTO aiseae Arar 200 expos vi cetenie aemwablamon ian COGoyounibrueinitactgme lane moan Ge FIO agree fied BSisissecttiee vm nie Seve headed DSM ics w teva cone AB iar and ads wih A6% of sn pe Semis tee evils va fe te Terms cs (2) Ths Fe gos west asd Snow phoaypernts ferme LEM don conti dona bx SDI al Naming eon o Fal an} Pannen Senomiytwnch try ane Ofer Gee fer Irate Speen fogs. Save efor 1? an ave Oh Ome omar ttripioyy, Atay ad Grete Une Os fat tne Oto Coon O€ SFL UX“ Cnty etl Resrt basin sta 82 000 Ue "or Ua Hagen Cee, he Ose ‘Stence fuk Or, Oi Of 404, UC “Oxo Corte Figs 2. Multiple sequence alignment of FTO fom human (ome sapien; Hs), meuse Mus musculus; toronto tecxcogy ad teats Chott Mn) and green agaelOsteocccs owt: OD) with Escherichia col CE) and Shewaneto woody Hepat Oxia. Onn 07370. Canc wach UK AB and human ABH and ABHS. A comparkon of OX FIO wih the enredundant poten sequence {oon msec mse Cte Hl aw Set) tonne at the Hatonal Gere fer Bwtecrolegy Ifomation wing PSLBLAST (29) reveled rn, O46 HD, UC abe Dest Sianeli (E< 0008) bene mat FO, baer AKB, as human Remo, ia saan mete Se sig oman, Cav yo rations,Consred ress highighted in red ae hie and crboofate (sp or) Fe Pimbagy stony a Gaets teniy of Oe bndingvesives of 206 ongensses Gf-220231,kp-230233, and H-308307 in marine Mofuman Se as Ra, Oi. Sx OX1 308 UR FO, respecte), a wel an agine 315/31), whith ins the 206 C5 carbonate Ina 206 “Te ahs cenit ely swt tnygrase subfamily U, 8, Te indrs and arews represent ces and stones, respecte, {Moe mtn ond cgay fs oo {signed as ina col strate of ABI3 29). Secondary suc nyelow represents re ternal eve mths coved cay 1 fu ek tego, vive sands repesont an asigned 09) substrate birng Ue for ABH, gon sands skled ice ston ened ea ro ttn oman ameras ery the eight Bonds at lon te conserved double ran ded beta of ita whom cevespnderce shold be addessed. Ema Me won connec Hd odteick COM the 206 axyyenases Gerinlo numbers: human FIO: 122937263; mouse Fo: 6753916: oren algae Gjagoracek (A) wIDagnecsMomacu GO} FTO: 116060758; human ABH2: 48717226; human ABH3: 21080275; E coli KB: 113638; S. woodyi estoperscheflég@ahematcal (C15) ‘AKB: 138070714 wunuscioncemag.org SCIENCE VOL 318 30 NOVEMBER 2007 1469PORTS ABI (AIKB homolog) family (Fig. 1). AIKB is We sereened potenti Ro subst, ine ere abo inhibitors in the 20G uncoupled 4 DNA repair enzyme that ropais cytotoxic cluding a syathetc single-stranded L-methyl tumover assays (Figs 20) Lanctiyladenine (IameA) and 3nethykytesine adenine (L-mcA) methylaied oligonucleotide, Using a guid chroraiography-mas spec (Game lesions by mathy! group hivdroxy Ly.9 methylated histene 13, Fyponia-inducie wmitry assay that dinccly. monitors. DNA followed by a reto-akiol mong the factorta (IIF-1a) subunit famens, IxBu, demetiylaton [without the ased for radilabelad various humm ABH, only «Wo, ABH2 and egenzyme A derivatives, and other known (G>-pubsres or coupled assays (A), we dene ABI, have boon shown to exhibit DNA de-subsertes of human 20G oxygensses (0) onstrated that Po catalyzes Fe(ll- and 206 methylation activity analogous 0 that of AIKB Only the I-meA methyl! oligonuclewide depen DNA cematyatcn, This acivty wa (U2, 13). ABIL 2 and ABUIS are ubiquitously 20G above contol levels stimulated by ascorbate, as observed forether 206 pressed. and ther expression is not rion to sivity was inhibited by N= oxygens (0) «Fig. 2). Signticamly rekicad be altered by physiological stimuli foxallelycine, fumarate, and succinate, which turnover was observed when the reaction was 7 z* 24 i. ie } La - a mer) tt “PRET ses wears Fae oe | % ava Fig. 2. Fto is a 2-oxogutarate-dependont DNA demethylase in vitro, (A) The oxygen contol reaction was carried out in an atmosphere of <1 Og: Denethylation of 1-meA is dependent on Fto in 206 decarboxylation assays; (© Inibition of Floatalyzed L-meA demethylation in 206 decarboxylation (B) Cofzctoxcosubstrate dependence of FTO activity on a Smet substate assays. all assays were performed in tipicate, at lest, wth error bars show by LC-MS, Data shown represent ratios cf thymine to 3-meT in s-DNA. dencting 45D. nar a] te 100) tm wpa Fig. 3. In vito activity of Fto with s-DNA substrates. (A) Demethylation of s-DNA substrates by Fe. UCANS esta for the inabation of syrthetic 18-nuclectde otigomers ofthe form 5'Timetyated bose TTTTTTTT-3 with A m= Fo, cofactors, and cosubstrates: positions of expected Tae Ties Tne Ties Te peaks for demethyated substrates are inated by dated bes Smaler peas at higher masses than vacant peaks probably are fom ta" and K* aus ofthe methylated oligonucleotides. mz = masso-charge ratio, shown i units of Dalton. (8) Stekhicmety ofthe to teacion.(C) Release cf formaldehye from metyited poyid ond pol). FTO and ABH were assayed foc demethyase ect by incbation wth [CF methylated poidA) or TCl-metiyated poly Rta counts per minste (9:1. 1000 or 800, respecte at 37°C for 25 min. Release of ethnel soluble Pchiermaldehyde was monitore. FIO -©>, -@; ABHS -2-, ~~ [C}metylated po¥ida) -C-, ~~ P*C-metiyated pay) -@-, An the absence of 206, ne significant activity was detected the ethanol soluble materia released by FTO or ABIES was at backgroud level (ess than 100 cpumd, 1009 sa ss: 1470 30 NOVEMBER 2097 VOL318 SCIENCE wwscioncemag.orgpened under reduced oxygen conditions. The production of suecinate was verified by 'H nucle magnetic resonance (44 MIL) analy ss and that of formaldehyde was Ustivaization with pentahuoropi “To tes! the predicted role of the assigned Feil) binding and 20G S.carboxylate binging resides, His¥04 and Atg-313 alanine subwttue tion mutants were constructed (10) (fig. $2) The His-304 mutant showed reduced 206, mover, shereas the Ang-313 masta ablated activity (Fig, 2B) consistent with observations DG oxygonases We next inves vty with sing stranded eligcnelsoties (s-DNA) matted 1a single pusition: L-methyladerine (IemeA), Leanethylguanine (LmneG, 3-methyleytosine GemcC ana 3nebyitynine QomeT) (Fig. 2B) Under the assay condions at pi 75, Fo exhib. ited uprerence for SaneTover enc or bine im SCDNA; ImeG was not an Flo subst ene sub tal Flo a (Fig. 3A), The preference of Fo for strates was ako observed in assays measuring the release of formakichye from randomly methyl ated poly(dA)anel pol(aT) (Fig. 3C). Consist with previous reports (4, 15) we found that recombinant ABI demethybvod Linc, with only very low levels of SneT demethylation observed under our conditions (Fig, 30) A proc with nc aid deta jt be expected to losalize to the ool Inca, emnoeal imagine of COS-7 cells «fem simian kidnoytanssed with a plasm encod: ing for yellow ferescent protein (YEP) tazgad Fi or with YEP along, revealed that VEP-Fo is concentra in tbenucieus, whereas YFP isprsent ‘only in the eytopksn (10) (Fig. 4A). YEP-Fo id not cobalize with mitohondia Fig. The FTO SNPs associated with ‘atx may exert fartinal eflosts dough ‘expression of FTO mRNA. IF FTO te ults nerey hornostai mgt be mone highly expressed in tissues involNed in the contol of ey balance and be influenced by nutritional REPOR signals. We examina Fo mRNA levels in mu tiple murine tise by quantitaive reverse tran scription polymerase chain reaction (Fig. 4B), ‘Consistent with human data (2), mouse Fo mRNA was detected in all tues Studd, with the highes expression in the bran. Within the Iai, high Ieyels were feud inthe hypethalams, an area tha plays 2 key sole in the contol of omaostasis In stu hybridization of hypo nie sices revealed that Fro mRNA was highly express in acuste (ARC), praventicule {PVY), dorsomedial (DMN), and ventomedia (VMN) nucle, all sites of ene importance foe the control of ener To determine whaiher Flo mRNA expres: subted, we examina Fro sion is nuttonaly mRNA seralisected hypo tuck (ARC. VMN. ax! PVN) fin the groups ‘of mice (ively fosting, fasta for 48 hears, and 48 hours fasted ueated with daly intapertoncal injections of leptin, respectively). The kin supplemented group was included to examine we art B 5 @ ® GS > to Fos s fou 2 oz ° ast Leptin NeB NSS 4. pression stties of Fto protein and mRNA in mice (A) Subceluiar tocalizaben of murine Ro in COS-7 ces Confocal fluorescence images of CO5-7 cells expressing YFP-Fto (or YEP show YFP-Fto localizing to the mucus. Nuclel nere visualized with 46-iamidino-z-phenyincole (DAPI) staining and. mitochondria ith MitoTacker. Coloclization of YFP (yellom) and APL (blue) in the merged images. produces a white signa. (B) Relative expression of Fio mRNA in diferent tissues. Ber giephs show the relative expression of fto mRNA noemalited to fs acin across 2 pane ofciferent tissues. Data are represented 3s the mean (SE) of sx independent mice per tisue. (©) In sit hybridization of murine Fto in brain. PUN, paraventricular nucleus; VIAN, ventromedial nucleus; DMN, dorsomedial nucleus, ARC, arcuate nucleus scale bar = 500 ym. (D) Nutritional regulation a Fio mRNA exression in ARC. Bar graphs show the change in expression of fto mRNA inthe arcuate nucleus of the hypothalamus inte fed, fasted, are leptin-treated-while-fasted state. Resporse is expressed in tems of fold induction of the fasted and leptinveated expression over the fed expression. The F value was calculated using a two-tailed disvibutin unpaired Students t tet. "P < 0101. Data ave represented asthe mean (eSB) of at least sx independent mice per group. SCIENCE VOL318 30 NOVEMBER 2007 1471 wunuscioncemag.org1472 whether any changes asocited with fasting were ‘caused by the redaction of ercuking leptin that ‘occurs duriag starvation (16). fa the arcuate 1a cles, Fo miRNA kevls were rstused by 0? in fistod mice and wore not rescued by epin supplementation (Fig. 1D). Fro mRNA level in the PVN and VMN were unchanged (/0) 206 oxygenase-caalyzed postransstional hydroxylation i central to tanseriptional neat Jaton in the hypoxic response 7, 18), and 20G oxygenases cnalyze histone demethylation (U9). The catalytic activity of FTO may simi larly regulate the transcription of genes involved in moubolism by nuckic acid demethylticn, Altraativels, it is possible thx FTO, as pro Posed fr ABIDD, can at asa nucleic acid repair ): There is evidence that breaksown ‘of genomic repr preceses leads tw obesity and metabolic syrrome (27-23) Under our assay conditions at physilosi- cal pl, the prefered subsrate of FTO was methybthymine in DNA, 2 minor but stable lesion thats genezted upon expose of DNA to mee ylang agents, Verification of whether smithy ated hase in DNA, or an aset-aninvestigted modification of DNA or possibly RNA, isthe physiologically wlevant substrates) of FTO is 2 key abjoctve of fiture work, Ks noteworthy that alhough both Zc AIKB and hun ABI late both DNA and RNA (24, 25), i a whether RNA demethylation is ally wlevant Icis now impostant o determine whether altered FTO demethylase activity undertcs the cnhancad ft tgs asoxiated with the FTO gene Yariant and whother this alteration in achivity is associated with increased food intke, decreased nergy expenditure, oF both, The maxnity of Thums wencic dees eswovated with obesity Ihave thoi principal impact the fintion of the hypothalamus (26), and ow findings wit reget 0s bypethakimic expression snd re tritional regulation suggest that this may aso be thecaxe for FTO, Fio is iibited by Krebs eyele intermediates, in purticular fumarate (Fig, 20), as proposed forthe HIF-le hydaoxslases (27). Disease states with elevated fumarstosuccnate levels anay therefore moxlulate PTO activity Father functional studies of Flo may lead 10 now imsighis into the pathogens of obesity and possibly new avenues for teatment Roteronces and Notes 1 Cm ot, Ret Gat 39,724 2007, 2 Lanting ao, Scece 236, 899 (007. BLL Seat eto Stence 316, 381 2007, 4 Setar el, Pas Cees 3, 215 2007) Soe A Amar, ep. U. Ree Horn enone 13, 186 2002. 6 LT ttton oo, J roe. ecw, 20,684 (006, 2. eal tl, Nour 394, 805 (958 5. LAr € Kasi, Gene Bot 2 este O007.1 cou. 9 EP using (rex Becher. Wo a 39,21 0008, srt method se apnea ct re 1 SC Trek Henig, P Howse, Ts, 1 See ture 49,174 2002, {UL unc at ly Pr: Met Ad Se USA 99, 16660 (eo Expression and Function of Junctional Adhesion Molecule-C in Myelinated Peripheral Nerves Christoph Scheiermann,*# Paolo Medo,? Michel Aurrand-Lions,* Rime Madani,” viangos Viangou,* Peter Coffey, Thomas €. Sal,* Dominique Ducrest-Gay,? Dorathée Caille? Onain Howell” Richard Reynolds,” Alexander Lobrinus,? Ralf H. Adams,* Alan 5. L. Yu,” Praveen Anand,® Beat A. Imhof ™ Sussan Hourshargh™?t {AMC is an adhesion motecae that is expressed on ces within the vascular compartment and ‘epithelia cells and, to date, hes been largely studied in the context of inflammatory events. Using immunolabeling procedures in conjunction with confocal and electron microscopy, ne show here thet JAN-C is also expcessed in peripheral nerves and tht this expression is localized to Schwann ‘els at junctions between adjoining myelin end loops. Sciatic nerves from JAIM-C™detcient [having the JAM-C gene hnocked out (KO)) mice exhibited loss of integrity of the myelin sheath end defective nerve conduction as indicated by morphological and elecrophysolegical studies, respectively. In addition, behavioral tests showed motor abnormalities inthe KO animals. JAM-C was also expressed in human sural nerves with an expression profile sinllar to that seen in mice These results demonstrate that JAM i {2 component of the autotypic junctional attachments of Schwann cells and plays an important role in maintaining the integrity and function of myetinated peripheral nerves. saifamily of junctional adhesion molecules, ramposed (a8 far a6 fs known) of JANA, JAM, ESAM, and CAR, which are pe is. member of an immunoglobulin B, 30 NOVEMBER 2097 VOL318 SCIENCE specially evict at ight junctions ele contacts (J 2. To dats, human JAM-C has been tepored to be egpresd on surface of pickets and cern leukocyte subtypes, as well 14 PF. fbi i 8. Seg J Bo Cher 29, 0170 (200, 35. C Oey, |W sigan Pe Nel od ek USA 4, 1051 (000, 36, RS. ins ey Ne 382, 250 990. BW. M han et oly Sence 292,464 QEOD, 11, Potato, Scene 392 68 (2000 BRL Keo, hare 4, 92 2300. 22 | fog etal, E4B0 J 25, 2309 (2000. 21 W.Varion a re Ae end. Se USA 103, 164 2000, ean de la et ol las Bt 5,2 (2007 Meteor el, 328,315 2000) I Dag ty aL El 1,207 G00, D Wetec a,j Bot chem 260, 39848 2005 1S Fray Ty, Are Re a 86, 4305, WA Skt Center Cl 7,77 2005). 28 AA Steel, Mae Acs Re 29,298 (2000) 29,0. Servi et as BO f. 25, 3339 (205). 30 Me honk) Mave ¢ Noeine, 2 Hosea, and ‘A iotrdey br wn dass 8 Fel and 6 Got tS alls the PE rec, nd tdi sc aed D finniegtn ard. se le abn terial nel. Tr wot wor ppd yt Boch ed BlogclResesrh Curl te Medi esearch Cone, Cancer Resesren UK tapan Covmuiy Pe (SMCTSOS04US1015512013, ed the Weleame Tat FARA 6 3 Rl Sey Red Peles C1. lord af Oe a carpany that ass ela Beppe feo 2. 2. 2 ‘upperting Ontne materat swonscinamag eget! IS17 0003 Totter ete figs sito 22 gus 2007; aot 30 Ober 207 Fada th nls ie ting pp ‘al junctions betwexn endottelial ells (ECs) and intestinal epthoial cells, and hes tanly boon investigated inthe contest of inflammatory nd vascular events (/-8), In addition, JAM-C plays an important role in establishing coll poltrty and the formation of endothelial tight snctions 1-3, 3, 9, AAs part of our investigations into the fae tional role of AM-C in eakosyte wansmnigeaticn, ne detected in vivo, using immunofluoresoonce analysis of cremastor muscles fiom wildtype AWD aise, low-level expression of JAM-C in micmessel at BC junctions colocaizing with the EC marker platelet endothelial coll ahesion molecule-I (PECAM-I) (10) (Fig. 1A), In adie tion, a strong and specific expression of JAM-C was detected at discrete sites. within nerve bundles (Fig, LA and fig. SI), Another member ‘ofthe JAM family, JAMA, was also found wo be expressed in EC junctions and lealized to Jjuetions of perincual cells surounding JAM- ‘C pitive nerves (Fig. 1B). The costning of mouse spl conds (CNS) and its yental wots lie. peripheral nervous system (PNS)} foe JAM-C and neurofilament or the CNS- and PNS.spectic myelin peowins, myelin ofgoden- diceyte ehscopotcin (MOG) or pric ze (Po) respectively, demonstrated thal neural JAM-C ‘vas restricted to the PNS (Fig, IC), ua scioncemag.orgIn the PNS, myclinung Schivanna cells wrap axonal membre into distinct domain know as nods of Ranvier (24, 12), ses important for rapid saltaory condustion. To i ‘condection pep heenween the axon and the sil cells at res that flank the nodes of Ranvier (avoglal par nodal junctions) and between layers of individual glial ells (12). Our obser ste ficient posnt membrane of teased cic JAM-C and stain anni 71 idicted that JAM.C presse in Schwann ff noncompact myelin. Tes sos awe paranodal " the node of Ranier, mosaxonal bands, most likely th ws either side irom wher inwxr mesason, could be seen connec itemodal Seimit-Lamsman incites ( and schematically st S2AMI2 ‘nalysis of JANEC expression ring de ment indicate localization at paranodal janctions fom possatal day PS onward fig. $3) Costaining with nearofasein 155, a molecule olen he formation of axo-glal paranodal junetions (1), revealed 2 broader disbution Prater of JAM-C atthe paranodal regions (Fi ), Funthormors, JAMC was more distally lo cated fom the node than E-cadherin, a marker of adheres junctions (13), but cokealized with the Tight junctional mokule caudin-19 (16) (Fig ). None of the molecules analyzed were Jocalizad in JAML-C knoekout (KO) mie Band fig S28) or ‘connexin 32 (/4) ara. for (MAG) and The node of Ranvier is organized on cher sie by two Schwann cell, whose cytoplasm increases at poranodal regions (noncompact mye Fin) to frm tenminal loops tht chal ime with the svon (at paranodal junctions) and the kecral mytin kimellac (fig, $44). Immun ld shining of loagitadinal sections of WT sciatic ap junetion component myelin associated nerve fibers showed that JAM-C was located a the lateral sides of adjacent myelin kxnelie of terminal paraneal loops. However, JAMEC was not expressed in the axon or regions of compact iyelin and could not be dotted at avo-shial pparadal junctions cr ight junctional demain ‘atonal Wega np ate, mpl Clege tendon, Loe UK Wiar Havey earch te, a Seu Laon ECM 80, UK. Cea chat Unbersae (np, ede Fay, Unvasty of Geneva Gere, asele, Fane Peper ep Unt, Ovo of Meroe and Neal ath, Ine Cle Lend {onden,tomion. UK "Deparment of Celular ard Moll ewesiewe, Dvsn ef tera apd Neva Heath, Inger Caliege Landon tener, UK. Nowlgn Reach Ia Ger Reeth UX. lander, UR. “epic, Diva, Departent of eine, tech Soo of edie, Utes cour Cai Ls Ate, US “Tye authors contin equaly his wort flo wher covespendence shou be addresed Ema S.noushergh@geel ack wunuscioncemag. ig, 24 and fig. $4, A10 ©) Its intresting hat the findings of of JAM-C akg the whoke largth of prardal terminal oops a dstbution ptr that has pot been reported for other tight junctional markers which siggests that colocalization between IAM and cluuin-19 is only porta, (soe Fig. IE) Expession of JAM-C jwwetion has ao ban ported in othr ell ype (Qh which implies the the characteristics of junctional localizaion of JAM-C may be cell The ligand with which JAM-C is at present unkno'n, se sti indicated expression specific imeracting bt itis highly probable that the imraction is mediated by JAM-C/JANLC ‘erwen adiacent terminal loops, because expec sion of JANE, # major binding portner in sndothoil cel was not detected. storypi abe JAN The above inumunoclection stacy also id tified an important, though mild, morphol cal defect in JAM-C-KO sciatic nerve fibers, Perk fixed samples indicated a profile of sequential terminal myelin loops that were mis aig resting a defective adhesive contact between these structures in. ibers of simiby caliber (Fig, 28 a A789 ofthe terminal parmodal loops closely interact with the axon, in WT a spectively: for four termina 1 JANEC KO mice, e- mice with 168 and 279 Ranvier counted for WT and KO, respectively). Those findings suggest an irmportant roe or JAW-C in maintaining the organization of the paranceal ‘erminal loops but the relatively lo incidence of as of JAM-C the nota defect under cont PECAN. ET amc Fig. . JAC is expressed in junctional regions of Schwann cell in peripheral nerves. (A) Confocal images of WT cremaster muscles immunostained for PECAN (red) and JAM-C lereen) show ‘expression of JAC in nerves () and vascular EC junctions (0. (8) Cremaster muscles stned for JAMA 1 (eed) and JAU-C (green) show that JAVA locabzes to EC junctions and surrounds JAMMC- postive nerves (arrows). (C) Longitudinal sections of mouse spinal cord (CNS) and its ventral roots (PNS) immunostained for Jl (jeer) and, in red, newoflamert (NF, Pe (marker for PRS), or NOG (marker for CNS} showing restcton of neural JAM-C expression to the PNS, (0) Teasod scatic nerve bers stained for JAM-C (green) and laminin 74 (ted. JAC is restricted to junctional regions of Schwann calls, te, the paranodes (arrows) surounding the nodes of Ranvier Gsterist), Schmidt-tanterman incisute single arrowheads), and mesanonal bands (oublearrewheads). (E) Teaed sciatic nerve fibers stained for JAM-C (green) and. in ved, neurofascin 155 (NF 155), E-cadherin (Ecad, or claudia-19 (ls-29) in WT and JAM-<-KO mice shoning normal sistebution of te latter molecules in KO mice Seale bars: (A,B, D, ) 10 ym, (©) 20 ym, vouss 3 9 SCIENCE NOVEMBER 2097 1473Bc 1474 ORTS Fig. 2. JAN-C deficiency alters the inteariy ofthe myelin sheath, (A) Immureelecon microscopy of longitudinal sections of WT sat neves revealed JAN-Cexpressionat lateral sides of adjacent myelin lamelize in noncompact paranodal myelin, but not in the axon (ax) oF compact myetn (cm). (B) Ultestuctuel anatyss of longitadinal sections of Sciatic nerves fom INT and JAMN-C-KO mice (16 weeks ole) prepared for immunoelectron microsco y (without antiboces to JAN-O indicated hat a proportion of KO terminal loops exiaited an apparent looser adhesion to adjacent loops and ‘axon, (€ and D) Ultrastructural analysis of transverse sedions of eatic nerves from WIT and JTAN.C-KO mice (16 neeks old) revealed abnormalities in te myetin sheath ofa coportion of KO fibers (7%) exiting layers of loose retin that ferm mild and severe tomacula. Scale bars: (A) 190 nm (8) 120 nm, and (Cand 6) 10m. Ye Conduction velocity (ns) > ‘Control (+7) JAM-C KO Fi AMC KO Ld 4 Tomacui (severe) B E & — = JAM-C = * . + KO 2 :, 8 & = 2 > 2 2 21 i é i : THs WANE KO Wi uae Ko 3. JAN-C-deficient mice exhibit elecrophysiologicel end behavioral reduced amplitudes compared with control nerves. (B) JAN-C-KO mice defects. (A) Isolated sciatic nerves from contol itermatas (4/-) and exbiited significantly reduced siride length (*P < 0.05, n= 13 to 18 TANN-C-KO mice revealed significantly reduced conduction velocities in mice) and grip strength (*P < 0.05, m = 8 to 9 mice). Experiments were the KO (°P < 0.05, 0 = four to eight nerves) and lag in thelr CAPS with performed by 2 blided examiner mechanisms in the KO mice, such as enuanced ‘expression and/or function of eter adhesion pathways. Furhsrmere, ulrastrctural analysis ‘of transverse sections ffom static nerves 1 vealed hat, whereas most KO fibers showed a normal phenotype (Fig. 2C), a proportion ‘exhibited accumulnied layers of loose myelin Within the periasoral space Forming tomacula [12 0.8 aad 69 + 2.1% tomacula formation in WT and KO mice, aspectively, P< 0.08, for seven fo nine mise with 157] WT and 4227 KO ‘bers countel (Fig. 2D). This thickening of the niyeln sheath isa characteristic feature of nerve Aber thn patients with herediary newo ‘with baby to peossure plsis (HINPP), Togeth cr these obmervations suggest tha the abnormal myelin phenotypes detected #1 JAMLC-deticient, mice ac caused by te los of JAMEC: mediated Autotypc iterations between mycin lamellae atthe paranodal regions. as well as potentially at SchmidhLarerman incisures, OF now, whereas JAN-C KO mice ean eshibita number of defects Such as mego-csophagus oc high suscepsiility to Jung infections (offen ft, and some of ther are —=S Fig. 4. JAME-Cis expressed inhuman peripheral nerves. Doublestaining of human sural nerves for JAN-C (Glad) and neurofiament (ed) obiained from a healthy tim (A) and a patient with a dincal and histepathologcal diagnosis of chronic inflammatory demyelinating polyneuropathy (B). JUNC is ‘expressed at paranodal regions (arr) in healthy nerves, ad the number ofJAN-C-positve paranades vas significantly reduced in nerves from patient in accordance wth the denyelinated site ofthe fibers, <0.01, fori healthy subjects and five patients with neuropathy; the images shoan are representative of al samples analyed). Sate bar, $0 um, smaller in size during early phases of their life Because detective JAM-C-medined adhesion span (<3 0 8 wooks) (7), Uae Young adult mice ly or ingiretly Tead to redo pat chesen forth studies detailed bere (ages 8 0 16 2 we assessed whsiher JAMEC KO weeks) were of normal heath sts neurmlogical defects. KO sciatic smuiched by weight withthe contol nenes displayed redueed conduction locity 30 NOVEMBER 2097 VOL318 SCIENCE wwscioncemag.orgerm mn Teter A DELICATE TOUCH The notion ofa cell membrane serving to contain alarge mixture of proteins randomly ‘moving about ina cytosol may once have been the grace school introcuction tocellu: lor biology. However, when one considers the nature and coordination of cellular ac tivities, it becomes evident that rancom colisions between proteins could not result in the spatial and temporal reaction and information-caurying cascades now known to occur in cells. Proteins must interact in very specific ways in order to coordinate nearly all cellular processes including ONA replication and transcription, RNA splicing, ‘and translation, protein modification anc secretion, cell cycle control and apogtosis, as woll as signal transduction and gene exoression. Therefore, a disruption in the interaction of proteins is ikely to contribute to the onset of disease ands the reason so much research effort is focused on understanding the nuances and implications of such interactions. By Alfrad Doig rotein-protein interactions (PPIs) occurring on the exterior ofthe cell mom brane provide signals as to the cell's external envisonment. Within the cell ‘membrane such signals are propagated by other specialized PPIs that serve to deliver the message to one or more of the compartmentalized cell struc: tures, such as the nucleus oF mitochondria, which might result in, for example, 2 change in gene expression or ATP production. PPIs are very diverse but all protein interactions occur ina highly specific manner ‘determined by structural and physiochemical properties of the interacting proteins. {Ac the molecular level PPIs can be characterized by their binding strength (permanent or transient), specificity (specific or nonspecific) the location of interacting segments (within one oF mare polypeptice chains), and the degree of similarity between inter acting protein subunits. Experimental Approaches PPis are affected by a numberof variables including protein concentrations ~as de termined by protein synthesis anc degradation —and the location of the interacting protein participants within the cell. In recent years, innovations in software, reagents, and instrumentation as well as improvements toexperimental protocols have given 3 ‘clearer understanding ofthe biological roles of many PPIs. Methods available to study PPIs extend from very qualitative approaches to highly quantitative measurements. The methods of choice are determined by the nature of the experimental study and can range from the desire to discover new PPIs to de: termining the cissociation kinetics between wel-establishea interacting pars. There exist a number af in vivo and in vitro methods used to identify and characterize PPI. The techniques are based on a variety of biological, biophysical, or physiochemical ‘measurements and some lend themselves to high throughput format development. Biological Methods Ifa yes/no answer regarding the significance of a suspected two-protein interaction is required, a synthetic lethality approach may be appropriate. This method involves the construction of cells containing two mutations, one in each of a suspected PP! pair. Neither ofthe mutations alone results in loss of cell viability, but if both shoul ‘occur in the same cell, death results, Another established method used in PPI re search isthe two-hybrid approach, first demonstrated using yeast strains and subse. quertly adapted for use with mammalian and human cell ines. The yeast two-hybrié methodology utilizes a nutrient-cependent yeast strain into which separate bait ang prey plasmids are introduced. One plasmid produces a known protein (the bait) with ‘a fused DNA-bincing domain (BD) fragment while the other plasmid produces a pro: tein product in which an activation domain (AD) fragment isfused onto continued» 61n recent years, innovations in software, reagents, and instrumentation as well as improvements to experimental protocols have given a clearer understanding of the biological roles of many PPIs.99 Look for these Upcoming Articles Roboties/Automation — January 18 ‘Mass Spectrometry — February 22 Proteomics 1 — March 7 Inclusion ofomponies in is article ces net indicate endorsement by ether AAAS orScienes, nor it meant {oimply tat ther products rsorvices are superior to these ofether companies. 1477the so-called prey protein, The prey protein can be either a single known protein or a library of knewn or unknown proteins. ifthe bait and prey proteins bind, transcription ofa reporter gene takes place, indicating the formation ofa PP. This “bait and prey" approach has been incorporated into a vari ety of commercial reagent formats. BD Biosciences markets the ED Matchmaker Two-Hybrid System 3, an enhanced GAL4 two-hybrid system The product uses yeast strain AH1O8, in which four reporter genes are integrated in the host genome. A similar system, DUAL: membrane from Dualsystems Biotech, is specifically designed to detect interactions involving integral membrane proteins with other such proteins, membrane-associated proteins, or soluble proteins. A human cell-based “beit-prey” PPI assay system, known as GRIP. is produced by Biolmage (part of Thermo Fisher Scientific. The GRIP technology is based on the translocation of human cAMP phos: phodiesterase PDE4AA and provides a high throughput method to screen for inhibitors of protein interactions. ‘Another in vivo method for visualizing PPIs involves use of the fluorescence resonance energy transfer (FRET) approach, In this technique, two cilferent fluorescent molecules (Nuorophores)—the donor and acceptor —are genetically fused to the two proteins ofin- terest. Regular fluorescence occurs when the protein-bound fluoro pphores emit energy atthe emission frequency. When the two labeled proteins interact end are stimulated by light energy atthe excitation Frequency forte donor fluorophore, some ofthis energy transfers to the acceptor, which then re-emits the light at its onn emission wave: length. The result is that the donor partnerin the PPL emits less light energy, while the acceptor emits more. FRET equipped microscopes and fiuorescence-based cell sorter systems are used in conjunction with these FRET reagents to quantity the PPIs. Featured Participants Agilent Technologies vmwrwagilentcon Invitrogen wa wmvitrogen.com tena ‘wor attana.com Micrcat wnurmierocatcom 0 Biosciences \wuru bobioscinces.com Panomies wnwpanomics.com Pierce Biotechnology (pat of Thermo Fisher Scientific) wnwpiecenet.com Siema-Aldcich wnwusigmaalerich.com ‘lotmage (part of thermo Fisher Scientific) ‘wr bioimage.com Bio Re ‘our bioad com Dualsystems Biotech mura duet Stratagene watatag TIRF Technologies wnatrtechnologies.com Yale University wyaleeds (GE Hoaltncare/Biacore mura biacore.com Hyprometrix or yprometebcom Ingenuity Systems ur Ingenity.com 1478 Isolating Protein Pairs In studies where the isolation of proteins involved in a PPI is desired, “pul down” assays can be used. Pierce Biotechnology (now a part of Thermo Fisher Scientific) offer its ProFound PPI pull down {its for this application. The product is based on either GST-or His- tagged fusion proteins. The tagged usion protein isusedas the “bait” protein and the pulldown processis based on a bead-based, affinity purification technique. The captured PPIs are eluted for analysis by Western blot For recovering interacting proteins from mammalian cells, Stratagene, an Agilent Technologies company, produces is InterPlay ‘Mammaiian TAP System. The method is based on expression of 2 protein of interest fused to two afinity tags: a streptavicin binding peptide (SBP) and a calmodulin binding peptide (CBP). "A two step tandem afinity purification protocot yields exceptionally pure and intact interacting proteins through gentle elution and elimination ofa protease digestion step,” says Benjamin ricer, the product manager for functional biology’ at Stratagene, “The isolated proteins,” he continues, “can be identified using Western biotting or mass spectrometry Array Screening Protein arrays are another, more recent addition to the PPI toolbox. A in the two-hybrid approach, a “bait and prey” strategy is used The methods involve incubeting an array of surface anchored proteins with cell supernatant, washing the assay surface, and analy2- 18 for PPIs. A variety of anchoring methods and formats is used cluding glass slides, polymeric beads, and chromatographic media, Hypromatrix supplies 2 general purpose PPI screening array called the AntibodyArray, which comprises membrane tethered an: tibodies against hundreds of well studied proteins. The antigenic protein binds to the antibody, thus capturing any PPIs between the antigen protein and its interacting partners. Similarly, Initrogen'’s rotoarray Human Protein Microarray contains approximately 8,000 ‘unique human proteins, selected from multiple gene families ancl ar rayed in duplicate on a 1 inch x 3 inch nitrocellulose -coated glass slide, To provide maximum flexibility, these microarrays are compat- ‘ble with fluorescent, chemiluminescent, radioisotopic, and other detection methods. ‘Specialty arrays for PPI screening are also available and include Sigma-Aldrich’s Panoram Human Cancer v1 Protein Avy used to Screen for interactions with 130 fully functional cancer proteins, ‘Also, Panomics? TF Protein Array is useful for determining how a particular protein interacts with the 140 known transcription factor proteins spotted on this array. Instrumentation for Bound Proteins ‘Over the past 15 years, specific instrumentation has evolved to mea~ ‘sure the physiochemical properties of known interacting proteins. ‘These instruments are based on crystal resonance, surface plasmon energy measurements, microcalorimetry, scanning tunneling micros copy, and total internal refection fluorescence. ‘The principle of crystal resonance has been applied by Attana to develop a quartz crystal microbalance (QCM) technology to ob tain labelfree, real-time measurements of PI kinetics, affinity, and specificity. QCM uses a piezoelectric effect to oscillate a crystal at its resonance frequency. This frequency stifts when molecules are added to or removed fiom the surface of the crystal. By attaching a ‘specific bait proteinto the biotin treated crystal surface, continues » wow sciencemag.org/products“The AFM instrument employs a sensitive probe, the deflection of which is a measure of sample surface topography.” an interaction with an introduced prey protein can be detected due ta the resulting change in protein mass. Taking a slighily different tack, (wo companies, GE Healthcare/ Biacore Systems anc Bio-Rad Laboratories, market instrumentation based on surface plasmon resonance (SPR). These instruments use 2 gold foil detector surface and reflected light energy to generate surface electron charge density waves (plasmon) PPIs forming between surface-tethered proteins and introduced proteins on the detector surface interact with these plasmons resulting in measurable refractive index changes that are proportional to changes in surface mass. Stefan Léfis, chief scientist, Biacore Systems, GE Healthcare Bio-Sciences AB, explains that “the combination of label-ee SPR detection, sophisticated microfluidies and a broad range of surface chemistries allows characterization of almost any type of biomo: lecular interaction at a very detailed level. The kinetic information obtained greatly facilitates the characterization of molecular mecha nisms and biological processes.” The introduction of higher throughput screening capacities for SPR instruments is a focus for Biacore and Bio-Rad. As Cathy Mainini, senior product manager, protein function division, Bio-Rad Laboratori, points out, “We have combined surface plasmon resonance technology with an innovative 6x6 microfluidic design that at lows one to measure an array of 36 biomolecular interactions simul taneously. This parallel approach can generate a complete kinetic profile ofa biomolecular interaction in a single experiment without regeneration.” TTIRF Technologies’ instruments are based on total internal reflec: tion fluorescence (TIRF). The process uses light propagating within a quartz crystal which, when it reaches an interface with a less dense aqueous solution, although fully reflected, generates an evanescent fiolé that extends beyond the interface and into the aqueous solu: tion. The fluorophores adsorbed, adhered, or bound to the surface ‘will fluoresce while fluorophores in bulk solution will not. The sensi Uivity ofthe T1RF approach is reported tobe 10,000times higher than SPR-based instruments. Based on the scanning tunnoling microscopy —the inventors of ‘which received half of the 1986 Nobel Prize in Physics—the atomic force microscope (AFM) is an imaging and measurement tool that Provides a three.cimensional map of a sample's surface. The AFM instrument employs a sensitive probe, the deflection of which is @ measure of sample surlace topography, With the capacity to observe and manipulate biological surfaces under physiological conditions, ‘AFM can be used to explore biological structures atthe single mole: cule evel and measure mechanical liganc:-receptor interactions with 30 resolution and sensitivity down in the picoNewton (N") range. 'W. Travis Johnson, senior scientist at Agent Technologies, explains, “We are combining AFM with surface chemistry and bioconjugation chemistry in order to stucy individual, discrete ligane:-receptor inter. actions fer from equilibrium. Thisis increasing our understanding of hhow biological systems work atthe single molecule level.” Instrumentation for Proteins in Solution ‘MicroCal has found a way to adapt ultrasenstive calorimetry to the study of PPts. Utilizing isothermal titration calorimetry ITC), the in Sstrument measures the heat that is absorbed or generated when a biomolecular interaction occurs. Uniquely, the method does not re- {quire thatthe target protein be labeled or bound to a surface, allow: ing the proteins to be studied in solution in a native state. Ernesto Freire, Henry Walters Professor, Biology and Biophysics, Johns Hop- kins University, reports his laboratory uses “ITC in all our projects involving protein-protein interactions. TC not only provides the most accurate determination of binding afinty, but also itis the only techy nique that reveals the nature and magnitude ofthe forces involved in the binding process by being able to measure the binding enthalpy andentropy : Bringing It All Together Understanding of the physiological and disease associated impor tance of protein protein interactions continues to expand. For exam pile, researchers recently reported using a Biacore system to generate binding kinetics measurements toidentifya site on the HIN-1 Env proteia that may be a target for vaccine development. PPI research is ‘moving forward aided by innovations in instrumentation, avaiabi ‘of reagents, and the generation of robust data sets. PPI researc is also benefiting from advances in knowledge software such as Ingenuity Systems’ Pathways Analysis. Mezan Laurance, senior scientist at Ingenuity Systems, points out “our soft ware application contains a comprehensive network of protein-pro- teininteractions and regulatory events [transcriptional effects, post: translational modifications. epigenetic events} manually curated from the scientific literature.” This enables researchers to put the particular PPL under investigation into a broader context of normal ‘and abnormal celular function. As Laurance puts it this allows them to “rapialy understand their experimental system asa whole, based ‘on what was detected at the protein interaction level.” Emphasizing the importance of depencing on multile technola- sles, Michael Snyder of Yale University points out that in his laboratory “we use a number of methods including two-hybrid, protein arrays, affinity chromatography, and SPR to investigate the biologi ‘al significance of PPIs. All of these methods have thelr inherent limitations; however, by integrating the resulting datasets we goin confidence in our understanding of the function and importance of specific PPis.” These technologies are enabling exciting new re ‘search on the characterization of multiprotein interactions involving, “scaffold” proteins responsible for bringing other proteins together sotthey can interact Continued advancements in the ability to measure the various parameters associated with PPIs coupled with knowledge based software will uncoubtedly provide the understanding required for systems biology moceling and serve to uncover new targets fr ther peutic developments. Alfed Doig i freelance bioscience writer and eitor Bung in Natick, A, DOL 10.1126/science.opms.p0700020 roduc 1481New Colorful Laboratory Instruments The new HydroFlex 3in-1 platform and the popular Sunrise microplate reader are available in bright orange, blue, red, end green. 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