cec,ZgzgUiest,4 1 lSaka Egypt. El-Sharkia, 519 44 University, Zagazig Science, Peetades elCceadCne ilg,Olhm eia Research USA. Medical 73104, Oklahoma OK Biology, City, Cancer Oklahoma and Foundation, Cycle Cell address: *Present France. Montpellier, Ho 34396 (CNRS), Scientifique Recherche la de National Re complexes synaptonemal Drosophila ARTICLE RESEARCH 4658 2014 August 26 Accepted 2014; May 16 Received " § the between 2 link -I-specific the physical of by segregation 1 a sister accurate the with maintained the ensures chromatids, segregation conjunction of homologous structures is in organization specific the on cohesion which side-by-side relies in The it kinetochores, step haploidy; molecules. key to and leads the the replication. that is DNA halve process of eventually I round single that Meiosis a follow rounds result number this segregation achieve chromosome To successive gametes. haploid that form two process to segregation cells chromosome germ drives specialized the is Meiosis INTRODUCTION Hira Recombination, Cohesin, Yem, complex, Yemanuclein, Synaptonemal Meiosis, Oocyte, Drosophila, WORDS: KEY the and complex synaptonemal between interaction the genetic here A report with complex. We cohesin Hira. associates with the Yem complex in in required that chaperone acts H3.3 is it histone where It pronucleus, a male reproduction. as the of sexual assembly chromatin for maternal for crucial zygote a structure is is (Yem) Yemanuclein proteinaceous that meiosis. factor of tripartite I a to by specific complex, prior mediated is the synaptonemal which requires axes, the chromosome formation homologous chiasma of chromatid of species, sister segregation of most for maintenance In allow the cohesion. to on rely turn, chiasmata In homologs, chromatids. chiasmata, between as (DSBs) homologous such breaks double-strand structures, the of reduction key repair by on formed Chromosome relies are replication, It which DNA formation. I. of meiosis genome round at single occurs haploid a in follow rounds which resulting segregation II), and chromosome I two (meiosis by characterized is Meiosis ABSTRACT fet rsmbytruhdsuto fHr–e complex. Hira–Yem of of disruption impact through in presumably the effect, by role supported early further an yem is has This Yem recombination. that meiotic suggesting distribution, crossover the uis Aı Ounissa hs uhr otiue qal oti work this to equally contributed authors These U1040/ INSERM (IRMB), Biotherapy and Medicine Regenerative of Institute Ge de Institut uhrfrcrepnec ([email protected]) correspondence for Author ˆ 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,45–66doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. gsE Meyer E. ´gis ia an-liCR,325Mnplir France. Montpellier, 34295 CHRU, Saint-Eloi pital Spo11 uain nDBkntc.A kinetics. DSB on mutations homolog ´ ne ¨ ´ t-Ahmed iu uan,Unite Humaine, tique 1, mei-W68 * ,§ he Algazeery Ahmed , eauli soitswt h oei and cohesin the with associates Yemanuclein 1,2, " oiida modified , ´ rped ehrh 12 Centre 1142, Recherche de Propre Hira ` rsn drs:Fclyof Faculty address: Present yem uaingv similar a gave mutation 1 1, oiatefc on effect dominant yem ` ,§ Miche , 1 V7E and (V478E) l Capri `le 1 ,He rti opee.Tefrtcmlxwoesrcueand structure of whose Resolution complex 2011). first The al., specific measured on complexes. et called relies genetically protein crossing-overs mark as be (Phadnis cytological intermediates can recombination a crossing-overs events as the break meiotic as observed of double-strand These be for use the chiasma. can template the connects repair, a from that (DSB) as references link results chromatid and physical which homologous 2005 the chromatids, Watanabe, species, homologous most 2003; In al., cohesion therein). et meiosis-specific recombination- held the Petronczki to a are (see owing centromeres II of anaphase sister until I the together Importantly, anaphase meiosis. at proficient centromeres homologous oei uuis oee,n ucini oeinhsbeen (Cona), Corona has non-SMC Later, cohesion the 2004). al., in kleisins, et elements) (Heidmann function to C(2)M no for related lateral found however, distantly C(2)M subunits; of be 2001). cohesin Hawley, to component and appear Page (a does 2003; McKim, transverse C(2)M and of (Manheim component and (a C(3)G crossover filaments) were and acid reported structure was amino complex formation the synaptonemal for at its In requirement conserved structure, level. necessarily complex the sequence not of synaptonemal spite are the In components of 1999). Kleckner, conservation and high Page Zickler for 2012; 2004; basis Hawley, Hawley, a and elements, and considered (Lake are assembly axial complex elements organize synaptonemal Axial the which chromatids. and from localize sister proteins the and Kleckner, cohesin originate and which and element to elements Zickler (Lake structures 2004; central element Lateral Hawley, central a and 1999). the Page elements, 2012; cross Hawley, that lateral filaments two transverse of conserved highly composed is complex yeast synaptonemal 2005). from the al., of et structure (Thomas the for The strategy meiotic different their a of use males segregation segregation meiosis; complex female synaptonemal to chromosome specific the is accordingly and the In recombination, (Page undergo recombination for 2004). homologous required on the Hawley, relies the is which of holds therefore, process, integrity process; complex assembly recombination synaptonemal the its and is during The together it 2001). structure homologs Stack 2006; Anderson, particular, the Jessberger, and and the in (Revenkova to for complex chromosomes; synaptonemal essential linked is meiotic intimately complex of cohesin al., et of organization the Wood aspects 2000; Importantly, many (Hirano, for 2010). function important the and is structure which is chromosome complex, structure complex cohesin other recognized the The proteinaceous 2006). is Moses, was zipper-like 1969; Moses, 1975; a recombination (Carpenter, complex, meiotic synaptonemal in function ´le n Brazier `ne .cerevisiae S. 1 hitn Ferry Christine , Drosophila Drosophil ommas ti rpriestructure tripartite a is It mammals. to ,ol oyechromosomes oocyte only a, h is opnnswhose components first the , 1 and

Journal of Cell Science opnn hti eurdfrsse hoai oein As looks complex cohesion. early synaptonemal chromatid in the sister complex staining, normal for synaptonemal C(3)G a by required formation Ord, evidenced is to complex contrast that synaptonemal in component 2008), for al., been required et also (Page be has element, to central a reported as described component another ARTICLE RESEARCH otefu-igcnlcls(aldpoocts.Yem pro-oocytes). (called cells complex synaptonemal canal restricted The well-characterized being and four-ring with before 1Ad,e). three- cyst (Fig. colocalization the 16-cell the the complex inside of to cells forms structures synaptonemal canal initially four-ring ribbon-like of the complex are of revealed magnification synaptonemal stages of drawing Higher chambers 1970). the various typical the egg King, in The and of from mid-prophase Yem-stained expression 2a 1997). (adapted to mRNA 1B region al., up Fig. its as shown et with 1A, early Fig. schematically line (Capri as in in from shown localization is whole-mount detected As which be Yem. germarium, immunostaining against could antibodies was Yem by in question with function this its ovaries Yem experiment, cytologically first investigate and a to In addressed nucleus aimed prophase. we meiotic oocyte work, in early present recognized the the been In already (Aı to work has previous meiosis Yem our female of for importance localization protein The synaptonemal-complex-associated new a is Yem RESULTS e,uviigfrtefrttm oeo h iaYmcomplex Hira–Yem the of of role meiosis. a localization female time in germarial first the the for recombination, unveiling of Yem, disruption meiotic through of formation. presumably DSB aspects of timing various the A and for distribution crossover required is namely, Yem is that it interacts show it that we and Here, vivo complex male synaptonemal 2005). the the of with al., associated assembly et chromatin in (Loppin be player to pronucleus complex major is chaperone a Hira H3.3 as 2013). Yem–Hira al., recognized the et of (Orsi member Hira first chromatin with the forms pronucleus it paternal complex that the the discovery through remodeling the in by 2010). functions al., supported Yem et further (Meyer were offspring, zygote the observations the parthenogenetic in These in defects syngamy in defective segregation and result chromosome oocyte are can there Yem that we reproduction, suggesting sexual of work in previous role mutations essential our an as has In Yem prophase. that shown meiotic have Yem- early as during known 1992) also yet synaptonemal (Yem, not Yemanuclein are the that and issues they complex axes understood. how are fully chromosome elements, and lateral subcomplexes, complexes the complex these of to (Gause How existence 2007). complex contribute possible the Bickel, synaptonemal the and SMC3, the Khetani form, and to and 2008; all Takeo al., localize SMC1 have et 2012; homolog, to 2011). Scc2 shown al., the al., been et Nipped-B, et and Qiao cohesins, Takeo canonical 2012; centromeric and 2012; al., flies at in et Hawley, not both (Bisig Interestingly, completion, persist 2001). mammals recombination does beyond Hawley, components far and regions disassembly complex (Page 6 synaptonemal complex stage in before whereas 2004), occur synaptonemal al., et (Webber 1 wild-type stage by prematurely, occurs ntepeetwr,w aeivsiae h oeo the of role the investigated have we work, present the In Hira ihcmoet ftechsncmlx eas hwthat show also We complex. cohesin the of components with nl uainhdasmlrefc nDBkinetics, DSB on effect similar a had mutation -null ord nl emra yt,btisdisassembly its but cysts, germarial -null tAmde l,19;Myre l,2010). al., et Meyer 1992; al., et ¨t-Ahmed a )(Aı tAmde al., et ¨t-Ahmed in fYmwt () n C(2)M. and colocalization C(3)G show clearly with 1C Yem Fig. of in Results Page 2003; 2001). McKim, Hawley, and and (Manheim C(2)M against of antibody version an HA-tagged and a C(3)G against antibody monoclonal co-staining a by with assessed was C(2)M, and C(3)G as such components, c(3)G ihrmgiiaino the of magnification higher A ssoni i.2,Ymsann a togyafce in affected for strongly homozygous was are staining that Yem females 2A, of Fig. punctuate germaria 2003). in by McKim, and shown replaced (Manheim C(2)M As is of structure absence complex the threadlike in synaptonemal As structures the the 2005). staining, of for al., C(3)G typical indispensable et by be revealed (Anderson lateral to previously formation a shown complex C(2)M, been synaptonemal and have component, component, filament element transverse a filaments transverse C(3)G, and synaptonemal elements on lateral depends Yem complex of localization germarial The 92,w eie oivsiaeispsil neato with interaction compl possible cohesin its the investigate of to components decided (Aı properties we (this DNA-binding 1992), complex has synaptonemal also the and work) with associates Yem Because complex associates Yem rpaei eedn nteitgiyo h synaptonemal the of meiotic integrity early the during on chromosomes that dependent of suggests the is result component C(3)G This to a prophase 2008). is localization al., of which for et Yem (Page made protein, that element previously Cona central to been the the has of identical observation localization similar the Yem A 2B). of (Fig. distribution punctuate r,a ela () n M1 scniee ob a be the to 2003; to McKim, contrast the considered and In backgrounds, is 2004). Manheim al., 2007; SMC1, et Bickel, Webber and and in Khetani C(2)M elements and the 2005; lateral as Yem required of well Yem component 2A). longer (Fig. as pachytene, complex no Ord, in synaptonemal of similarly nucleus localize also the C(3)G oocyte end of the the presence to towards localization However, complex. o hsproe ecntutdafysokta a ohthe both had that stock fly 2004). a al., constructed et gSmc3-ha we (Heidmann purpose, SMC3 with this pro complex For a cohesin form to canonical known a the SMC3, tested We extracts. protein co-immunoprecip performed we oee,te r o ssal ste r nwild-type in material 5). (supplementary are Fig. complex S1; they synaptonemal 2004). Fig. the al., as of et integrity Webber stable that 2007; noteworthy as is Bickel, It structures. and not (Khetani threadlike are backgrounds complex they synaptonemal However, the of formation LGeioea eaiecnrl ssoni i.3,in 3A, was Fig. signal no in antibody, shown FLAG As the and using control. experiment immunoprecipitations negative control a the u the as we 2008). epitope of al., FLAG experiments, specificity et blot (Gause the FlyBase) western The in assess Vtd tags. as To antib (listed epitope an Rad21 with (SMC3) kleisin also HA and and antibodies [C(2)M] Myc immunoprecipitate the The and again 2004). antibodies with al., performed et (Heidmann 68 ora fCl cec 21)17 6846 doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal MnemadMKm 03 aeadHwe,2001). Hawley, and Page 2003; McKim, and (Manheim and gc(2)M-myc nvivo in ord eepoe ihtecorresponding the with probed were s yem akrudde o oal bls the abolish totally not does background uatbcgonshdn feto the on effect no had backgrounds mutant ihcmoet ftecohesin the of components with rngnsdsrbdpreviously described transgenes ord c(2)M nvivo in tto xeiet sn ovary using experiments itation ,imnpeiiain were immunoprecipitations n, tteedgnu e protein Yem endogenous the st e natbd gis the against antibody an sed x oaddress To ex. Drosophila en n (),wihis which C(2)M, and tein, uatbcgon Fg 2B). (Fig. background mutant d gis h endogenous the against ody EP2115 soito fYmwith Yem of association c(2)M emru hw a shows germarium and Adro tal., et (Anderson tAmde al., et ¨t-Ahmed c(2)M hsquestion, this c(3)G EP2115 mutant 4659 or

Journal of Cell Science EERHARTICLE RESEARCH ie nadto a2 a ossetyfudin 4660 found complex. the consistently of and part antibodies complex is cohesin data Rad21 meiotic was Our that the different 3B). with associates (Fig. Yem protein that two Rad21 suggest Yem endogenous the with against addition directed obtained In immunoprecipitates first the complex for time. here the meiotic reported in same result found also a immunoprecipitate, the was C(2)M–Myc Rad21 within Interestingly 2004). be al., et to (Heidmann described have been C(2)M–Myc and SMC3–HA already C(2)M–Myc. as and SMC3–HA Yem hand, for other bait the a on and, SMC3–HA, and a Yem as for C(2)M–Myc hand, bait one the on using, experiments by reciprocal confirmed the were results and These C(2)M–Myc. Yem and of SMC3–HA also amount substantial a precipitated antibody polyclonal anti-Yem the whereas fraction, immunoprecipitated the in observed etdterl fYmi eobnto ygntclyanalyzing genetically nonexchange by of recombination effect in associated Yem the is of of Yem first role (3) We the components. and tested cohesin increase 1968), and complex al., synaptonemal an the et (Baker with the Sandler reflect 1976; backgrounds might Hall, recombination-defective and of which in 2010), chromosomes consequence al., et indirect (Meyer 4 chromosome netl Myre l,21) etse eoi eobnto in recombination meiotic tested we 2010), al., et (Meyer infertile sseiial oaie oteoct (Aı that oocyte protein in the role DNA-binding (2) to potential a localized a is specifically has Yem is it (1) that recombination: suggest meiotic Yem of properties Several in distribution Crossover eas eae htaehmzgu o the for homozygous are that females Because yem ora fCl cec 21)17 6846 doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal 1 oye rqetyso bomlsgeainof segregation abnormal show frequently oocytes yem 1 uaini h rgn fmtn mothers. mutant of progeny the in mutation yem 1 anfctoso h germaria. the of magnifications higher displays shown. panel is Lower (red) staining C(3)G C(2)M and with (green) staining Yem of of Projection deconvolved (C) 1970). nurse (King, NC, cells FC, oocyte; (S6). OO, 6 cells; stage follicle to up ovariole an of representation 3 Schematic stage (B) the oocyte. in and 2a chamber region egg germarium a of two proocytes the in Yem of localization e ribbon-like and typical d the pictures show The stacks. the software to Volocity of application after obtained e reconstructions and 3D c show germarium. the of represent regions of R3 to projection R1 a sections, b, confocal In a. in shown germarium a the of display magnification c higher and b ovariole Images (wt) (a–e). wild-type a in stages early at localization Yem (A) and Methods. Materials the in described as stained were ovaries Whole-mount germaria. wild-type in Yem-alpha protein of Localization 1. Fig. +oocytes /+ tAmde l,1992); al., et ¨t-Ahmed z sre.Colocalization -series. yem 1 uainare mutation

Journal of Cell Science h etrsls rsmbybcueaplcoa nioydrce gis h noeospoenwsmr fiin hnat-a niois (B antibodies. immunoprecip anti-tag consistently than was Rad21 efficient antibody. more polyclonal was rabbit a protein columns). with non-r (Yem endogenous (A) the antibodies the in anti-Y anti-Yem represents described with against different complexes that immunoprecipitation directed immunoprecipitated that two (FL) antibody the noteworthy through in is polyclonal The flow It assessed a was analysis. antibodies. the because blot presence tag (E), western presumably anti-Myc extracts to results, submitted and protein best were anti-HA The fractions the antibody, antibody. (IP) polyclonal monoclonal immunoprecipitated the AS2 anti-FLAG and anti-Yem an fraction with with performed performed were was immunoprecipitations immunoprecipitation The (A) proteins. transgenic ci ta. 98 aeadHwe,20) efrtdetermined first We 2001). 1976; Hawley, and Mason, Page 1966; 1998; Hinton, al., (Carpenter, et 1974; dosage McKim Sandler, and to Carpenter 2003; recombination meiotic of sensitivity EERHARTICLE RESEARCH i.3 e associates Yem 3. Fig. of progeny the yem 1 eeoyosfmls aigavnaeo the of advantage taking females, heterozygous nvivo in ihC2M M3adRad21. and SMC3 C(2)M, with vr xrcswr rprdfo le htepesbt M3H n C(2)M–Myc and SMC3–HA both express that flies from prepared were extracts Ovary h fetof effect the eaeaeadevrnetlcniin,sc stemperature, to as experimental same sensitive such the in conditions, determined was is frequency environmental recombination and recombination age meiotic female Because chromosome. ora fCl cec 21)17 6846 doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal e tiigi akn ntegrailgr ience of nuclei line germ germarial the in c(2)M lacking is staining Yem e in a Yem from projection full a represents for mutant components. ovaries complex in synaptonemal localization Yem 2. Fig. obcgon hnuigteYmantiserum. Yem the owing using probably when cyst, background the to throughout observed be can staining The 2004). e tiig pera rgetdadsot inl in signals spotty and c(2)M fragmented as appear stainings Yem owl-yei h oyencesb tg (arrowheads). from 3 germaria stage of by magnification nucleus High oocyte (B) the in wild-type to eet r oprbet hs rvosydsrbdfor in described C(3)G previously and These those C(2)M to (arrow). comparable spots both are as oocyte, appear defects 2 stainings stage C(3)G the In and germarial Yem fragmented. later looks at it disturbed where severely stages early is at then forms and complex C(3)G, stages, of synaptonemal that that to shows similar which staining a displays Yem panels), (right aes and panels) yem EP2115 EP2115 c(2)M 1 ord emra(ro) nthe In (arrow). germaria n c and ncosvrdsrbto ln h X the along distribution crossover on ord and eiinyue eei f2)-0 Some Df(2R)3-70. is here used deficiency 5 /Df (3)G c(3)G rgtpnl)mtns ohC3Gand C(3)G both mutants; panels) (right ord 68 5 /Df uatadwl-ye(t ovarioles. (wt) wild-type and mutant uat u t nest a similar was intensity its but mutants uatgrai Wbe tal., et (Webber germaria mutant z sre.()Lclzto of Localization (A) -series. ord 5 ahimage Each c(2)M /Df matbd gave antibody em uatgermaria mutant EP2115 Rad21 ) (left ttdby itated mock etained 4661

Journal of Cell Science hsnbcueo t ml ie hsrdcn h rbblt of probability the was reducing chromosome, thus size, X small the its of of region because subtelomeric chosen the in located The is backgrounds. mutant different in interval a of and mutation regions Because region. centromeric central and the telomeric in at decrease increase an namely EERHARTICLE RESEARCH 4662 in shown tested, intervals is chromosome mothers the of all wild-type progeny In in 4A. the that Fig. in to tested mothers frequencies heterozygous recombination of ratio The wild-type, for conditions peodto’caso eoi ee CretradSandler, and (Carpenter meiotic definition1974). of by class crossovers, ‘precondition’ of distribution the W68 ete hs osoercmiainfeunyfrthe for frequency recombination score to chose then We 1 /+ ohr a iia feto rsoe frequency, crossover on effect similar a had mothers mei-W68 and yem uatbackgrounds. mutant y–w yem yem nevl which interval, al nothe into falls yem 1 /+ and affected mei- y–w 30 shg.I conclusion, In high. is (3.0) hc stesrnetefc fthe of increase, 50% effect nearly strongest a the was recombination interval is this the which in Moreover, scored events. frequency recombination multiple uat C.Wl-ye sdtrie noreprmna odtos is and conditions, genotypes experimental precondition our white, in in determined indicated trans-heterozygous as and Wild-type, (B) (C). mutants mutants meiotic heterozygous of offspring backgrounds rqece bevdi h igemtns h discrepancy The if value expected mutants. the frequency and recombination (0.3) single determined experimentally the the between in observed frequencies the for obtained result the contrast, In 0.8). (expected ( 03.T drs hsqeto,w oioe h Sswt an with DSBs the monitored we question, this address To 2013). as estimated was for interval cM same same 1.7 the in the being whereas wild-type the conditions for cM experiments, experimental 1.2 being our as In estimated was 4A). (Fig. heterozygous xhnemttosbhv ncmiainwith combination in behave mutations exchange 1998). Hayashi-Hagihara, for and as McKim formation, 2002; DSB al., in role et its with consistent material is that (supplementary finding Our earlier S1). in published Table are results those Our 4B). with (Fig. agreement decrease dramatic a revealed mothers oteepce aus h xeietlvlei Mws2.1 was cM in value experimental for The 2.8) values. (expected expected the close to were frequencies recombination determined experimentally mei-218 elc oeo e nDBmetabolism. DSB in Yem of might role which a here, reflect analyzed mutations other the unlike interaction, BC eobnto rqec c)aaye o the for analyzed (cM) frequency Recombination (B,C) efre naayi ftercmiainfeunyi the in frequency recombination for the interval of analysis an performed P22 A rsigoe itiuinaogteXcrmsm for chromosome X the along (cM). distribution centimorgans 5 over in than Crossing less expressed (A) was is age frequency whose Recombination females days. with and conditions mutant experimental in controlled chromosome X the mothers. on wild-type frequency Recombination 4. Fig. ogi ute nesadn nYmrl ntemeiotic the in role the Yem in DSBs on analyzed we understanding pathway, further recombination gain To in modified is dynamics DSB ohr eeoyosfor heterozygous the mothers in observed yem yem akrudue ntegntceprmnsaogwith along experiments genetic the in used background ;yem +; te rn-eeoyoscmiain ihthe with combinations trans-heterozygous other c)i niae o ahitra.Saitclsgiiac sasse with assessed is significance * Statistical test. distance interval. Chi-squared genetic each the wild-type for the indicated proximal; is the to (cM) along distal intervals from experimental genetic listed our represents chromosome genetic in axis the (determined abscissa over type The genotype wild conditions). mutant the the in in observed observed distance distance genetic of ratio eobnto aein rate test. recombination Chi-squared in the is with frequency determined * recombination was in significance decrease Statistical the a and black. gray, displays in that indicated mutant are exchange frequency recombination the in increase ry and gray) P yem , nodrt ana nesadn fhwteepeodto and precondition these how of understanding an gain to order In .5 ** 0.05, 1 1 103 2 /+ /+ and ) ncnrs,teaayi fpoeyfrom progeny of analysis the contrast, In . 1 ora fCl cec 21)17 6846 doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal /+ 1 eoye h bevdrcmiainfeunyws0.8 was frequency recombination observed the genotype, akrudwsntepce rmterecombination the from expected not was background mei-W68 + yem /+; P eoye.Terslsaesoni i.4.I ohthe both In 4C. Fig. in shown are results The genotypes. , mei-218 Hira- .1 n *** and 0.01, 1 /+ ul( null 1 l h eobnto et eecridoti well- in out carried were tests recombination the All /+ mei-218 mei-W68 y–w 1 P dr ry eae.Teodnt xsrpeet the represents axis ordinate The females. gray) (dark + yem /+; yem , n the and .5 ** 0.05, Hira P 1 nevlwe nlzn rgn of progeny analyzing when interval /+ , 1 1 mei-W68 .0.()Nt h ihdces of decrease high the Note (C) 0.001. + yem /+; mei-P22 HR1 + yem /+; mei-W68 1 P /+ ohr.Asmlrices was increase similar A mothers. yem , uatbcgons(rie al., et (Orsi backgrounds mutant ) mei-9 yem , .1ad*** and 0.01 mei-W68 and 1 1 /+ 1 /+ al ntepeodto class precondition the in falls and , a 1 + yem /+; mei-218 hra o the for whereas , mei-W68 and uat ncmaio othe to comparison in mutants yem Hira 1 yem + yem /+; P yem yem , 1 0.001. /+ 1 1 1 mutant 1 mutation. 1 y–w , /+ eeindependent were htdslyan display that ipa genetic a display c(3)G uainwhen mutation 1 eoye the genotypes /+ nevlin interval yem mei-W68 mei-W68 y–w yem yem and 1 1 mei-9 /+ mei-9 and 1 yem 1 interval /+ mei-9 mutant (light ,we mei-9 -null mei- (Liu 1 a y–w a /+; /+; /+ a /

Journal of Cell Science hfe oerircssaddslydwae inlintensity. signal weaker displayed and cysts Hira earlier to shifted the In staining. epnet Ss ec tcnb sda akrfrthe and formed, for As fully is marker 2002). complex al., synaptonemal a the et after as in Madigan 2a, DSBs used region 2003; 5A, Fig. al., be in et shown can schematically (Jang it DSBs of hence presence DSBs, to response as (denoted diin e inlwsntdtce ntegrailmeiotic germarial the on detected not was signal Yem a addition, ( hshrltdi h rsneo Ssbt nmttcand 1999). al., mitotic et in Rogakou both 2001; al., DSBs Similarly, et of (Mahadevaiah presence cells meiotic the in phosphorylated anti- ARTICLE RESEARCH oc n ci,21;Lk n aly 02.()Woemutoailswr tie o N bu) () gen and (green) C(3)G (blue), DNA for stained (wt), were wild-type ovarioles analyzed: Whole-mount were (B) genotypes 2012). Hawley, and Lake c 2010; McKim, and Joyce of Effect 5. Fig. C(3)G-negative The of in including role nuclei. formation, the the DSB underscoring activity, in nuclei, activity Mei-W68 the Mei-W68 all of from disappeared absence staining is In It anti- 2b. was nuclei. region an negative and staining 2a that region the anti-C(3)G with noting late of worth germaria, cysts an DSBs in wild-type observed with essentially for In antibody. complex and polyclonal synaptonemal backgrounds antibody al., genetic the et the various monoclonal Webber of of for 2006; germaria 3 McKim, shows stained and region 5B stage Mehrotra Fig. this in 2003; at 2004). pachytene, repaired al., fully et late are (Jang by DSBs that absent suggesting germarium, are foci H2Av fetof effect yem HA tiig r hw eaaeyfrec eoyea lc-n-ht mgs l mgsrpeetafl rjcino a of projection full a represent images All images. black-and-white as genotype each for separately shown are stainings -H2Av c HR1 nl)oaiswsi tiigcnrs ihtewild-type the with contrast striking in was ovaries -null) HA nioy nmmas h 2Xvratis variant H2AX the mammals, In antibody. -H2Av yem (a null Drosophila Hira- c c HA,popoyainocr nsrn 3)in 137) serine on occurs phosphorylation -H2Av, and HA tiigin staining -H2Av yem yem ul uainhdasmlrefc Fg B.In 5B). (Fig. effect similar a had mutation null) Hira c and HA tiigwsas eetdi C(3)G- in detected also was staining -H2Av uatbackgrounds, mutant null Hira 2vudrossc modification a such undergoes H2Av ntesnpoea ope.Saebr:20 bars: Scale complex. synaptonemal the on uain on mutations yem yem 1 c /Df3450 1 HA foci. -H2Av /Df3450 Drosophila c HA tiigwas staining -H2Av , yem or A ceai iwo emru ihtemi vnso h al eoi rpae(dpe from (adapted prophase meiotic early the of events main the with germarium a of view Schematic (A) 2 yem D35 (yem /Df3450 omi late in form 2 c c /Df3450 -H2Av -H2Av c - null m m. ,Hira ), hoooe nthe in chromosomes ee ta. 00.Tepoete fYmrie h usinof prophase. question the meiotic raised early Yem in of role properties its The 2010). al., et Meyer feto yatnmlcomplex. synaptonemal on effect the activity Mei-W68 of absence the In antibody. same the with e sanwsnpoea-ope-adcohesin- and synaptonemal-complex- new a is Yem genomic the the in fall contains which that a in mutations, region recovered female-sterile was allele for mutant V478E screen expressed The 1987). maternally al., et for Ahmed screens was (Aı Yem differential meiosis. genes early in in Yem identified of function first the on here report We DISCUSSION u eut lal hwta e soitswt the with associates Yem to similar that very is that show pattern a clearly following complex synaptonemal results Our protein associated ciiy the the activity, whether verify To and anti- the complex. by Hira–Yem revealed DSBs the precocious DSBs on on Yem dependent of function meiotic is the that suggests which S2), Fig. oedsucinsc surpie elcto ok Fg B.It that 5B). noting DSBs (Fig. worth forks is replication precocious unrepaired as the such dysfunction therefore some observed, the in not observed were foci H2Av HR1 Hira ( Hira ora fCl cec 21)17 6846 doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal tAmde l,19;Aı 1992; al., et ¨t-Ahmed null uatbcgonswr eedn nMei-W68 on dependent were backgrounds mutant ), mei-w68 mei-W68 yem Hira Hira 1 1 uatbcgon antb trbtdto attributed be cannot background mutant yem ; , mei-W68 nl swl as well as -null HR1 yem 1 /Df3450 akrud(upeetr material (supplementary background 1 ou (Aı locus yem ; tAmde l,18;Aı 1988; al., et ¨t-Ahmed 1 obemtn a assessed was mutant double c /Df3450 HA nioyi the in antibody -H2Av yem c HA rd.TeC3Gand C(3)G The (red). -H2Av z tAmde l,1992; al., et ¨t-Ahmed sre.Tefollowing The -series. nl uain a no had mutations -null oeteasneof absence the Note . 4663 yem ¨t- c -

Journal of Cell Science la nmmas is,teewsacnrvrya owehror whether to as controversy a was there First, mammals. in in clear elements axial the of component cerevisiae S. a is it Moreover, 1999). Nurse, both cohesion meiotic for in essential is which that Rad21/Scc1 kleisin cohesin Rec8, a meiosis-specific in to equivalent Rec8, the no similar homologs. and with functional function paralog by complex a obscured has synaptonemal be it might the what whether determined in and be complex, has to for remains 2005; Yem It required al., role 2003). et is McKim, (Anderson that and complex been component Manheim synaptonemal has element the Yem, axial as of an complex formation contrast, same be the In to looks in antibody. reported found anti-C(3)G complex we the which synaptonemal with C(2)M, probed the when antibodies), normal anti-Yem with ta. 03.I h aeo e,isrl nchso and cohesion in role its Yem, elusive. of still is of case effect maintenance No the or assembly In complex synaptonemal 2013). (Calvente al., meiosis during exist et roles might different components controversial fulfil cohesin double and with still simultaneously complexes this of is of variety a impact cohesion fact, the chromatid However, sister (Herra 2012). on al., deficiency et knocked (Llano simultaneously are a Rad21L out in and substantially Rec8 affected both where get context only elements the protein axial paralogous with Rad21L; Rad21 called solved meiosis-specific of partly new component a been of a has identification controversy is This complex elements. cohesin axial meiotic mammalian the not eoi tgsi eedn nteitgiyo h synaptonemal the of integrity an is the Yem Interestingly, on complex. dependent is early at stages chromosomes the meiotic to localization its Therefore, 2004). for al., et mutant are that is germaria in that ord C(3)G pattern of a that follows 2001). to Yem similar Hawley, described, and C(2)M previously Page and as 2003; Moreover, C(3)G McKim, well-characterized and two (Manheim the proteins for described that ARTICLE RESEARCH vrdsrbto a ergltda w ees 1 tteDSB the at (1) 4664 levels: two at Crossing regulated regulated. be is can crossovers distribution of over over distribution crossing spatial one the least crossing obligate over, the at Beyond pair. that homologous each ensure between forms to stringently particular, be in must regulated, formation in crossover support the that Therefore segregation mechanisms randomly. crossovers, segregate of chromosomes absence homologs, In I. the the meiosis at segregation link for their in they resulting because essential cells recombination-proficient are Crossovers in formation DSBs and distribution Crossover be would such for as null maintenance. and are that or elements mutants formation in axial even complex the However, synaptonemal of for part essential is assume to tempting Yem is it that Revenkova features these 2001; From 2006). Orr-Weaver, Jessberger, the the and and of of (Lee elements component lateral complex the essential synaptonemal build an eventually that is axes Cohesin chromosome complex. cohesin the eoi n h ayuasee usin a enpublished been 2013). has al., questions et unanswered (McNicoll recently many the during complexes and cohesin on meiosis knowledge current our of account understood. An fully our being complexity, before investigations their further Given require results complex. any work synaptonemal phenotypes present the the mutant in on show the effect we of could neither analysis 2010), detailed al., et a (Meyer of spite in observed oocytes .pombe S. MnemadMKm 03 aeadHwe,20;Webber 2001; Hawley, and Page 2003; McKim, and (Manheim ´ ta. 01 siuoe l,21;Laoe l,21) In 2012). al., et Llano 2011; al., et Ishiguro 2011; al., et n yem Kene l,19) h ucin fRc r a less far are Rec8 of functions The 1999). al., et (Klein and 1 .cerevisiae S. uaino itrcrmtdchso ol be could cohesion chromatid sister on mutation Drosophila Kene l,19;Wtnb and Watanabe 1999; al., et (Klein nvivo in ate fcmoet of components of partner a ensont ea be to shown been has yem n tiiga all at staining (no yem 1 c(2)M mutant and uain Cretr 03.W eete bet hwthat show to able then recombination-defective were We of 2003). effect (Carpenter, mutations dominant the of advantage took rsigoe vnsdpns npriua,o crossover from recovered on be can progeny 2011). from few particular, Boulton, a crossover and only (Youds As new crossover another in a to close prevents too depends, forming that versus phenomenon events over a non-crossing interference, of over number non- relative crossing versus over The crossing over. of resolution crossing in (2) and level, formation ta. 00,t etteefc of effect the test to 2010), al., et o sal uhrzd(hgte l,20) ohin are Both they Drosophila 2004). where al., et place (Bhagat authorized a usually back-up at not a formation backgrounds, their mutant allow the would in mechanism crossovers of decrease number a the to response in In indirect. is distribution crossover on effect of W68 diverse regardless [ as formation functions 1974) complex synaptonemal with as as genes Sandler, includes classified group and This function. are their (Carpenter distribution genes crossover precondition affected alleles whose genes mutant All 1974). Sandler, and (Carpenter mutation exchange heterozygous in is recombination of in frequency the It the tested on effect mutations chromosome. same the meiotic had X conditions the the all that along noteworthy distribution crossover affected o e eeto,imnsann a efre ihapolyclonal Yem- a with against performed developed was immunostaining antibody detection, Yem For the and Yem, complex of synaptonemal detection for immunocytochemistry S2. Table Whole-mount material supplementary in given are work this in used stocks Fly METHODS AND MATERIALS the in chromosomes meiotic the on Yem of homozygotes.Hira absence in the with affected in observed is is effect frequency over Interestingly, what experience to crossing clear not is is extent our might it distribution However, in regions. centromeric observed and to crossover telomeric be 4 response can where in As 1978). data, chromosome context Szauter, and (Sandler a McKim, aberrant even and in Joyce 2006; recombination Indeed, al., et (Carlton 2010). occur not they do where locations normally chromosome in even form to crossovers allow rti opee tesnpoea ope n cohesin meiotic and the meiosis-specific complex on synaptonemal in metabolism. structure DSB chromatin (the and status complexes) of issues, complexes chromatin impact new protein raises the of work as gain this role such to Moreover, order the process. recombination in first investigations into the further for deserve here insight reported H3.3 data is Hira–Yem These kinetics the DSB time. of the function in complex The histone complex. Hira–Yem suggest dependent the is data prophase on meiotic early these during together, Yem of Taken function the partner. that Hira its on dependent lo 8 n lx lo 5 MlclrPoe)o lorbs546 Fluoprobes or Probes) Alexa (Molecular to 555 either Fluor coupled Alexa (Interchim). ImmunoResearch were and antibodies Jackson 488 secondary Fluor from other The were Inc. anti-rabbit-IgG Labs antibodies FITC-conjugated 1:400 anti-mouse-IgG or at Rhodamine- and antibody, used (Sigma). tag 1:200 anti-HA antibody, monoclonal at the anti-C(2)M used and Additional 1:500 rabbit 2003), 2010). McKim, at 2001), and al., used (Manheim et Hawley, antibody, Meyer (Aı and anti-C(3)G 1997; mouse (Page previously al., included et described antibodies Capri primary as 1992; al., were et antibodies Ahmed secondary fluorescent y–w n S ear(hgte l,20) ugsigta the that suggesting 2004), al., et (Bhagat repair DSB and ) HR1 ora fCl cec 21)17 6846 doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal akrud hrfr,terl fYmo S ieisis kinetics DSB on Yem of role the Therefore, background. nevlwt h xeto of exception the with interval hcpithsbe eotdta easpcyeeto pachytene delays that reported been has checkpoint a , yem uain fettetmn fDB.Asimilar A DSBs. of timing the affect mutations Hira yem 1 HR1 uain oecosvr omi the in form crossovers more mutation, c uatoais eutta si line in is that result a ovaries, mutant -His2Av yem a ahn n nuainwith incubation and washing ; 1 c(3)G nmitcrcmiainwe recombination meiotic on ,DBfrain( formation DSB ], mei-9 yem 1 h archetypal the , eae (Meyer females .elegans C. yem mei- and ¨t- 1

Journal of Cell Science ** h ifrn uatsok ihsok otiigappropriate containing are that stocks females generate with to markers stocks for chromosome heterozygous X crossing mutant by of age-dependent chromosome combinations avoid X different the to for discarded. used assayed were the was were parents the overs period laying, Crossing egg-laying egg effects. of 0–5-day the days the 5 exactly After Only mated in day. were same out females hatched the carried Freshly on experiments. were test experiments as conditions control same All over. crossing EERHARTICLE RESEARCH eiiain.W cnweg uinCuadNcl Lautre test Nicole statistical and as Cau for Julien input Montpellier) acknowledge their (INSERM, We for Artero verifications. Giard Sylvaine and/or Laure and stocks and students fly Schoenhals masters providing Matthieu for thank USA), UK) We Center, Edinburgh, antibodies. Research of (Doisy (University Dorsett Heck Lehner Dale Margarete Christian Switerland), USA), Zurich, Institute, of (Stowers (University Hawley (Dartmouth Scott Bickel Kim Sharon USA), USA), USA), College, University, (Indiana, Kathy (Princeton center thank Schupbach stock We Trudy (Rutgers Drosophila manuscript. McKim, McKim the Bloomington Kim the on and and comments work Matthews valuable her this his for of for UK) stage USA) Cambridge, early University, of an (Unversity at Carpenter suggestions Adelaide insightful to grateful are We Acknowledgements 50 extracts with cleared procedure diluted immunoprecipitation the The to experiments. was submitted blot then homogenate western were the cleared in input the as use centrifugation, 1 After to adjusted lysis final added. ice-cold mM of was 1 volume a buffer equal to an added homogenization, also previously Before was described concentration. PMSF tablets). as (Roche essentially cocktail inhibitor was 250 buffer in Lysis ice (Ja on buffer. manually lysis dissected cold were ovaries 100 indicated. protocol (Ja a previously used essentially described we experiments, co-immunoprecipitation analysis For blot western and Immunoprecipitations 25 at raised were flies The analysis frequency Recombination ihaBoa eieo es S 1 microscope. 510 LSM Zeiss a or device Biorad for either a used performed with was was microscopy microscope or Confocal DM600B epifluorescence. Probes) Leica conventional (Molecular A 555 (Interchim). Fluor Secondary 546 Alexa Antibody). Fluoprobes to pS137 either coupled H2AvD were Anti-Histone antibodies (Rockland 1:500 anti- rabbit at and mouse 1:500 included at antibodies used Primary antibody anti-C(3)G 2001). Hawley, and (Page previously 9 sesdwt h-qae et o l xeiet,* experiments, all For test. Chi-squared a with was significance Statistical S2). assessed Table material (supplementary used were white ehrs ed Sga r-nuae ihtepiayatbde [AS2 antibodies (10 primary polyclonal the rabbit with pre-incubated (Sigma) beads Sepharose nicMc(2 anti-c-Myc ope nimueIGadat-abtIGatbde (1:10,000; antibodies horseradish-peroxidase- anti-rabbit-IgG goat and were Beckman). antibodies anti-mouse-IgG et (1:400; (Gause Secondary coupled 1:500 at 2008). used anti-HA were by USA) al., used Center, provided Research (kindly Sigma), were Doisy antibodies Dorsett, antibodies polyclonal Dale anti-Rad21 polyclonal rabbit (1:400; rabbit and anti-Flag 1:100 AS2 at The M2 anti-c-Myc Yem used: supplier. Sigma). were monoclonal Sigma), the tags epitope by the (1:1000; was against recommended antibodies blotting as Western Western following Immobilon Western ECL substrate Millipore Pierce device. or Chemiluminescent either Scientific) Bio-Rad (Thermo using Substrate procedures a Blotting standard on with with out carried gels performed was acrylamide electrophoresis the SDS-PAGE 8% analysis. assess blot to western experiment to mock a reactions. in immunoprecipitation the used of was specificity antibody anti-Flag The a gre l,20)btwssplmne iha DAfe protease EDTA-free an with supplemented was but 2001) al., et ¨ger P For h rti xrcsadtertie rcin eete submitted then were fractions retained the and extracts protein The n eeoyosfor heterozygous and , nteohreemres ifrn e p fmre chromosomes marked of ups set different markers, eye other the on .1ad*** and 0.01 c HsA eeto,fmlswr isce n ie sdescribed as fixed and dissected were females detection, -His2Av m m nlssbfe.Toaiut f30 of aliquots Two buffer. lysis in l ;Sga,rbi hPgaeat-A(2 anti-HA grade ChIP rabbit Sigma), l; yem P , m 1 , 0.001. ) 2mueat-lg(2 anti-Flag mouse M2 l), mei-W68 vvfcar f v cv w y gre l,20)wt oemdfctosas modifications some with 2001) al., et ¨ger ˚ o l h rse eindt measure to designed crosses the all for C 1 , mei-P22 ocp ihteeittcefc of effect epistatic the with cope To . 103 , mei-218 c m HsA nioyused antibody -His2Av ;Sga,monoclonal Sigma), l; m du(otele RIO (Montpellier ´dou eestaiefor aside set were l 1 , c(3)G m ;Abcam)]. l; m 1 fice- of l P or , m mei- 0.05 lG- alo,P . argi,A .adDrbr,A F. A. Dernburg, and P. A. Farruggio, M., Aı P. and Carlton, M. Thomas-Delaage, J., J., M. Page, Santoni, A., M., J. Capri, Suja, R., Fuente, la de T., M. Parra, A., Viera, A., Calvente, C. J. Hall, and S. B. Baker, Herra Lehner, and K. Nasmyth, A., Schleiffer, S., Heidmann, S., Horn, D., Heidmann, apne,A T. A. Carpenter, Ho H., Scherthan, A., Kouznetsova, F., M. Guiraldelli, G., C. S. Bisig, K. McKim, and E. D. Sherizen, A., E. Manheim, R., Bhagat, and A. M. Lilly, A., Lai, S., K. McKim, L., S. Page, M., S. Royer, K., L. Anderson, Aı iao T. Hirano, W. C. Hinton, Eissenberg, A., R. Rollins, G., Haller, Z., Misulovin, A., H. Webber, M., Gause, Aı Aı References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.152520/-/DC1 online available material Supplementary material Supplementary Egyptian the from fellowship training A.A. PhD year). 4-year (1 Government. the ARC a from Fondation by and fellowships supported years) held (3 was scientifique R.E.M. program recherche training O.A.). PhD la to national de (grants French national ARC Centre Fondation the and by (CNRS) supported was work This Funding and read authors All manuscript. and manuscript. the A.A. final wrote R.E.M., the O.A. experiments. approved data. the experiments. the performed the C.F. interpreted and designed O.A. H.B. O.A. M.C., and A.A., A.A. R.E.M., R.E.M., study. the conceived O.A. contributions Author interests. competing no declare authors The interests Competing apne,A .adSnlr L. Sandler, and T. A. Carpenter, T. A. Carpenter, cngah ta al hs ftemanuscript. the of phase early an Patrick Menthie at analyses; de iconography deconvolution Sarrauste and Cyril microscopy and confocal Atger with help for Imaging) ¨ ¨ ¨ -he,O,Blo,B,Cpi . olt .adToa-eag,M. Thomas-Delaage, and C. Joblet, M., Capri, B., Bellon, O., t-Ahmed, -he,O,GifnSe,R,Vl,C,Cpi . hmsCvli,M and M. Thomas-Cavallin, M., Capri, C., Vola, R., Griffin-Shea, O., t-Ahmed, R. Rosset, and M. Thomas-Cavallin, O., t-Ahmed, ats .L,d aVg,C . abr,J .adRfs .S. 5 oocyte. J. a Drosophila Rufas, of and L. Implication J. divisions. Barbero, meiotic G., grasshopper C. in Vega, subunits 122 cohesin la of de Dynamics L., J. Santos, divisions. F. C. 66. eoi rpaeporsinadcosvrcontrol. crossover and progression prophase meiotic in pairing centromere homologous for required spermatocytes. are mouse and J. centromeres R. at Pezza, persist and S. D. Dawson, Press. Academic London: 352-434. pp. In segregation. Drosophila chromosome and recombination complex. USA synaptonemal Sci. Drosophila of Acad. region central the within C(3)G S. R. Hawley, specific protein nucleus. binding oocyte DNA Drosophila the new for a yemanuclein-alpha: The (1992). e.Biochem. Rev. melanogaster. Drosophila of mutant exhibits and synapsis fertility. meiotic in in dimorphism functions sexual RAD21L subunit A cohesin de The (2011). L., J. Barbero, A., cells. meiotic and somatic in cohesin D. and Dorsett, and Nipped-B E. S. Bickel, C., J. fdfeetal xrse aenlgns(eagn region). cluster a gene contains (yema which genes 153-162. genome maternal Drosophila expressed the differentially of of region a of characterization eobnto oue ntoallso h rspiamitcmtn mei- mutant meiotic Drosophila the of alleles two W68. in nodules the recombination of change temporal wild-type. and in arrangement, complex Structure, synaptonemal I. females. melanogaster Drosophila in over crossing of distribution females. the and mutants crossover-specific on melanogaster. D. Pardo, uat nDoohl melanogaster. Drosophila in mutants n . Gutie Y., ´n, 77-91. , 20) h rspiamitckesnC2Mfntosbfr h meiotic the before functions C(2)M kleisin meiotic Drosophila The (2004). Genetics ora fCl cec 21)17 6846 doi:10.1242/jcs.152520 4658–4666 127, (2014) Science Cell of Journal 20) hoooechso,cnesto,adseparation. and condensation, cohesion, Chromosome (2000). yoee.Gnm Res. Genome Cytogenet. Chromosoma 18) slto fdvlpetlyrgltdgnsi Drosophila in genes regulated developmentally of Isolation (1988). e.M sbre,E oisi .L asnadJ .Thompson), N. J. and Carson L. H. Novitski, E. Ashburner, M. (ed. 16) nacmn frcmiainascae ihtec3G the with associated recombination of Enhancement (1966). 20) utpsto fC2Madtetases iaetprotein filament transverse the and C(2)M of Juxtaposition (2005). re-aalr,C,Sa C., ´rrez-Caballero, il Cell Biol. 69 163 102 115-144. , 17) lcrnmcocp fmissi Drosophila in meiosis of microscopy Electron (1975). 20) omlsnpoea ope n abnormal and complex synaptonemal Normal (2003). 9 1337-1356. , eh Dev. Mech. 4482-4487. , oigsqec ntreigmtra RAt the to mRNA maternal targeting in sequence coding ´ LSGenet. PLoS eh Dev. Mech. 113 62 aa . eRoj .G,Sj,J . ln,E tal. et E. 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Journal of Cell Science ciol . tvne .adJsbre,R. Jessberger, and M. Stevense, F., McNicoll, ig .C. R. King, E. S. Bickel, and S. R. Khetani, S. S. K. K. McKim, McKim, and and F. A. E. E. Manheim, Joyce, R., Bhagat, E., D. Sherizen, K., J. Jang, Ja ee,R . eag,M,Rse,R,Cpi .adAı and M. Capri, R., Rosset, M., Delaage, E., R. Meyer, S. K. McKim, and S. Mehrotra, C., Steinberg, G., Chin, J., J. Sekelsky, L., B. Green-Marroquin, S., K. A. McKim, Hayashi-Hagihara, and S. K. McKim, M. S. J. Mason, K. McKim, and A. E. Manheim, siuo . i,J,Fjym-aaua . ao .adWtnb,Y. Watanabe, and S. Kato, S., Fujiyama-Nakamura, J., Kim, K., Ishiguro, ARTICLE RESEARCH 4666 Buono M., Galova, P., Mahr, F., Klein, aaeaa,S . unr .M,Bua,F,Rgku .P,d or P., Boer, de P., E. Rogakou, F., Baudat, M., J. Turner, K., S. Mahadevaiah, L. R. Glaser, and L. H. Chotkowski, P., J. Madigan, S. R. Hawley, and M. C. Lake, opn . onfy . nem,C,Laurenc C., Anselme, E., Bonnefoy, B., Loppin, S. Herra K. E., McKim, Llano, and N. Kato, K., J. 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