Journal of Cell Science bec ftebuut S eartkspaenvrhls.Mroe,rcmiainlDBrpi,a oioe by monitored as repair, DSB recombinational not is the bouquet Moreover, the in that impeded nevertheless. conclude is we pairing place Therefore, homologous stage. although takes bouquet that the show repair from we exit DSB study after present only bouquet, the place in takes that incorporation, the However, suggesting (DSBs), bromodeoxyuridine repair. breaks of DSB double-strand DNA for meiotic absence needed by triggered is is bouquet that response the damage DNA meiotic (ATR)-dependent related vratraierpi pathways. repair alternative over repair DSB for required recombination interhomolog promotes and centromeres by organized The Article Research rvddta h rgnlwr spoel ie n l ute itiuin ftewr or work the of distributions further all terms. License and Commons cited Creative properly same medium the is any to work in subject are reproduction original adaptation and the distribution (http://creativecommons.org/licenses/by-nc-sa/3.0/), that use, Attribution provided non-commercial Commons License Creative unrestricted the permits of Alike terms which the Share under distributed article Non-Commercial Access Open an is This (see movements and the On elongation of 2009). nuclear form sophisticated Kleckner, includes a which and absolutely possesses bouquet, Koszul yeast not fission 2004; hand, al., is al., other et the et but (Lee Harper recombination facilitates, it see and 2012; maize, pairing bouquet in homologous (see the and for, well required yeast that as budding formation, found In provided was 2009). been Kleckner, its have and Koszul explanations or chromosomes, accompany alternative homologous configuration, of but bouquet coming-together that the the to contribute that movements is is clustering movements. chromosome telomere view chromosomal far, conventional so telomere-assisted The studied cases by the In accompanied 2001). al., and et 2009), al., et (Phillips in For cluster the appearance. in canonical participates chromosome the species, from in animal deviations and nuclear example, few plant the fungal, a of at only arrangement range with wide area This a 2001). limited in Scherthan, conserved a see is in review, a cluster (for telomeres periphery prophase, all meiotic in which arrangement in chromosomal a is bouquet The Introduction words: Key In pair. of must formation in the and for chromosomes yeast meiotic crucial budding a is homologous in bouquet, function pairing the centromere chromosome meiosis, of that form in show exaggerated centromeres during We an is eukaryotes. segregation organisation among This conserved pole. Tetrahymena reductional widely opposite is the undergo that at arrangement centromeres and chromosome to pole one and at assembled telomeres crossovers form Tetrahymena to order In Summary 10.1242/jcs.112664 doi: 5873–5880 125, ß Science Cell of Journal 2012 August 15 Accepted ( correspondence for *Author 2 1 Loidl Josef eateto hoooeBooyadMxF euzLbrtre,Cne o oeua ilg,Uiest fVen,A13 ina Austria Vienna, A-1030 Austria Vienna, Vienna, of A-1030 University Laboratories, Biology, Perutz Molecular F. for Max Center (CIBIV), Laboratories, Vienna Perutz Bioinformatics F. Integrative Max for and Center Biology Chromosome of Department 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. Arabidopsis Tetrahymena eoi,Cnrmr,Buut hoooepiig Recombination pairing, Chromosome Bouquet, Centromere, Meiosis, ssrthdbuutad hrb,frhmlgepiig hsfnigad opeiu eot fteiprac of importance the of reports previous to adds finding with This arranged pairing. are homologue chromosomes for nucleus, thereby, and, elongated bouquet the stretched within ’s and, immensely, elongate nuclei prophase meiotic , 1, anradtselegans Caenorhabditis eoee lse ttencels(Armstrong nucleolus the at cluster telomeres , ,AnezaLukaszewicz Agnieszka *, [email protected] e se per u a encsayfrteaineto ooooslc nodrt rmt ooooscrossovers homologous promote to order in loci homologous of alignment the for necessary be may but , ) nyoeedo each of end one only eoi hoooebuutis bouquet chromosome meiotic 1 ahlA Howard-Till A. Rachel , Drosophila uayts eudrokasuyo eoi centromere meiotic of study a and undertook pairing in among behaviour prevalence (Subramanian meiotic its we investigate the bouquet to been eukaryotes, understand and the has centromeres better of clustering function for To centromere 2011). substitute bouquet, Hochwagen, to a and Tsai feature proposed (see 2009; of not pairing meiosis Shaw, the does and chromosome in Moore Particularly, of 2011). 2008; McKee, initiation Dawson, the and Stewart in 2006). Smith, centromeres and of (Davis extent large recombination a and to pairing it and on 2001), depend Hiraoka, and Yamamoto eroeedo h lnae eoi ulu Liland (Loidl nucleus meiotic the elongated at assemble 1976; centromeres the Notably, 1J). al., of clustered (Fig. of are 2004) end et Scherthan, Telomeres polymerization 1A–I). one Wolfe (Fig. the 1985) near 1956; dramatic al., by et (Ray, a Kaczanowski propagated undergoes microtubules is nucleus intranuclear which the prophase, elongation, different latter 2005). of meiotic Gorovsky, and the cells Collins During 2000; two Karrer, Only when (see fuse induced mitotically. types mating is which divides which micronucleus, meiosis, germline, the performs whereas the the process, division, amitotic represents an centromeres) cell possess by During not splits do micronucleus. chromosomes and (whose macronucleus generative macronucleus vegetative diploid polyploid a a nuclei, two possesses . hr sicesn vdnefrapeiul irgre role disregarded previously a for evidence increasing is There erhmn thermophila Tetrahymena Tetrahymena 1 n iaKoestler Tina and erhmn thermophila Tetrahymena sbuuti naai eagetsa n RAD3- and telangiectasia- ataxia an is bouquet ’s sauiellrclae rts.It protist. ciliated unicellular a is 2 . Drosophila which , 5873 Journal of Cell Science oadTl ta. 01.Ltr ulisotnadchromatin and shorten nuclei Later, 2011). During al., Dmc1 2010; et al., crescent. et Howard-Till Lukaszewicz protein become 2009; Mochizuki, chromosomes and recombination so-called homologous (Loidl and juxtaposed chromatin, formed, before on the foci are spindle-shaped, forms in DSBs then will elongation, cells drop-shaped, culminates of become nuclei pairs elongation meiotic first types, The meioses. mating will synchronous different initiate of and conjugate mixing cells In Upon 1. spherical. starved Fig. is in of nucleus shown germline is the dynamics cells, nuclear nonmeiotic meiotic of course time A prevents clustering Cna1 centromere protein centromeric of Depletion Results an play centromeres pairing. the chromosome that homologous that in that and role find function, show active also centromere we on We Here, depends bouquet. was DSBs. not do a repair that it nuclei in to form repaired are Hence, required DSBs expectation, this is to response 2009). contrary damage or DNA Mochizuki, helps a is that and elongation nuclear that (Loidl sensor hypothesized damage DNA ATR the on dependent kinase also al., and is et bouquet (Mochizuki The lesions elongate 2008). a clustering DNA induced in centromere artificially to DSBs by and restored of are elongation fail absence nuclear the However, nuclei in mutant. cluster because not do (DSBs), centromeres breaks strand nucleus. attaches elongating or the elongation drives to either chromosomes itself centromere-proximal centromere a the either or that of region tip suggests the 2006; nucleus at elongated centromeres Gorovsky, of the clustering and The 2008). (Cui al., et juxtaposed Mochizuki regions poles two homologous the between and parallel in stretched organisation bouquet arms extreme chromosome an with to centromere-speci leading 1J), the 10 (Fig. of bar: pole immunostaining Scale opposite by probe. detected telomere were a Centromeres with micronuclei. FISH elongating by in telomeres green) and (Tel, Cna1, telomeres and red) (Cen, centromeres 1. Fig. 5874 epciesaei otaudn seMciuie l,20;Lkseize l,21) cl a nA 10 A: in bar stages Scale meiosi Meiotic 2010). of process. al., induction the et after in Lukaszewicz time, participate the 2008; not indicate al., does bi panels et (MAC) forms the Mochizuki macronucleus underneath and (see Numbers vegetative (F) abundant 1974). The shortens Hiwatashi, most division. then and is (I) (C–E), (Sugai second stage Hiwatashi elongates and respective and (MIC) (H) Sugai micronucleus first to generative a according the classified in prophase, separated During are nuclei. which generative (G), their of meioses synchronous The Tetrahymena Tetrahymena ora fCl cec 2 (23) 125 Science Cell of Journal meiosis. oqe stigrdb eoi N double- DNA meiotic by triggered is bouquet ( A–I AIsandsae.()Goigclspirt ojgto.()Savn el fdfeetmtn yeascaeadinduce and associate type mating different of cells Starving (B) conjugation. to prior cells Growing (A) stages. DAPI-stained ) Tetrahymena bouquet spo11 m m. D eutn eoi ar,tewl-yeprnr ipae the displayed partners the of wild-type most whereas for the nucleus, Cna1p elongated pairs, of characteristic depleted meiotic been had resulting that seen cells was to cells staining background cells, unspecific S1E,F). 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(Fig. the where Immunostaining chromatin at nucleus cluster cells its wild-type 2A). centromeric (Fig. present, of the premeiotic highlighted still spherical minority during was the remained nucleus nucleus in generative generative the Importantly, lost growth. had partners xrsino sN a nue yteadto fCdCl of addition the by induced was dsRNA of expression n n 5 5 o tdigncerbhvori eoi,w ae wild-type mated we meiosis, in behaviour nuclear studying For osuyapsil oeo etoee nncerelongation, nuclear in centromeres of role possible a study To 0 el) ntennidcdcnrl 0%o cells of phenotype 100% RNAi control, This nucleus. non-induced normal-looking the a had In 100) cells). 200 m nAI ( A–I. in m MTT1 ealtini rmtr and promoter, metallothionein J xmlso h itiuinof distribution the of Examples ) CNA1 eein(Cervantes deletion , 0h nthe In h. 30 cna1 ,we the when s, i histone fic valents – are I–V CNA1 RNAi 2 to Journal of Cell Science hoooe otetpo h lnaignces u o for not but nucleus, elongating the of elongation. connecting tip driving for the required of to an are chromosomes centromeres delineated extension 1976; Therefore, also 2C,F). al., tubulin (Fig. chromatin-free et Indeed, Wolfe 1992). elongated 1975; Fleury, and LaFountain, Gaertig and (Davidson end. nuclei one against at immunostained behind we left interpretation, a is this chromatin for of support mass add between To a stretch and poles, not two do the centromeres disabled with chromosomes AI(le nB n .Saebri :10 D: in (F) chromatin. bar of Scale staining F. background strong and a C is B, There in staining. Cna1p (blue) (D,E) DAPI nucleus. the of (arrows) part 1) ttebgnigo ula lnain centromeres elongation, nuclear of beginning Fig. the material supplementary At therein; references (Mochizuki S1E). and telophase 2008, poleward al., preceding the et of during the consequence orientation reflects a are probably centromere This orientation, centromeres meiosis. Rabl cells, of onset chromosomal untreated the (cf. In to nocodazole prior 1985). inhibitor clustered al., microtubule et the possibility with Kaczanowski this cells tested of to We treating due failure microtubules. is by capture the tip to nuclear that inability the to their possible attach is to centromeres it Cen1p-depleted Therefore, and kinetochore, Santaguida 2009). (see the microtubules Musacchio, to complex, chromosome protein the links a which assembles centromere regions chromosome The intercalary and of microtubules pairing on promotes dependent is clustering Centromere nucleus. probably the of appendix that portion seems This elongated it Therefore, chromatin-free 2B,D,E). (Fig. a ectopic represents the nucleus Instead, from 2006). projecting structure Gorovsky, vegetative thread-like occurs and a only along Cui deposited this free (see Cna1p since phase of centromeres, S into transfer during However, incorporated the 1966). not (McDonald, was to partner it due wild-type the is from protein which partner, Cna1p-depleted portion nuclear chromatin-free and chromatin-containing the as 10 well in A: as expression in partner, the Cna1p wild-type bar of In the explanation Scale (arrow). in an partner. nucleus (For nucleus elongated end. wild-type the one outlines the at (orange) clustered of staining remains tip chromatin the the while in nucleus, centromeres elongated clustered reveals (green) Cna1p cna1 wild Conjugating 2. 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RNAi feogtdnce n h eutn aallarneetof arrangement parallel ends that intercalary of resulting opposite pairing regions. homologous confirms the the at supports and in arms telomeres This chromosome nuclei frequency and elongated 3C,D). the centromeres (Fig. of of of non- clustering in nuclei 10.0% the pairing to bouquet that reduced clusters found elongated was centromere We disrupting nuclei 2009). on elongated Mochizuki, effect nocodazole, and using stronger result (Loidl a this 2009). has extended Mochizuki, fluorescence and which and confirmed (Loidl a we MICs Here, is non-elongating of region in chromosome pairing reduced intercalary (FISH)-marked that hybridization benomyl inhibitor 3B). to was centromere (Fig. cells centromeres clustering nocodazole-treated Therefore, of telomere in abolished Notably, ability also centromeres. microtubules. the with on of that associate depends indicates clustering probably This clustering also the but 3A). elongation nuclear (Fig. for for required wild 2009) cluster only the not to are Mochizuki, in failed microtubules centromeres 59% and Moreover, to stage). compared (Loidl same as the completely scored induction, at cells type meiosis was and conjugating efficiently after 200 added elongation h in 3.5 Loidl nuclei induction, was nocodazole elongated meiosis (no nocodazole 2008; suppressed expected, after When al., min fully elongation. 110 As the nuclear et in prevented 2009). (Mochizuki re-assemble Mochizuki, and nucleus dispersed elongated transiently become nmitctlmr tahett h ula eihr and periphery 2007; Scherthan, nuclear (see organisms the of role range to a crucial in a attachment formation play bouquet telomere to found meiotic been have Mps3, in proteins Ndj1/Tam1, related Rap1, and as Taz1 such proteins proteins Telomere-associated bouquet-related for search A twssonpeiul ytetetwt h microtubule the with treatment by previously shown was It cna1 Niprnr n1 eietstecnor fteempty the of contours the delineates Cna1p partner, RNAi eoi etoeecutrn 5875 clustering centromere Meiotic a tblnsann oag) hoai ssandwith stained is Chromatin (orange). staining -tubulin cna1 Nicls e h antx. (C) text.) main the see cells, RNAi nthe in s cna1 a nsitu in -tubulin RNAi Journal of Cell Science xeiet ih2 ulieautdpreprmn.Errbr niaes.d. indicate 10 bars C: Error and experiment. three B per from evaluated A, average nuclei in the 25 bars represents with Scale column experiments Each C. pairing. and of hence B Quantification burst, in (D) section) seen Methods size. and are Materials larger nuclei the their isolated (see by FISH nuclei for non-meiotic prepared non-elongating from Cells in discriminated signals are in unpaired These signals of nuclei. paired Examples of Right: Examples nuclei. Left: elongated non-elongating (C) and nuclei. elongated meiotic in (nocodazole-treated) (pink) region chromosomal ( intercalary and panels). clustering (lower panels). telomere elongation (upper and nuclear cells centromere untreated prevents the in treatment at nuclei Nocodazole clusters bouquet (Tel) elongated telomere of and ends arrows) (Cen, centromere highlight (green) Ss n ehpteie htteprle arrangement parallel the that hypothesized we to response and a is formation DSBs, bouquet that shown previously have We is formation bouquet when prevented even repaired are DSBs known this to similarity However, distant a 2010). proteins. with hits telomere-associated only al., produced domains also http:// et protein search (FACT; Koestler compared Tool Comparison also fact.cibiv.univie.ac.at; Architecture sequence we Feature poor the the 2009), generally using Since Price, is any. and proteins detect (Linger telomere-associated to of failed conservation similar we by However, 1997). periphery the of cerevisiae homologues we to nucleus, for attached the the proteome BLAST-searched are we 2008; of Therefore, proteins. al., end they telomere-associated et centromere-distal if Conrad the wondered 2007; at in al., clustered Since et are 2009). Bupp Dernburg, and 2007; Hiraoka al., et Schmitt and formation bouquet prevents microtubules pairing. of Inhibition 3. Fig. 5876 ( A muotiigo rti n1 rd n ( and (red) Cna1p protein of Immunostaining ) ora fCl cec 2 (23) 125 Science Cell of Journal d1o a1o ua r rtis(lshle al., et (Altschul proteins Trf human or Rap1 or Ndj1 C .pombe S. , D arn faFISH-labelled a of Pairing ) Tetrahymena a1o a1and Rap1 or Taz1 B eoeeFISH telomere ) telomeres , m m. S. hra hoooe htaefamne ymitcDSBs meiotic by fragmented are well, loading that previously the in chromosomes have stay chromosomes whereas We intact by PFGE, long monitored in (PFGE). that were found confirmation, electrophoresis repair unduly independent gel and DSB-formation from an pulsed-field of as dynamics proteins Therefore, the are recombination DSBs. DSBs persisting of that indication detachment strong form. not were a does bouquet they a is when that even This found repaired 4A). and nocodazole-treated (Fig. the in induction, culture and control untreated meiotic the in after both Dmc1p reduced of h presence the 5.5 for assayed foci next We when formed. induced is efficiently bouquet are no DSBs that (170 indicates This control 2011). al., the et of nuclei elongated n in (179 foci nuclei of bouquet number non-elongated The in suppressed 2011). foci al., n et Dmc1p we Howard-Till immunostained (see of indeed DSBs then number Again for We marker is a nocodazole. 2008). as repair. bouquet to Dmc1p al., exposure facilitates DSB the by et formation whether nucleus for (Mochizuki investigated elongated required DSBs we the of Therefore, repair within recombinational chromosomes of sadtoa ro htDB r eardi h bec of absence the in repaired are DSBs This that formation. 4B). bouquet (Fig. proof that, meiosis found additional nocodazole-treated in we is disappear points Here DSBs time of 2010). late diagnostic at bands al., meiosis, et untreated to (Lukaszewicz similar gel the enter tgs u xrml aeyi aial lnae nuclei elongated subsequent maximally and 1) in Fig. rarely in 4C). extremely (Fig. V but Stage shortening in stages, to foci BrdU (corresponding at observed cells we nuclei In cells, conjugation, 1987). of al., conjugating labelled et stages early heavily Allis later (see or observed S-phase unconjugated fixed We delayed of undergo and initiation. which minority elongate) after a to h in start 5 nuclei or just h nuclei 4.5 when of cells initiation time the a after (at h meiosis 2–2.5 (BrdU) bromodeoxyuridine to In the cells about vague therein). were place. authors references takes these but it 1987), when and al., stage et 2007; (Allis al. et al., et Prensky, Tetrahymena DNA and Terasawa Wimber (cf. repair. 1963; analogues base detected recombinational of DNA be (immuno)labelling for accompanies can assayed we To synthesis that window, 2011). time al., this synthesis et within (Howard-Till we repair emerge gone pinpoint virtually bouquet, bivalents and distinct nuclei the are elongated when foci, maximally exiting g-H2A.X in observations and Dmc1p abundant for Previous DSBs, most of place. signal markers takes that of shown the repair have repair DSB likely the when that not wondered thus, for and required is DSBs not DSBs of is repair formation recombinational bouquet the that recognized we bouquet Since the of during collapse place the takes synthesis DNA Recombination-related opee earcnntb h inlt xttebuutstage. bouquet therefore the exit shortening, to signal started the are be have of not DSBs nuclei can absence that repair the after completed demonstrates in only experiment labelled This repaired not 4C). 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(Takeo but exists centromere, sites al., initiation situation et synapsis Tanneti analogous are sites and An prophase initiation 2008). synapsis al., Drosophila preferred were et be centromeres (Tsubouchi to yeast, budding the reported In among consistently synapse. often are to any they regions therefore, In earliest and 2009). centromeres regions, of Shaw, adjacent pairing and/or homologous and the (Moore promotes clustering wheat case, in in pairing pairing 2008). centromere centromere Dawson, autonomous and be Drosophila Stewart may of recognition exceptions (see the Possible themselves by than centromeres and rather homologous regions the the (Obeso arm by adjacent driven formation of of be assortment pairing the to DSB cases, seems most centromere centromeres In on 2010). homologous to al., dependent contrast et Falk In 2010; is Dawson, 2012). al., this et coupling, Ines Da 2008; Dawson, opigwsas ouetdin of documented succession centromere also a pairwise was Recently, nonhomologous coupling 2008). and clustering Dawson, centromere and Stewart Roeder, and 2005; 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al., et r CNA1 Arabidopsis psri.The strain. hp D nse al., et Ines (Da spo11 knockout Journal of Cell Science oynmeso netdtasei N,anoyi eitnegn,adthe and gene, resistance neomycin a DNA, transgenic inserted Cd of numbers copy el yboitctasomto t1 r otmxn oalwclst rcs the process to cells allow to mating post-mixing ( into hrs Transformants 10 vector. introduced at rDNA was transformation biolistic JLTet3 by JLTet3[CNA1-HP]. cells creating thereby vector, The Nikokonof knockdown administered RNAi and medium starvation 100 in of solution concentration stock final mg/ml a 2 at a as prepared was in induced were starvation meiosis by types. and 10 mating Conjugation conjugation h. different for 16 of least competent cultures at starved made for mixing 7.4) were by (pH they Tris–Cl mM and 10 2000), al., et eefxdadpeae codn ooeo h olwn ehd.()5ml 5 (A) methods. and following tube, centrifuge the a of to transferred one were 250 suspension to cell cells according (conjugating) FISH, of prepared or aliquots immunostainings and different for Southern fixed slides of were use by further the on staining Depending gel) nucleus-borne and preparation cytological the Fixation, vegetative al., et along (Lukaszewicz the nucleus from all generative of the Chromosomes to scattered specific distinguished 2010). band. probe a are distinct with were hybridization a (which as chromosome nucleus minichromosomes appear DSB-dependent whereas sizes gel, where generative different the conditions of under enter PFGE not previously fragments by do described separated was chromosomes as DNA plugs intact 2010). agarose al., in et prepared (Lukaszewicz was DNA Chromosome-sized (PFGE) electrophoresis gel Pulsed-field is which rDNA, pD5H8-MTT- contains vector vector (hp) This the hairpin 2006). restriction in RNAi Yao, appropriate amplified the and add into to (Howard-Till back-to-back primers SerH3-HP cloning PCR using for DNA sites genomic from amplified a create To n. ulnae A)splmne ih05m/lDP Hwr-ile al., were et (Howard-Till preparations DAPI the mg/ml Laboratories 0.5 (Vector and with secondary agent supplemented 2011). anti-fading previously fluorescence-labelled CA,) Vectashield Burlingame, described and in Inc., coverslip Primary as a under min). applied mounted (5 were X-100 antibodies Triton 0.05% + and slide a to (methanol, by applied were fixative collected suspension resuspended this of and was of washed drops ml pellet were and 1 cells ethanol, cell the 70% in air-dried. h of the 1 ml resuspended After and 1 in 6:3:2). was tube acid suspension acetic pellet centrifuge cell chloroform, (conjugating) of a The aliquots ml to centrifugation. 5 (D) transferred air-dried. and were slide a to 50 applied in resuspended methanol, 120 of random presence to the due in CdCl copies of absence grown the rDNA in were starved CNA1hp-carrying then were cells cells of These mitoses, paromomycin.) loss during minimize assortment (To h. 30 120 ouin rpo hsmxuewssra noasieadardid B ml 5 (B) air-dried. and tube and centrifuge slide a a to onto transferred were spread suspension 500 was 1,000 cell mixture at (conjugating) this min of 500 2 aliquots of for drop in A centrifuged resuspended solution. was suspension was cell pellet fixed the min, 30 transformation by re-created repeatedly be the to had of pJLTet3. 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Yao, and Howard-Till (see promoter metallothionein CNA1 m 0 Triton+50 10% l m CNA1 m /l oices h ubro DAcopies rDNA of number the increase to g/ml, eoeb uooosrpiain nuighigh ensuring replication, autonomous by genome fmtao,addoso hssseso were suspension this of drops and methanol, of l m 2 /lt ojgtn cells. conjugating to g/ml a de oepnnilygoigclsfrca. for cells growing exponentially to added was m f4 aaomleye+34 sucrose 3.4% + paraformaldehyde 4% of l p eeslce ntal nmdacontaining media in initially selected were hp) m m m f3%fradhd eeadd After added. were formaldehyde 37% of l f3%fradhd eeadd After added. were formaldehyde 37% of l fprilShuinsfxtv (saturated fixative Schaudinn’s partial of l a tbln(:0,muemonoclonal, mouse (1:300, -tubulin m 7 omleye eeadded. were formaldehyde) 37% l m 6 /l o fiin depletion efficient For g/ml. B (2 PBS SERH3 MTT1 6 i)ad1 and min) 5 rget nthis in fragments CNA1 2 rmtr the promoter, n ae to mated and g R was ORF n the and , 6 m m g/ml PBS lof rjce speiul ecie eg od n ohzk,20;Howard-Till 2009; Mochizuki, and and Loidl 2011). deconvolved (e.g. al., recorded, et described were images previously of as stacks projected 3-D documentation, For filters. Cna1 procedure. simultaneous immunostained, FISH then the For FISH, to for 2004). subjected as subsequently prepared Scherthan, and were slides and FISH, telomere Loidl and with staining detected (see and hybridized avidin produced, were FITC-labelled repeats oligonucleotides subtelomeric denatured biotinylated see and telomeres, details telomeric were detecting (for of For Cy3-labelled 2009). probe probes was Mochizuki, PCR-generated region and chromosomal a DNA Loidl intercalary pairing, kb testing formamide. 22.1 For a slides. 70% against the to in hybridized and denatured separately and thiocyanate u,B .adGrvk,M A. M. Gorovsky, and W. B. Cui, L. S. Jaspersen, and S. E. Stensrud, E., A. Martin, M., J. Bupp, aIe,O,Ae . obl,C,Gleo .E n ht,C I. C. White, and E. M. Gallego, C., Goubely, K., Abe, O., Ines, Da A., Shinohara, H., Kosaka, M., Shinohara, G., Chao, C.-Y., Lee, N., M. Conrad, A. M. S. Gorovsky, H. and Malik, K. and Collins, C. M. Yao, D., Vermaak, X., Xi, D., M. Cervantes, Ho H., Scherthan, A., Kouznetsova, F., M. Guiraldelli, G., C. H. Bisig, G. Jones, and H. C. F. Franklin, J., S. Armstrong, Scha L., T. Madden, F., S. Altschul, Acknowledgements 2 a (from synthesis BrdU DNA recombination-related of Detection li,C . oaioSeasi .adGrvk,M A. M. Gorovsky, and M. Colavito-Shepanski, D., C. Allis, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.112664/-/DC1 online Deutsche available material Supplementary the immediate by for PMC in supported Deposited Vienna Haeseler]. number release. von of [grant Arndt is to 1174 University 1628/9 SPP HA the program T.K. by priority Forschungsgemeinschaft and I031-B. P23802] [grant Initiativkolleg (FWF) and Fund Science P21859 Austrian the numbers by supported was work This Funding antibody. Research Cna1 Cancer the Hutchinson (Fred for Malik Seattle) Harmit Center, to grateful are We rUfdclspeae ypoeue()wr ahdwt ae 5mn and min) (5 90 water at with thiocyanate darkroom washed sodium of were Slides red M meiosis. (A) 1 under of procedure with induction done after by incubated min prepared were 15 cells h BrdU BrdU-fed 4 to harvested were exposed Cells illumination. cultures with manipulations 2 nioy(:0 ba,Cmrde K a ple t4 at applied was UK) Cambridge, Abcam, (1:40; antibody isdwt 2 with rinsed loecnelble eodr nioyo h etday. next the on antibody secondary fluorescence-labelled eauaintetetwssopdb rnfrt c-odwtr olwdby 2004). followed Scherthan, water, and ice-cold to Loidl transfer (cf. by antibody stopped 1 the was to treatment Denaturation nucleotides labelled expose 6 6 ldswr vlae ne loecnemcocp ihteappropriate the with microscope fluorescence a under evaluated were Slides sodium M 1 with pretreated were (D) procedure by made slides FISH, For rpaei rmtdb D1 P3 n S4adi ouae by modulated Tetrahymena is in conjugation during and elimination DNA CSM4 thermophila. for and and division cell vegetative MPS3, NDJ1, by promoted recombination. is E. M. prophase Dresser, and J.-A. Conchello, R317-R318. domain mouse Sad1-UNC-84 yeast in budding the pairing requires Mps3. periphery protein nuclear centromere the at homologous anchoring for required J. are spermatocytes. R. and Pezza, centromeres and S. D. Arabidopsis in synapsis chromosome meiotic thaliana. precede pairing and clustering telomere eurmnsfrRD1adDC nmitcpiigo etoee and centromeres of pairing meiotic in DMC1 thaliana. Arabidopsis and in arms RAD51 chromosome for requirements chromosome for essential is micronucleus. thermophila germline Tetrahymena the ciliate in segregation the of histone CNA1 programs. search database J. D. Lipman, Tetrahymena. conjugating in development Biol. during synthesis DNA unscheduled 10 B (2 PBS 2 4 124 ocnuaigcls2o fe nuto fmiss All meiosis. of induction after h 3 or 2 cells conjugating to M 6 469-480. , .Cl Sci. Cell J. i)ad1 and min) 5 6 o.Cl.Biol. Cell. Mol. S n eaue n7%fraiefr2mntsa 65 at minutes 2 for formamide 70% in denatured and SSC 6 .Cl Biol. Cell J. 19) apdBATadPIBAT e eeaino protein of generation new a PSI-BLAST: and BLAST Gapped (1997). LSGenet. PLoS Cell 10 2 1 133 eoi etoeecutrn 5879 clustering centromere Meiotic 114 tc nDS)wsaddt ia ocnrto of concentration final a to added was DMSO) in stock M 1175-1187. , 6 4207-4217. , B .5 rtnX10( i) a anti-BrdU Rat min). (5 X-100 Triton 0.05% + PBS uli cd Res. Acids Nucleic 21) yatnmlcmlxcmoet ess at persist components complex Synaptonemal (2012). 179 8 26 e1002701. , 845-854. , 4499-4510. , fe,A . hn,J,Zag . ilr .and W. Miller, Z., Zhang, J., Zhang, A., A. ¨ffer, 20) erhmn thermophila. Tetrahymena (2005). 20) etoei itn 3i seta for essential is H3 histone Centromeric (2006). 20) ai eoeemvmn nmeiotic in movement telomere Rapid (2008). o.Bo.Cell Biol. Mol. LSGenet. 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