RESEARCH ARTICLE

Conservation of chromosomes syntenic with avian autosomesin squamate reptiles revealed by comparativechromosome painting

Martina Pokorná & Massimo Giovannotti &Lukáš Kratochvíl & Vincenzo Caputo & Ettore Olmo &

Malcolm A. Ferguson-Smith & Willem Rens

Received: 21 January 2012 /Revised: 5 April 2012 /Accepted: 10 April 2012 /Published online: 18 May 2012# Springer-Verlag 2012

Abstract In contrast to mammals, birds exhibit a slow rate ofchromosomal evolution. It is not clear whether high chromo-some conservation is an evolutionary novelty of birds or wasinherited from an earlier avian ancestor. The evolutionaryconservatism ofmacrochromosomes between birds and turtlessupports the latter possibility; however, the rate of chromo-somal evolution is largely unknown in other sauropsids. Insquamates, we previously reported strong conservatism of thechromosomes syntenic with the avian Z, which could reflect apeculiarity of this part of the genome. The chromosome 1 ofiguanians and snakes is largely syntenic with chromosomes 3,5 and 7 of the avian ancestral karyotype. In this project, weused comparative chromosome painting to determine howwidely this synteny is conserved across nine families covering

most of the main lineages of Squamata. The results suggestthat the association of the avian ancestral chromosomes 3, 5and 7 can be dated back to at least the early Jurassic and couldbe an ancestral characteristic for Unidentata (Serpentes, Igua-nia, Anguimorpha, Laterata and Scinciformata). In Squamatachromosome conservatism therefore also holds for the parts ofthe genome which are homologous to bird autosomes, andfollowing on from this, a slow rate of chromosomal evolutioncould be a common characteristic of all sauropsids. The largeevolutionary stasis in chromosome organization in birds there-fore seems to be inherited from their ancestors, and it isparticularly striking in comparison with mammals, probablythe only major tetrapod lineage with an increased rate ofchromosomal rearrangements as a whole.

Communicated by: Erich Nigg

Martina Pokorná and Massimo Giovannotti contributed equally to thepaper.

M. Pokorná : L. Kratochvíl (*)Faculty of Science, Department of Ecology,Charles University in Prague,Viničná 7,128 44 Prague 2, Czech Republice-mail: lukkrat@email.cz

M. PokornáInstitute of Animal Physiology and Genetics,Department of Vertebrate Evolutionary Biology and Genetics,Academy of Sciences of the Czech Republic,Rumburská 89,277 21 Liběchov, Czech Republic

M. Giovannotti :V. Caputo : E. OlmoDipartimento di Scienze della Vita e dell’Ambiente,Università Politecnica delle Marche,via Brecce Bianche,60131 Ancona, Italy

M. Pokorná :M. Giovannotti :M. A. Ferguson-Smith :W. RensCambridge Resource Centre for Comparative Genomics,Department of Veterinary Medicine, University of Cambridge,Madingley Road,Cambridge CB3 0ES, UK

V. CaputoIstituto di Scienze Marine, Sezione Pesca Marittima,Consiglio Nazionale delle Ricerche,Largo Fiera della Pesca,60125 Ancona, Italy

Chromosoma (2012) 121:409–418DOI 10.1007/s00412-012-0371-z

Introduction

In contrast to mammals, birds exhibit a slow rate of changein chromosome number and interchromosomal rearrange-ments (e.g. Ferguson-Smith and Trifonov 2007; Griffin etal. 2007; Ellegren 2010), although their rate of intrachromo-somal rearrangements is substantial (Skinner and Griffin2012). Ellegren (2010) referred to conservatism in aviankaryotypes as a notable case of evolutionary stasis ascribedto their small genome size. The decrease in genome size inthe ancestor of the avian lineage was associated with theextensive elimination of repetitive sequences includinggenome-wide interspersed mobile elements (Organ et al.2007; Chapus and Edwards 2009). As chromosome rear-rangements tend to occur at sites of repetitive elements (e.g.Kejnovský et al. 2009), it was speculated that the conserva-tism of genome organization in birds could be related to therelatively low frequency of repetitive elements in theirgenomes (Ellegren 2010; cf. to the high content of inter-spersed repetitive elements in mammalian genomes, e.g. inOrgan et al. 2007). Nevertheless, it is not clear whetherchromosome conservation is an evolutionary novelty ofbirds, or whether it is typical of all sauropsid lineages(Sauropsida is a clade containing birds, crocodiles, turtles,tuataras and squamates). The strong evolutionary conserva-tism of macrochromosomes for instance between birds andthe Chinese soft-shelled turtle (Pelodiscus sinensis; Matsudaet al. 2005) and a low variability in the number, morphologyand G banding pattern of chromosomes in turtles and croc-odiles (reviewed in Olmo 2008) suggest that chromosomeconservation might not be restricted to birds. However, withthe exception of birds (Griffin et al. 2007), the ancestralkaryotype has not been reconstructed by molecular cytoge-netic methods for any sauropsid lineage so far and theconservation of chromosomal synteny within particular rep-tilian lineages is still largely unknown.

The determination of the rate of chromosomal changes inSquamata, the clade encompassing lizards, snakes andamphisbaenians, could be especially informative in this re-spect. In contrast to the 300 turtle species and the 23 species ofcrocodiles, this ancient lineage currently contains about 8,200described species that exhibit great karyotype variability(reviewed in Olmo 2008). However, the knowledge on syn-teny of squamate and avian chromosomes enabling the esti-mation of rates of chromosomal rearrangements is restricted toa single species (green anolis, Anolis carolinensis; Alföldi etal. 2011) and only particular parts of genomes in a few otherspecies (e.g. Matsubara et al. 2006; Srikulnath et al. 2009).Whole-genome sequencing revealed relatively high conserva-tion in the organisation of most parts of the genomes in thelineage leading to A. carolinensis after its split from the avianlineage (Alföldi et al. 2011; http://www.ensembl.org). Unfor-tunately, at present, there are only a fewmolecular cytogenetic

comparative studies across or within basic squamate lineages(but see Giovannotti et al. 2009 for a demonstration of chro-mosomal conservatism in the family Scincidae and Trifonovet al. 2011 in selected species of the family Gekkonidae).

Recently, we conducted a comparative study on the partsof genomes homologous with the avian Z sex chromosomein reptiles (Pokorná et al. 2011). We observed a strongconservation of the chromosomes syntenic with the avianZ in a turtle and a crocodile species as well as across mostmajor squamate lineages, despite the divergence of thelineages leading to extant birds and squamates, which oc-curred about 275 Ma (Shedlock and Edwards 2009). Theconservation cannot be attributed to the function of the Zchromosome as a sex chromosome because it is autosomalin most reptilian lineages (Pokorná et al. 2011). Neverthe-less, conserved synteny may still be an exclusive property ofthis chromosome. A similar study devoted to parts of squa-mate genomes syntenic with avian autosomes is needed torefute this possibility.

The largest metacentric chromosome of two species ofiguanian lizards, the agamid Leiolepis reevesii (Srikulnath etal. 2009) and the iguanid A. carolinensis (Alföldi et al. 2011),and the colubrid snake Elaphe quadrivirgata (Srikulnath et al.2009) is largely syntenic with chicken (Gallus gallus, GGA)chromosomes 3, 5 and 7, which are syntenic with thecorresponding chromosomes in the avian ancestral karyotype(Griffin et al. 2007). The annotated genome of A. carolinensisshowed that the largest chromosome of this species also con-tains a part syntenic with a small region on GGA13 micro-chromosome (http://www.ensembl.org). In all three squamatespecies, an arm of the largest chromosome is largely syntenicwith GGA3, while the proximal and distal parts of the otherarm are syntenic with GGA7 and GGA5, respectively. How-ever, this conservation may be explained by the fact thatiguanian lizards and snakes seem to be members of the samephylogenetic lineage, Toxicofera (Vidal and Hedges 2005).Here, using comparative chromosome painting, we test howwidely this synteny is conserved across nine families coveringmost of the main lineages of Squamata.

Materials and methods

Species studied

We selected 10 species of squamate reptiles from ninefamilies representing most of the main lineages in the taxo-nomic nomenclature and phylogeny suggested by Vidal andHedges (2005). We included members of the clades Scinci-formata (Scincidae), Teiformata (Teiidae), Lacertibaeniacomposed of Lacertiformata (Lacertidae) and Amphisbaenia(Trogonophidae), and Toxicofera (Iguania: families Agami-dae and Iguanidae; Anguimorpha: families Varanidae and

410 Chromosoma (2012) 121:409–418

Anguidae; Serpentes: Colubridae). For the list of the speciesand sex of the individuals examined, see Table 1.

All squamate individuals were captive bred animals main-tained in the laboratory breeding rooms at the Faculty ofScience, Charles University in Prague, Czech Republic(accreditation No. 24773/2008-10001) and in the Diparti-mento di Biochimica, Biologia e Genetica, Università Poli-tecnica delle Marche, Ancona, Italy. The blood sample ofAnguis fragilis was kindly provided to MG and VC by Dr.Raffaele Gattelli (Aquae Mundi–Ravenna, Italy).

Preparation of metaphase chromosomes and probes

Metaphase chromosome spreads were prepared from cultures ofwhole blood following the protocol described in Ezaz et al.(2005) with slight modifications. The paints from chicken auto-somes were prepared from chromosomes sorted with a duallaser cell sorter (Mo-Flo, Dako) at the Cambridge ResourceCentre for Comparative Genomics, Department of VeterinaryMedicine, University of Cambridge, Cambridge, UK, as previ-ously described (Yang et al. 1995). Sorted chromosomes wereused as templates for DNA amplification by degenerateoligonucleotide-primed polymerase chain reaction (DOP-PCR, Telenius et al. 1992). Primary DOP-PCR product wasused as a template in a secondary DOP-PCR to incorporatebiotin-16-deoxyuridine triphosphate (Roche).

Fluorescent in situ hybridization and signal detection

Fluorescence in situ hybridization (FISH) was performedusing the protocol described in Yang et al. (1995) and Renset al. (1999, 2006) with several modifications. Briefly, slideswere dehydrated through an ethanol series, aged for 1 h at65 °C, denatured in 70 % formamide/0.6× saline sodiumcitrate (SSC) at 70 °C for 1–4 min (time depending onspecies and metaphase preparation) and dehydrated again.Eight microlitres of biotinylated probe were precipitated in

ethanol and resuspended in 11 μl of hybridization buffer[40 % deionized formamide (v/v), 10 % dextran sulphate, 3×SSC, 0.05 M phosphate buffer, pH 7.3]. This mixture wasdenatured for 10 min at 75 °C, preannealed at 37 °C for30 min and applied to each slide. Hybridization was carriedout at 37 °C for three nights. Posthybridization washes wereperformed in 40 % formamide/1.8× SSC twice for 5 mineach, followed by 2× SSC twice for 5 min each and 4× SSCwith 0.05 % Tween-20 (4× T) once for 4 min. Washes werecarried out at 42 °C. Probe detection was carried out using200 μl of diluted (1:500) Cy3-Streptavidin antibody (Amer-sham) per slide at 37 °C for 30 min. After detection, slideswere washed in 4× T three times for 3 min each at 42 °C andmounted in Vectashield Mounting Medium with DAPI(VECTOR Laboratories).

Microscopy and data analyses

Images were captured using the Leica DMRXA microscopeequipped with CCD camera (Photometrics Sensys). LeicaCW4000 FISH software (Leica Microsystems) was used tocapture grey-scale images and to superimpose the sourceimages into colours to visualize the results of the FISH.Image enhancement was performed with a custom-madesoftware module written in Perl. This module distinguishesthe signal (red) from nonspecific hybridization (dim red)and from relatively bright spots (presented in green) causedby the low temperature wash. The final composition of theimages was performed in CorelDraw X5 software (CorelCorporation).

Results

The images in Fig. 1 are ordered according to the phyloge-netic position consistent with Table 1 and Fig. 2. TheGGA3, 5 and 7 probes hybridized to the same regions ofthe largest biarmed chromosome pair in six species: Varanusacanthurus (Fig. 1a–c), Pogona vitticeps (Fig. 1j–l), Iguanaiguana (Fig. 1m–o), Trogonophis wiegmanni (Fig. 1p–r)and Chalcides ocellatus (Fig. 1β–δ). In all these species,the GGA3 paint covered the q-arm of the largest chromo-some, while the GGA5 and GGA7 paints hybridized to thedistal and proximal parts, respectively, of the p-arm of thelargest chromosome. In the skink Tiliqua scincoides(Fig. 1y–α), the GGA3 and GGA7 paints showed the samepattern, while the GGA5 probe hybridized to distal parts ofboth q- and p-arms of the largest chromosome suggestingintrachromosomal rearrangement. The signal after hybrid-ization with the GGA3 probe was present on one arm ofthe largest chromosome in the agamid P. vitticeps and alsoon the other chromosomes possibly as background. TheGGA3 and GGA5 paints hybridized to the same regions

Table 1 The list of species and sex (where known) of the individuals usedin the study

Species Family Sex

Varanus acanthurus Varanidae –

Anguis fragilis Anguidae Female

Hierophis viridiflavus Colubridae Female

Pogona vitticeps Agamidae Female

Iguana iguana Iguanidae Male

Trogonophis wiegmanni Trogonophidae Female

Eremias velox Lacertidae Female

Cnemidophorus deppei Teiidae Male

Chalcides ocellatus Scincidae Male

Tiliqua scincoides Scincidae Male

Chromosoma (2012) 121:409–418 411

respectively in the snake Hierophis viridiflavus (Fig. 1g, h)as they did in the species mentioned above. Unfortunately,we were not able to localize a clear signal with the GGA7paint in this species. Nevertheless, previously publishedanalyses based on gene mapping confirmed the associationof the avian ancestral chromosomes 3, 5 and 7 in agamidsand snakes (Srikulnath et al. 2009).

The probes hybridized to three different pairs of acrocentricchromosomes in Cnemidophorus deppei (Fig. 1v–x). In A.fragilis (Fig. 1d–f), the GGA3 probe covered the fourth larg-est, subtelocentric chromosome, while the GGA5 and GGA7paints hybridized to the distal and proximal parts, respectively,of the third largest, subtelocentric chromosome.

In Eremias velox (Fig. 1s–u), a species with an all acro-centric karyotype, the GGA5 and GGA7 paints hybridizedto the distal and proximal parts, respectively, of the largestchromosome. The GGA3 probe covered another pair ofacrocentric chromosomes.

Discussion

Our results of chromosome painting confirm and largely extendthe previously published results obtained by physical genemapping in the iguanian lizard L. reevesii and the colubridsnake E. quadrivirgata (Srikulnath et al. 2009) and by wholegenome sequencing in the iguanid lizard A. carolinensis(Alföldi et al. 2011). We demonstrate that the synteny of theGGA3/7/5 is not restricted to the members of the Toxicoferalineage, but is widely distributed across most major squamatelineages. The phylogenetic distribution suggests that the chro-mosomes homologous to the ancestral avian chromosomes 3, 5and 7 formed parts of the largest metacentric chromosomealready in the common ancestor of the lineage Unidentata,the clade encompassing Serpentes, Iguania, Anguimorpha,Laterata and Scinciformata (Fig. 2). The conservation of thischromosome can be dated back to at least the Lower Juras-sic period, when the deepest split within Unidentata, i.e. thesplit of Scinciformata and Laterata + Toxicofera, occurredabout 190 Ma (Shedlock and Edwards 2009).

We found three independent fissions of the largestancestral metacentric chromosome within Unidentata: inthe ancestors of A. fragilis (Anguidae), C. deppei (Teiidae)and E. velox (Lacertidae). The family Anguidae isgenerally characterized by a large variability in karyo-types, with A. fragilis probably exhibiting a highlyderived karyotype (Gorman 1973). The large ancestralmetacentric chromosome split into two chromosomes, onesyntenic with GGA3 and the other syntenic with GGA5/7in the ancestor of this species. Metacentric chromosomesare common in basal teiids and members of their sisterfamily Gymnophtalmidae, while the karyotype characterizedby the numerous acrocentric chromosomes found inC. deppei

is derived fromwithin the family (Gorman 1973). The fissionsin the ancestor of C. deppei produced three separate chromo-somes, in a similar fashion as to that of the ancestral aviankaryotype. On the other hand, many lacertids have all acro-centric karyotypes with 2n038, which seem to be the ancestralkaryotype for the family (Olmo et al. 1987; data from Olmoand Signorino http://ginux.univpm.it/scienze/chromorepmapped to phylogenetic hypotheses by Arnold et al. 2007 orMayer and Pavlicev 2007). The lacertid species in our sample,E. velox, possesses this typical lacertid karyotype. The resultsof the chromosomal painting suggest that the split of theancestral metacentric chromosome into two acrocentric chro-mosomes syntenic with GGA3 and GGA 5/7 occurred duringthe formation of the ancestral lacertid karyotype.

We are not able to determine whether the chromosomeshomologous to the ancestral avian chromosomes 3, 5 and 7were already associated in the ancestral squamate karyo-type. Gekkota, the sister group to Unidentata, possesses anancestrally all-acrocentric karyotype (e.g. Gorman 1973;Pokorná et al. 2010), which is highly different from theputative ancestral karyotypes of other squamate lineageswhich have at least some metacentric macrochromosomes.The macrochromosomes are metacentric also in some mem-bers of the family Dibamidae (Gorman 1973), probably themost basal squamate lineage (Vidal and Hedges 2005). Aninclusion of dibamids would therefore be highly informativephylogenetically for the reconstruction of the ancestral squa-mate karyotype. However, members of this elusive tropicalgroup are difficult to collect. Tuataras (Rhynchocephalia),the sister lineage to Squamata, can also be important forreconstruction of the ancestral state of Squamata. In tuataraonly preliminary information on gene localization is currentlyavailable (O’Meally et al. 2009). The few genes mapped so farin the tuatara karyotype do not give any evidence for thefusion of the avian ancestral chromosomes 3, 5 and 7 in thislineage. The available molecular cytogenetic and wholegenome sequencing data from more distant outgroups toLepidosauria (Squamata + Rhynchocephalia) also did notclarify this situation. The avian ancestral chromosomes 3, 5and 7 are held intact as separate chromosomes in twoturtles (P. sinensis–Matsuda et al. 2005, and Trachemysscripta–Kasai et al. 2012); however, the turtle ancestralkaryotype has not been reconstructed yet. Although thereis some limited evidence suggesting that the avian ancestralchromosomes 3, 5 and 7 do not form a single chromosome

b

412 Chromosoma (2012) 121:409–418

Fig. 1 Cross-species FISH with GGA3, 5, 7 probes on metaphases ofVaranus acanthurus (a–c, VAC), Anguis fragilis (d–f, AFR), Hierophisviridiflavus (g–i, HVI), Pogona vitticeps (j–l, PVI), Iguana iguana (m–o,IIG), Trogonophis wiegmanni (p–r, TWI), Eremias velox (s–u, EVE),Cnemidophorus deppei (v–x, CDE), Tiliqua scincoides (y–α, TSC),Chalcides ocellatus (β–δ). Results of FISHwith GGA3, 5, 7 are in the first,second and third column, respectively. HVI results for GGA7 are lacking.Arrows indicate FISH signals

Chromosoma (2012) 121:409–418 413

Fig. 1 (continued)

414 Chromosoma (2012) 121:409–418

among crocodiles, the sister lineage to birds. One species,Crocodylus niloticus (CNI), shows fusion of the homo-logues of the ancestral avian 3, 5 and 7 to 1q, 4q and1p, respectively, represented by CNI 1, 2 and 3 (Kasai etal. 2012). Equivocal evidence is present in mammals, theclosest outgroup to sauropsids. The ancestral avian chro-mosomes 3, 5 and 7 show homologous regions on platypuschromosome 1 in the order 5/3/7, but homology is shownfor only a few genes (http://www.ensembl.org). The ances-tral avian chromosomes 3, 5 and 7 are not associated in theancestral karyotypes of the more derived mammalian line-ages (Marsupialia and Placentalia), although the homologof GGA 7 and a region on GGA 3 are homologous withhuman chromosome 2q and 2p, respectively. The chromo-somes homologous with the avian ancestral chromosomes3, 5 and 7 seem to still be largely syntenic, but form partsof distinct chromosomes in the clawed frogXenopus tropicalis(based on the localization of genes with their known positionin the physical map byWells et al. 2011 in the annotated draftof the chicken genome, http://www.ensembl.org) and theAmbystoma salamander (Voss et al. 2011). In conclusion, theavian ancestral chromosomes 3, 5 and 7 were likely to havebeen separate ancestrally in tetrapods and they or their partsfused independently in the ancestors of Unidentata and platy-pus. Within Unidentata, fissions of their largest ancestral

chromosome led to an independent evolution of chromosomeorganization resembling the avian ancestral state in C. deppei(Fig. 2). Furthermore, we reconstructed independent fissionsof the largest ancestral chromosome of Unidentata formingseparate chromosomes homologous with GGA3 and GGA7/5in the ancestors of E. velox and A. fragilis (Fig. 2). Theseevents show that homoplasy in the genome organization oc-curred during tetrapod evolution. More information on ances-tral genome organization, in particular sauropsid lineages, isneeded for a robust support of the scenario of chromosomeshomologous with the avian ancestral chromosomes 3, 5 and 7being separate in the ancestral karyotype of sauropsids.

Integrated analysis (Fig. 2) of our present results withpreviously published data on the synteny of the chromosomeshomologous with the avian Z chromosome across Squamata(discussed in Pokorná et al. 2011) showed that the parts ofsquamate genomes syntenic with GGAZ and GGA3, 5 and 7exhibit a similar history. Parts of genomes homologous withGGAZ, 3, 5 and 7 probably formed four separate chromo-somes in the karyotype of the ancestor of avian lineage, andthis state might be ancestral for turtles as well. The chromo-some homologous with the avian Z formed the p-arm ofthe second largest chromosome in the karyotype of thecommon ancestor of Unidentata (the q-arm of the chromo-some 2 is syntenic with several avian microchromosomes in

Fig. 1 (continued)

Chromosoma (2012) 121:409–418 415

A. carolinensis; Alföldi et al. 2011). This fused situationcorresponds to the association of the avian ancestral chromo-somes 3, 5 and 7 in the ancestor of Unidentata, i.e. a reductionin chromosome number compared to birds and turtles. Inter-estingly, the independent chromosomal fissions in the ances-tors of A. fragilis, E. velox andC. deppei concerned, in each ofthese lineages, both of the two largest ancestral chromosomesof Unidentata. These results suggest a long evolutionary stasisof the studied chromosomes followed by an increased rate ofchromosomal rearrangements in certain squamate lineages(for an analogous situation within a single lizard family, seePokorná et al. 2010).

In summary, the integrated information (Fig. 2) documentschromosome conservation for genome parts homologous to

both the avian sex chromosomes and the ancestral macro-chromosomes 3, 5 and 7 across a phylogenetically widespectrum of Squamata. Additional evidence suggests thatchromosomes syntenic with other avian macrochromosomescould be equally conservative in Squamata and other saurop-sid lineages (see, e.g. the synteny analysis of particular chro-mosomes in Leiolepis, Elaphe, Pelodiscus, Anolis; Matsuda etal. 2005; Srikulnath et al. 2009; Alföldi et al. 2011). Forexample, the Z chromosome of advanced snakes is homolo-gous to part of the GGA2 and GGA27 (Matsubara et al. 2006)and recent analysis has documented that the genes linked tothe snake Z chromosome also largely map to only two sepa-rate chromosomes in the frog X. tropicalis (O’Meally et al.2010). Similarly, the chromosome homologous with GGA3 is

Fig. 2 Phylogeneticdistribution of homologybetween GGA3, 5, 7 and Z andchromosomes in othersauropsids. Diagram combineshere described results with thepublished data from Matsuda etal. (2005), Giovannotti et al.(2009), Srikulnath et al. (2009),Alföldi et al. (2011) andPokorná et al. (2011). The datafor skinks are based oncomparison of chromosomesynteny within the family viachromosome painting(Giovannotti et al. 2009). Theancestral situation is forsimplicity depicted only inbirds (Griffin et al. 2007)

416 Chromosoma (2012) 121:409–418

syntenic with just the fifth chromosome of the frog (cf. Wells etal. 2011; http://www.ensembl.org). Therefore, it seems that theslow rate of chromosomal evolution is an ancestral character-istic of all sauropsids, if not tetrapods. The karyotype conser-vatism in sauropsids could be attributed to considerably lowercopy numbers of repetitive elements in comparison to mam-mals (Organ et al. 2007). For example, transposable elementscan form over 40 % of the human genome (Volff et al. 2003),but only around 20 % in green anole and a mere 9 % in thechicken (Kordis 2009). The large evolutionary stasis in chro-mosome organization in birds thus seems to be inherited fromtheir ancestors, and it is particularly striking in comparisonwithmammals, probably the only major tetrapod lineage with anenlarged rate of chromosomal rearrangements as a whole.

Acknowledgments We are grateful to Patricia O’Brien, Frances L.Dearden, Margaret Wallduck, Paola Nisi Cerioni and Šárka Pelikánováfor assisting in cell culture or FISH experiments. Petr Ráb and MarieRábová provided continuous support and encouragement. ChristopherM. Johnson provided many valuable comments. The studies weresupported by grants GAČR 506/10/0718 and GAUK 9942/2009 toMP and LK and PRIN 2009 provided by Ministry of Education,University and Research, Italy to EO.

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