The Journal (2003) 34, 283±291 The absence of Arabidopsis-type telomeres in and closely related genera Vestia and (): ®rst evidence from

Eva Sykorova1,2,y, Kar Yoong Lim1,y, Mark W. Chase3, Sandra Knapp4, Ilia Judith Leitch3, Andrew Rowland Leitch1,Ã and Jiri Fajkus2 1School of Biological Sciences, Queen Mary University of London, London E1 4NS, UK, 2Institute of Biophysics, Academy of Sciences of the Czech Republic and Masaryk University of Brno, Brno, Czech Republic, 3Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK, and 4Department of Botany, Natural History Museum, Cromwell Road, London SW7 5BD, UK

Received 2 October 2002; accepted 24 January 2003. ÃFor correspondence (fax ‡44 208 983 0973; e-mail [email protected]). yJoint ®rst author.

Summary Using slot-blot and ¯uorescent in situ hybridization (FISH), we found no evidence for the presence of the

Arabidopsis-type telomeric sequence (TTTAGGG)n at the chromosome termini in any of the Cestrum spe-

cies we investigated. Probing for the human-type telomere (TTAGGG)n also revealed no signal. However, polymerase chain reaction experiments indicated that there are short lengths of the sequence TTTAGGG dispersed in the genome but that these sequences are almost certainly too short to act as functional telomeres even if they were at the chromosome termini. An analysis of related genera Vestia and Sessea indicates that they too lack the Arabidopsis-type telomere, and the sequences were lost in the common ancestor of these genera. We found that the Cestrum investigated had particularly large mean chromosome sizes. We discuss whether this is a consequence of alternative telomere end maintenance systems.

Keywords: absence of TTTAGGG telomere, dicot, chromosome, FISH.

Introduction The ends of eukaryotic chromosomes frequently, but not of chromosome ends degraded by incomplete lagging ubiquitously, have TnGn rich minisatellite sequences occur- strand synthesis during the S phase of the cell cycle. ring in tandem arrays of many hundreds or thousands of However, the highly conserved minisatellite telomeric repeats. In higher , the consensus sequence cloned sequence is not universally found. Sahara et al. (1999) used from Arabidopsis thaliana is (TTTAGGG)n (Richards and Southern hybridization to show that the species in four Ausubel, 1988). Similar minisatellite sequences are con- orders of insects (Diptera, Coleoptera, Heteroptera, and served in vertebrates, as in human, in which the consensus Dermaptera) and a spider lacked the insect consensus sequence is (TTAGGG)n (Moyzis et al., 1988), whereas in sequence (TTAGG)n. Of the nine species of Coleoptera many insects it is (TTAGG)n (Okazaki et al., 1993). Telo- investigated, four lacked (TTAGG)n. If these numbers are meres distinguish the natural chromosome ends from in any way representative of Coleoptera, it could be pre- unrepaired chromosome breaks and are thought to per- dicted that there may be millions of beetle species without form the following functions: (i) prevention of end-to-end the insect consensus sequence. In Drosophila melanoga- fusions of chromosomes; (ii) binding of speci®c telomere- ster (Diptera), the telomeres contain Het-A and TART that associated proteins involved in DNA folding producing a preferentially retrotranspose to the chromosome termini, stable structure that may interact with the nuclear envel- whereas in D. virilis and other species in the virilis group, ope, be involved in chromosome disposition, and per- as well as in the Chironomus (Diptera), complex haps prevent chromosome fusions; and (iii) re-synthesis satellite DNA sequences, presumed to be elongated by a

ß 2003 Blackwell Publishing Ltd 283 284 Eva Sykorova et al. homologous recombination mechanism, are located at the appear to be absent from the chromosome termini and telomeres (Biessmann et al., 2000). therefore are not telomere sequences. Thus, this paper In plants too, there is evidence that the plant consensus provides evidence of an absence of Arabidopsis-type telo- sequence (TTTAGGG)n is lacking in some groups. Fuchs meres in eudicot plants. et al. (1995) showed that it was absent in Allium, Nothos- cordum, and Tulbaghia of the monocotyledonous family Results Alliaceae. Further analysis showed that the sequence was most likely replaced by rDNA, a satellite sequence or retro- Pich and Schubert (1998) and Adams et al. (2000) demon- transposable element(s) at the end of the chromosome strated an absence of Arabidopsis-type telomeric (Pich and Schubert, 1998; Pich et al., 1996a,b). Adams et al. sequences in the monocot genera Aloe and Allium using (2000) showed that Aloe in Asphodelaceae also lacked several methods: (i) ¯uorescent in situ hybridization (FISH)

(TTTAGGG)n.AsAllium and Aloe are distantly related using a PCR-generated concatemer of the Arabidopsis con- members of the order Asparagales, this led to an investiga- sensus sequence (TTTAGGG)5 as a probe; (ii) Southern/ tion of other related genera. Many other species were found slot-blot hybridization analysis using the TTTAGGG con- to lack the sequence suggesting that an absence of the catemer probe; and (iii) elongation or asymmetric PCR Arabidopsis-type repeat was common in Asparagales using the forward primer only of the oligonucleotide

(Adams et al., 2000) although the human-type of telomere sequence (TTTAGGG)4. Variations on these approaches

(TTAGGG)n occurs in some of the species in its place (Weiss were used on Cestrum. and Scherthan, 2002). For the purpose of this paper, it is important to clarify the Absence of Arabidopsis-type telomeres using FISH terms Arabidopsis-type telomeric sequence, which de®nes the minisatellite (TTTAGGG)n sequence, as distinct from Fluorescent in situ hybridization experiments using a bio- the Arabidopsis-type telomere, which describes a telomere tin-labelled concatemer of the Arabidopsis-type telomeric formed by this sequence. Here we provide evidence that sequence (TTTAGGG)5 (Cox et al., 1993; Ijdo et al., 1991), whereas short lengths of the Arabidopsis-type sequence detected by avidin-Cy3, revealed that there was an absence are dispersed in some species of the closely related genera of probe labelling to the ends of chromosomes in Cestrum Cestrum, Vestia and Sessea (Solanaceae), these sequences parqui (Figure 1a,b) and (Figure 1d,e)

Figure 1. FISH to metaphase spreads of (a±c), Vestia foetida (d±f) and Nicotiana sylvestris (g,h), stained with DAPI for DNA (blue (a,d,g)) and probed with avidin-labelled Arabidopsis-type telomere probe detected with Cy3 (red (b,e,h)). Note only N. sylvestris shows strong labelling at the ends of the chromosomes. For a positive control, the metaphases of C. parqui and V. foetida are probed with digoxigenin-labelled 18±26S rDNA (c) and 5S rDNA (f), respectively (yellow-green). The intensity of the FITC signal for 18±26S rDNA in C. parqui (c) has resulted in some light coming through the Cy3 ®lter block and is thus visible in (b). Scale bar ˆ 10 mm.

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 283±291 Loss of TTTAGGGn-type telomeres in a eudicot group 285 metaphase cell spreads. Ribosomal DNA (18±26S or 5S rDNA) was used as a positive control for the ef®cacy of the in situ methods (Figure 1c,e). Clear signals were observed using the Arabidopsis-type telomeric sequence on control metaphase spreads of Nicotiana sylvestris as expected (Figure 1g,h). FISH may be a low-sensitivity method for detecting sequences, as less than 1 kb of con- tiguous sequence may not be detectable (Gill and Friebe, 1998). For this reason, the results were complemented by slot-blot and PCR approaches.

Slot-blot analysis of genomic DNA Using slot-blot hybridization and the labelled ATSB probe, we analysed genomic DNAs from eight Cestrum species and a hybrid (Table 1) for the presence of Arabidopsis-type Figure 2. Slot-blot hybridization with radioactive-labelled telomere primer telomeric sequences (Figure 2a). The negative control used (a) ATSB or (b) HUSB as the probe. in the experiment was genomic DNAs of Allium cepa, and Control DNAs are Nicotiana sylvestris (S), Silene latifolia (R), Homo sapiens the positive controls were genomic DNAs of N. sylvestris (Q), Allium cepa (V) and N. otophora (Y). Species investigated in the Sola- naceae are (A), C. elegans (B), C. fasciculatum (C), and Silene latifolia, the latter having relatively short telo- C. nocturnum (D), C. psittacinum (E), C. purpureum (F), C. roseum (G), mere repeats with approximately 2.5±4.5 kb of Arabidopsis- C. parqui (T), C. parqui  C. aurantiacum (U), Streptosolen jamesonii (H), type telomeric sequence per chromosome terminus (Riha Solanum betaceum (I), Physalis alkekengi (J), Iochroma australe (K), barbarum (L), L. cestroides (M), Withania coagulans (N), Vestia foetida (O), et al., 1998). All membranes were re-probed with 18S rDNA Nicandra physaloides (P), and Sessea stipulata (X). to ensure transfer of genomic DNA (data not shown). Greater sensitivity may be obtained using Southern hybri- dization because the method can potentially separate telo- and control DNA from S. latifolia were analysed by this mere repeat sequences from the bulk of genomic DNA. technique for the presence of Arabidopsis-type telomeric Therefore, genomic DNAs of Cestrum parqui, C. elegans sequences. No signal was obtained in Cestrum DNAs, but

Table 1 The species used

Species Herbarium voucher specimen Seed/plant source

Allium cepa L. cv. Ailsa Craig RBG, Kew Arabidopsis thaliana L. cv. Columbia Queen Mary, London Cestrum aurantiacum Lindl. Chase 12216 (K) RBG, Kew (Brongn.) Schltdl. Lim 002 (BM) Ginkgo Nursery, London, UK (Schltdl.) Miers Chase 12218 (K) RBG, Kew L. Chase 12219 (K) RBG, Kew Cestrum parqui L'Her. Chelsea Physic Garden, London, UK Cestrum parqui  Cestrum aurantiacum Saikia 001 (BM) Ginkgo Nursery, London, UK Cestrum psittacinum Stapf. Chase 12220 (K) Chelsea Physics Garden, London Cestrum purpureum (Lindl.) Standl. Chase 12221 (K) RBG, Kew Cestrum roseum Kunth Chase 12222 (K) RBG, Kew Solanum betaceum Cav. Chase 12267 (K) RBG, Kew (ex. Cyphomandra betacea (Cav.) Sendtn.) Iochroma australe Griseb. Lim 025 (BM) Chelsea Physic Garden Lycium barbarum L. Lim 024 (BM) Chelsea Physic Garden Lycium cestroides Schltdl. Lim 023 (BM) Chelsea Physic Garden Nicandra physaloides (L.) Gaertn. Lim 021 (BM) Chelsea Physic Garden Nicotiana otophora Griseb. Queen Mary, London Nicotiana sylvestris Speg. & Comes USDA seed bank Physalis alkekengi L. Lim 022 (BM) Chelsea Physic Garden Sessea stipulata Ruiz & Pav. Nee et al. 51749 (NY) Bolivia. La Paz, Sud Yungas Silene latifolia (Mill.) Rendle & Britten Institute of Biophysics, Brno Streptosolen jamesonii (Benth.) Miers Chase 12223 (K) RBG, Kew Vestia foetida (Ruiz & Pav.) Hoffmanns. Chelsea Physic Garden, London and RBG, Kew Withania coagulans Dunal Chelsea Physic Garden, London and RBG, Kew

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 283±291 286 Eva Sykorova et al. there was hybridization to S. latifolia DNA (data not shown). As Weiss and Scherthan (2002) showed that Aloe paral- lelifolia and A. deltoideodonta var. variegata have the human-type telomeric sequence, we probed Cestrum, Ves- tia and Sessea with the human-type telomere repeat (HUSB) using Homo sapiens DNA as a positive control. No signal was generated to indicate that these plants have the human-type telomeric sequence (Figure 2b). In those species with an Arabidopsis-type repeat sequence, the proportion of the genome containing these sequences varies on a species-by-species basis, and calcu- lating the sensitivity of slot-blot hybridization depends upon haploid chromosome number, 1C genome size, mean and range of telomere repeat lengths per chromosome arm and uniformity of the array structure. The signal in slot-blot hybridization (Figure 2a) for Silene latifolia (2n ˆ 24) repre- sents approximately 10±20 pg of telomere repeat sequence in 1 mg of genomic DNA loaded onto the membrane (i.e. 2.5±4.5 kb telomere repeat length per chromosome arm  number of chromosome arms in haploid genome/ 1C genome size  1 mg genomic DNA loaded). Neither the Cestrum species (Figure 2, slots A±G, T, U) tested, nor the negative control Allium cepa (Figure 2, slot V), showed any signal in slot-blot hybridizations. The chromosome num- bers and genome sizes of three Cestrum species and hybrids were measured to be: C. parqui (2n ˆ 16;

1C ˆ 10.94 pg), C. parqui  C. aurantiacum (2n ˆ 16 plus Figure 3. Framework phylogeny of Solanaceae including only those genera 10 B chromosomes, 1C ˆ 12.09 pg) and C. elegans that were sampled in this study. (2n ˆ 16; 1C ˆ 9.76 pg). On the basis of genome size, chro- Telomere loss has been mapped onto the framework tree using MACCLADE version 3.06 (Madison and Madison, 1996) showing the most parsimonious mosome numbers and slot-blot sensitivity, we estimate states at each node. The topology of this tree should not be interpreted as that there is less than 0.6, 0.4, and 0.55 kb of Arabidop- the true phylogeny of the family, but instead as a heuristic device with which sis-type telomeric sequence per chromosome arm for each to examine the distribution of characters of interest in the family. Those interested in the more complex trees resulting from the molecular analyses of these samples, respectively. are referred to the original literature (Fay et al., 1998; Olmstead et al., 1999). Cestrum belongs to Solanaceae, which includes the well Lack of shading indicates absence of Arabidopsis-type telomere in the studied-genomes of Nicotiana tabacum (tobacco), Sola- Cestrum clade. num tuberosum (potato) and S. lycopersicum (tomato). They all have telomeres containing the Arabidopsis-type telomeric sequence (Ganal et al., 1991; Kovarik et al., 1996; dization signal was detected in Streptosolen jamesonii Suzuki et al., 1994; Tanksley et al., 1992). We investigated (Figure 2a, slot H), a species closely related to the Cestrum whether the Arabidopsis-type or human-type telomeric clade. The data indicate (i) loss of the Arabidopsis-type sequence, as detected by slot-blot hybridization, occurred telomere in the common ancestor of the Cestrum clade in genera related to Cestrum. Within Solanaceae, Cestrum consisting of Sessea, Vestia and Cestrum, and (ii) the belongs to a small clade, well-de®ned both morphologi- telomere was not replaced by a human variant at this point cally (Hunziker, 2001) and with DNA sequence data (Fay in evolution. et al., 1998; Olmstead et al., 1999). It includes the genera Cestrum, Vestia and Sessea (the Cestrum clade, see PCR analysis of genomic DNA Figure 3). Traditionally, the genus Metternichia was also included in this group, but recent phylogenetic work (Fay The Expand High Fidelity PCR system (Roche Diagnostics et al., 1998) indicates that it is only distantly related. GmbH, Roche Applied Science, Mannheim, Germany) Results from slot-blot hybridization using either ATSB includes Taq polymerase and a proof-reading polymerase, (Figure 2a) or HUSB (Figure 2b) probes showed that both which under the conditions used, enables the ampli®cation V. foetida (Figure 2, slot O) and Sessea stipulata (Figure 2 of PCR products up to 8 kb with a low expected error ratio. slot X) lacked hybridization signal whereas positive hybri- This length exceeds the telomere length of species with

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 283±291 Loss of TTTAGGGn-type telomeres in a eudicot group 287

Figure 4. Primer extension reactions on (a) con- trol DNAs and (b) Cestrum species. (a) Typical results of primer extension reactions with TEL21 (1,4), TEL3C (2,5) and TEL3G (3,6) primers are shown for Arabidopsis telomere- negative plant Allium cepa (1±3) and telomere- positive plant Arabidopsis thaliana (4±6). Markers (m) ± 1 kb GeneRuler (MBI Fermentas). (b) The same protocol was used for primer extension reactions on Cestrum parqui (1,5,9), C. parqui  C. aurantiacum (2,6,10), C. elegans (3,7,11) and C. psittacinum (4,8,12). Markers (m) ± 100 bp and 1 kb ladders (New England Biolabs).

short stretches of the Arabidopsis-type repeats at the chro- products were then analysed by Southern hybridization mosome termini (e.g. as in S. latifolia and A. thaliana). using pTEL as a probe. A smear of hybridization signal When this system was used with any single telomeric was obtained in A. thaliana re¯ecting primer extension at primer, i.e. TEL21, TEL3C, and TEL3G to A. thaliana,a the long tandem arrays of Arabidopsis-type telomeric smear of products was generated because the primers sequences (Figure 5, lanes 4±6). The reduced abundance can bind at any position across the large array of mini- and size categories of the product (compared with satellite tandem repeats and elongate into the genome (Figure 4a, lanes 4±6). In contrast, Allium generated no product with this reaction (Figure 4a, lanes 1±3). When the method was used on C. parqui, C. elegans, C. psittaci- num and hybrid DNAs, both a smear and discrete bands were generated (Figure 4b), the distribution of which varied with species and primer. Furthermore, the size and distri- bution of bands were highly sensitive to minor changes in PCR parameters. The pattern of bands seen in the Cestrum species is most likely generated by inverted repeats of the sequence scattered throughout the genome and not orga- nized in long tandem arrays. The smear is probably gen- erated by short interstitial direct repeats in the genome. To be certain that the products generated by the elonga- tion reaction of Cestrum were not derived from long tan- dem arrays of sequences as occurs at the telomere, the PCR reaction was carried out in the absence of the deoxygua- nosine triphosphate (dGTP) using the C-strand-speci®c tel- omere primer TEL21. If there are long blocks of the Arabidopsis-type repeats in Cestrum, the absence of dGTP should not terminate the extension reaction since the strand being extended would lack G in the sequence 50- 0 Figure 5. Primer extension reactions without dGTP in the PCR mix for (CCCTAAA)n-3 or variants thereof. However, if the Arabi- Cestrum parqui (1±3) and control DNA from Arabidopsis thaliana (4±6). dopsis-type sequences were interstitial and short, no pro- Reactions were performed with the C-strand-speci®c telomere primer TEL21 duct would be expected because dGTP would be expected on increasing amounts of template DNA (triangles) re¯ecting the larger genome size of Cestrum (5, 50 and 500 ng of genomic DNA) and Arabidopsis to occur in genomic DNA in one out of every four bases. In thaliana (0.1, 1 and 10 ng). Products were transferred to a membrane and our experiments, the electrophoretically separated reaction hybridized with the plant telomere probe pTEL.

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 283±291 288 Eva Sykorova et al.

Figure 4a) demonstrated that in the normal extension reac- Among unicellular plants (algae), the small genome of tion much of what is generated is intrachromosomal geno- Chlorella vulgaris (1C ˆ 38.8 Mbp) has been characterized mic DNA. No signal was detected when Cestrum DNA was in detail (Higashiyama and Yamada, 1991), and even in this ampli®ed with unidirectional primers in the absence of alga, telomeres of about 0.5 kb length are present at chro- dGTP (Figure 5, lanes 1±3). Thus, the absence of dGTP in mosome ends (Higashiyama et al., 1995). We conclude that the reaction mixture does terminate the PCR reaction in the TTTAGGG repeat arrays of up to 35 bp long that have Cestrum. been observed in Cestrum (data from sequenced clones) To gain conclusive evidence that Cestrum lacks Arabi- would not be capable of performing telomere functions, dopsis-type telomeres, we cloned the PCR products from even if some of them were located at the chromosome the entire pool of the extension reactions shown in termini. Figure 4. A large number of Cestrum clones (461) were An absence of Arabidopsis-type telomeres in Cestrum is a screened for the presence of telomere sequences. Approxi- surprising result because Solanaceae includes Nicotiana mately 50 clones were selected for sequencing. None of species in which telomere sequences are well characterized them had Arabidopsis-type telomeric sequences longer and shown to contain large blocks of Arabidopsis-type than (TTTAGGG)5, and usually they contained only telomere repeats (Kovarik et al., 1996; Suzuki et al., 1994).

(TTTAGGG)3, the length of the PCR primer. This detailed Furthermore, long arrays of these sequences also form analysis, and the information that it yields about the Ces- telomeres in the well-characterized genomes of S. tuber- trum genome structure is the subject of another paper. osum (potato) (Fajkus et al., 2002; Zhong, 1998) and S. lyco- persicum (tomato) (Ganal et al., 1991). Slot-blot analysis (Figure 2) indicates that Vestia and Sessea, both genera Discussion closely related to Cestrum, also lack the Arabidopsis-type telomeres. The closest relative to these three genera that was investigated is Streptosolen (Olmstead et al., 1999), a Loss of Arabidopsis-type telomere in the Cestrum clade member of a clade containing the genus Browallia. Strep- (Cestroidea: Cestreae) tosolen, as in all other non-Cestrum clade genera in Sola- Fluorescent in situ hybridization, PCR, slot-blot and naceae examined, shows hybridization signals using Southern hybridizations indicate an absence of long arrays probes for Arabidopsis-type telomeres in slot-blots. Thus, of the Arabidopsis-type telomeric sequence (TTTAGGG)n, the result indicates a loss of the Arabidopsis-type telomere forming a functional telomere in Cestrum species ± the ®rst in the common ancestor of Cestrum, Vestia and Sessea. such report for a eudicot. A similar absence of the `typical' The only other plant species so far reported without telomere has been demonstrated in some monocots Arabidopsis-type telomeres are in Asparagales, which are (Adams et al., 2001). However, the PCR reaction using all petaloid monocots and include diverse genera such as the unidirectional primers homologous to Arabidopsis- Aloe, Allium, Convallaria, Asparagus and others (Adams type telomeric sequences generates bands and smears, et al., 2001). The discovery of dicots without Arabidopsis- and this means that there are short, dispersed, direct and type telomeres now sheds considerable doubt on how inverted TTTAGGG repeats scattered throughout the gen- widespread this type of telomere sequence is in plants. It ome. This is supported by evidence from the inhibition of is clear that a thorough sampling of angiosperm taxa, with primer extension using unidirectional primers in the extensive phylogenetic coverage is required before any absence of dGTP. assertion of the generality of Arabidopsis-type telomeres Such short sequences, even if present at the chromo- in plants can be made. some ends, could not form `typical' functional telomeres. It has been shown recently (Riha et al., 2001) that telomerase- Replacement of the (TTTAGGG) by other T G repeats de®cient mutants of Arabidopsis thaliana loose 250±500 bp n n n of telomeres per generation. In the sixth generation, the Adams et al. (2000) reported that some species of Aspar- plants displayed chromosomal abnormalities (end-to-end agales, a group of monocots, lacked the Arabidopsis-type chromosome fusions, reduced ef®ciency of seed germina- repeat. Later, Weiss and Scherthan (2002) showed that in tion) when the shortest telomeres were approximately Aloe the typical plant telomeric sequence had been

900 bp long. An independent estimate of minimal telomere replaced by the human-type (TTAGGG)n. For this reason length can be inferred from analysis of products of chro- the Cestrum clade was analysed by slot-blot with the mosome healing in wheat, in which de novo synthesized human (TTAGGG)n repeat, but no evidence of these repeat telomeres are several hundred base pairs long (Tsujimoto sequences were found (Figure 2b). Perhaps in Aloe and et al., 1997, 1999). Yet another source supporting a mini- other Asparagales the RNA template of telomerase has mum functional telomere length comes from the shortest changed. If a similar process has happened to members natural telomeres described in unicellular eukaryotes. of the Cestrum clade, the new template is unlikely to be of a

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 283±291 Loss of TTTAGGGn-type telomeres in a eudicot group 289 human-type. It is known from early analyses of telomeres in chromosomeÀ1) by slot-blot (Figure 2) and FISH (not Arabidopsis and tomato that variants of TTTAGGG can shown) and found that it does have the Arabidopsis-type occur in their genomes (Ganal et al., 1991; Richards and telomeric sequence. Therefore, where an over-active repla- Ausubel, 1988). However, in these cases it is generally cement hypothesis may be true in the Cestrum clade, it accepted that variant sequences come from the following would only be one of a number of mechanisms leading to places (i) degenerate subtelomeric sequences (Richards enlarged chromosomes. and Ausubel, 1988; Richards et al., 1993), (ii) interstitial telomeric sequences (Cheung et al., 1994; Presting et al., Experimental procedures 1996; Richards et al., 1991) and (iii) proof reading errors derived from telomerase activity (Fitzgerald et al., 2001). It Plant material has never been reported that there is more than one RNA template for any telomerase-based systems. If a telomer- The plant species used are shown in Table 1. Voucher specimens ase-based system does exist in the Cestrum clade, the RNA for plants are either at Queen Mary University of London, or deposited in herbaria at the Royal Botanic Gardens, Kew (K) or template is probably a novel variant for plants. the Natural History Museum, London (BM). A molecular phylo- geny of Solanaceae based on plastid DNA sequences and restric- tion length fragments (Olmstead et al., 1999) was the basis for Replacement of the (TTTAGGG) by other systems n generic sampling (see Figure 3 for details). Minisatellite telomere repeat sequences are found across eukaryotes. Their absence in some groups (e.g. Drosophila DNA extraction and Chironomus in insects and Allium in plants) must DNA was extracted from fresh leaf material using standard pro- therefore be a derived condition that has arisen indepen- cedures (Dellaporta et al., 1983) or using CsCl (Sambrook et al., dently in these groups. The mechanism that replaces the 1989). Control DNAs and DNA for PCR experiments were isolated elongation function of the `typical' telomere is best under- using DNeasy Mini kits (Qiagen Ltd, Gatwick Road, Crawley, UK). stood in D. melanogaster, where retrotransposition of Het- A and TART to chromosome termini balances DNA loss DNA probes and primers associated with lagging strand synthesis (Mason et al., The following synthetic oligonucleotides and DNA probes were 2000). In yeast and human cells, maintenance of telomeres used for PCR or Southern hybridization experiments: 0 0 in the absence of telomerase can occur as a result of re- (1) TEL21: 5 -ACCCTAAACCCTAAACCCTAA-3 , an oligonucleotide designed to prime DNA strand synthesis from the telomere combination-dependent exchanges between imperfectly towards the centromere. matched sequences (Dunham et al., 2000; Lundblad (2) TEL3C: 50-TAAACCCTAAACCCTAAACCC-30, a primer of similar and Blackburn, 1993; McEachern and Blackburn, 1996; melting temperature to TEL21 but the primer sequence is Nakamura et al., 1998; Teng and Zakian, 1999). shifted. This was used to vary primer annealing at the 30 end It is possible that when a telomerase-independent end of the oligonucleotide to search for product variation if the sequence occurred in short arrays. synthesis does not precisely balance end-replication loss, (3) TEL3G: 50-TTTAGGGTTTAGGGTTTAGGG-30 which represents over time inadequate replacement would lead to chromo- the Arabidopsis-type telomeric sequence designed to prime some reduction and death, whereas over-active replace- DNA synthesis from internal sites towards the distal ends of the ment would lead to chromosome enlargement and genome telomere. 0 0 size gain. We found that C. parqui (Figure 1a±c) and V. foe- (4) ATSB: 5 -GGTTTAGGGTTTAGGGTTTAGGGTTTAG-3 , used as a probe in slot-blot experiments to detect the Arabidopsis-type tida have particularly large chromosomes for Solanaceae telomeric sequence. It was [32P]-end-labelled by T4 polynucleo- (Figure 1d±f) and we measured the genome size of two tide kinase (New England Biolabs Ltd, Wilbury Way, Hitchin, Cestrum species. We calculated mean chromosome size in Hertfordshire). Solanaceae (1C DNA amount (pg) divided by the haploid (5) HUSB: 50-TTAGGGTTAGGGTTAGGGTTAGGGTTAG-30,used chromosome number (n); data were presented in the pre- as a probe in slot-blot experiments to detect the human-type telomeric sequence. It was [32P]-end-labelled by T4 polynucleo- sent study and Bennett and Leitch (2001)). We found that tide kinase (New England Biolabs Ltd). C. parqui has the largest mean chromosome size (1.36 pg (6) pTEL: a cloned double-stranded fragment of Arabidopsis-type À1 chromosome ) of any Solanaceae species so far reported. telomeric sequence containing (CCCTAAA)21 used for South- The average chromosome size for all Solanaceae species ern hybridization to membranes with digested genomic DNA 32 is only 0.26 pg chromosomeÀ1. Outside the Cestrum samples or primer extension reactions. This was P-labelled using DecaPrime kit (Fermentas INC, 7520 Connelley Dive, clade the largest mean chromosome size in Solanaceae Hanover, MA, USA). is Solanum sibundoyensis (ˆ Cyphomandra sibundoyen- sis) from Solanum section Pachyphylla (ex. Cyphomandra) Probes for in situ hybridization with a mean chromosome size of 1.28 pg (Bennett and (1) The Arabidopsis-type telomeric sequence probe was prepared Leitch, 2001). We analysed the genome of S. betaceum by PCR concatenations of (TTTAGGG)5 primers using methods (section Pachyphylla, mean chromosome size 0.65 pg described in Ijdo et al. (1991).

ß Blackwell Publishing Ltd, The Plant Journal, (2003), 34, 283±291 290 Eva Sykorova et al.

(2) pTa71: a 9 kb EcoRI fragment of 18S-25S rDNA and the inter- Acknowledgements genic spacer cloned from Triticum aestivum (Gerlach and Bed- brook, 1979). This work was funded by the Leverhulme Trust, the Grant Agency (3) pTZ19-R: a 120 bp fragment of the 5S rDNA unit isolated from of the Czech Republic (project 204/02/0027) and the Czech Ministry Nicotiana rustica (Venkateswarlu et al., 1991). of Education (MSM143100008). We thank the Chelsea Physic The probes were labelled by nick translation using either biotin- Garden for providing some of the plant materials, Ms L. Hanson 11-dUTP (Sigma±Aldrich Company Ltd, Dorset, UK) or digoxi- for the DNA measurements of Cestrum and Mr J. Clarkson for genin-11-dUTP (Roche Molecular Biochemicals, Bell lane, Lewes, technical support. East Sussex BN7 1LG, UK).

Slot-blot hybridization References

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