Telomere sequence localization and karyotype ... - Springer Link

Plant Systematics and Evolution

P1. Syst. Evol. 196:227-241 (1995)

© Springer-Verlag 1995 Printed in Austria

Telomere sequence localization and karyotype evolution in higher plants* J. FucHs, A. BRANDES, and I. SCHUBERT Received September 29, 1994; in revised version January 20, 1995

Key words: Angiosperms, gymnosperms, bryophytes. - Telomeres, fluorescent in situ hybridization, karyotype evolution. Abstract: Data for chromosomal localization of the Arabidopsis-type of telomeric sequence repeats (TTTAGGG)n are compiled for 44 species belonging to 14 families of angiosperms, gymnosperms and bryophytes. For 23 species and seven families this is the first report. Species of all families, except the Alliaceae, revealed these sequences at their chromosome termini. This indicates that Arabidopsis-type telomeric repeats are highly conserved. It is inferred that they represent the basic telomere sequence of higher plant phyla. In the Alliaceae, a deviating sequence (and mechanism?) for the stabilization of chromosome termini has possibly evolved secondarily. Nine species revealed interstitial telomeric sequences in addition to the terminal ones, in three species (Vicia faba, Pinus elIiottii, P. sylvestris) also at centromeric positions. Interstitial telomeric sequences may indicate karyotype reconstructions, in particular alterations of chromosome numbers by chromosome fusion - or inversions with one breakpoint within the terminal array of repeats. They may contribute to stabilization of chromosome breaks, especially centric fissions, and increase the frequency of meiotic and illegitimate recombination. Telomeres like centromeres of eukaryotic chromosomes are essential for a stable maintenance and distribution of the nuclear hereditary material. Centromeres are responsible for the correct transmission of chromosome complements during mitosis and meiosis. Telomeres prevent degradation of the ends of linear chromosomes by nucleases, illegitimate fusion of natural chromosome ends with each other or with artificial ends created by chromosome breaks, and shortening of linear D N A molecules due to incomplete replication at the 5 '-ends by D N A polymerases. Both chromosome "organelles" consist of specific D N A sequences and proteins organized in higher order structures deviating from the remaining chromatin. The evolution of telomere structures is considered to be the most important prerequisite for stability and propagation of linear DNA molecules and thus, for the origin of eukaryotic chromosomes. Circular prokaryotic genomes do not require any of the known telomere functions. * Dedicated to Prof. Dr R~GOMARR~ECERat the occasion of his

65 th birthday.

228

J. FtJcHs & al.:

The DNA of eukaryotic chromosome termini is usually formed by tandemly arranged short simple sequence repeats followed, in many cases, by larger and polymorphic subtelomeric repetitive sequences (for plants see BEDBROOK& al. 1980, BROUN& al. 1992, KILIAN& KLEINHOFS1992, R6DER & al. 1993). The terminal repeats form no nucleosomes or structurally altered ones (for review see TOMMERUP& al. 1994) and are often associated with specific proteins. G-rich overhanging 3 '-ends are supposed to form quadruplex structures via hydrogen bonds between guanines promoted by telomere-binding proteins (FANG & CECIl 1993). These ends may be prolonged by telomerase, a revertase with an internal RNA-template (GREIDER& BLACKBURN1989). The same enzyme may in some cases also stabilize broken ends by addition of telomeric repeats (WILKIE& al. 1990, MORIN 1991). The hexanucleotide TTAGGG, which already forms the chromosomal termini of some protozoa (Trypanosoma), was found in all tested vertebrates, in some avertebrate (PELLICCIA& al. 1994) and slime mold species (FORNEY& al. 1987). The pentanucleotide TTAGG forms the telomeres in Bombyx mori and was found to be present in several species of eight of eleven tested orders of insects (OKAzAKI& al. 1993). A heptanucleotide, TTTAGGG, originally isolated from Arabidopsis thaliana (L.) HEYNI-I.,Brassicaceae (RICHARDS& AUSUBEL 1988), has been shown to form telomeres in several angiosperm species (Table 1). The octanucleotide TTTTAGGG forms the telomeres of the alga Chlamydomohas reinhardtii (PETRACEK& al. 1990). Other variants of this simple sequence repeat have been reported for several protozoan species. Irregular G-rich sequence repeats (TG1 3 or AG1-8) form the complex telomeres of some yeasts and slime models (SHAMPAY& al. 1984, EMERY& WEINER 1981). Such sequences may be prolonged at the chromosome ends or added to them via gene conversion (WANt & ZAKIAN1990). A telomerase activity specific to these sequences has not yet been proved by in vitro experiments, although recently SINGER & GOTTSCHLING(1994) found a RNA sequence presumed to be the template sequence of a yeast telomerase. In several orders of insects short telomeric simple sequence repeats have not been detected. Instead, Drosophila melanogaster chromosomes revealed truncated so-called HeT-A sequences of - 6 kb (YOuN~ & al. 1983) at their very ends. These sequences which are also able to stabilize terminal deficiencies represent retroposons and transpose preferentially (or exclusively) from their original locus within the ~-heterochromatin to chromosome termini with a frequency sufficient to compensate for replication-mediated shortening of the terminal sequences (BIESSMANN & MASON1992). Large repetitive sequences were also found at the ends of the polytene chromosomes of chironomid species (SAIGad~; EDSTR(3M1985, CARMONA& al. 1985). Almost half of the more than one hundred tested species of vertebrates revealed telomeric repeats not only at the chromosome termini, but also at various interstitial loci; most frequently at the centromeric regions, in some cases at the nucleolus organizing regions, and, more seldom, at other, mostly heterochromatic, interstitial loci (MEYNE& al. 1990).

T e l o m e r e s e q u e n c e l o c a l i z a t i o n in h i g h e r plants

229

Table 1. P l a n t s p e c i e s i n v e s t i g a t e d b y in situ h y b r i d i z a t i o n for l o c a t i o n o f t e l o m e r i c s e q u e n c e s ( T T T A G G G ) n Species

Chromosome number

Terminal signals

Nonterminal signals

References

n = 9

+

this p a p e r

2n = 18

+

this p a p e r

2n = 24 2n = 24

-(?) +

+

2n = 24

+

+

2n = 18

+

+

2n = 34

+

this p a p e r

2n 2n 2n 2n

+ + + +

Bryophytes

Pelliaceae Pellia epiphylla (L.) CORDA Gymnosperms

Cycadaceae Zamiafurfuracea L . f . Pinaceae Picea abies (L.) KARSTEN Pinus elliottii ENOELM. Pinus sylvestris L.

this p a p e r A. KAMM & J. S. HESLOPHARR*SON (pers. c o m m . ) this p a p e r

Angiosperms

Chenopodiaceae Beta vulgaris L. Rosaceae Malus domestica BORKH. Fabaceae Lathyrus sativus L. Phaseolus coccineus L. Pisum sativum L. Viciafaba L. V. narbonensis L. V. sativa L. Vigna radiata (L.) R. WILCZEK Apiaceae Daucus carota L. subsp, sativa (HOFFM.) SCHUBL. • MART. D. montevidensis LINK ex SPRENOEL

= = = =

12 22 14 12 (14)

T. SC~MDT & J. S. HESLOPHARRISON (pers. c o m m . )

2n = 14 2n = 12

+ +

2n = 22

+

Cox & al. (1993) NAGL (1991) RAWLINS & al. (1991) SCHUBERT (1992), SCHUBERT & al. (1992) this p a p e r Cox & al. (1993), this p a p e r Cox & al. (1993)

2n = 18

+

this p a p e r

2n = 22

+

this p a p e r

24 20 24 14

+ + + +

GANAL & al. (1991) PAROKONNY & al. (1992) C o x & al. (1993) J. L. OUD (pers. c o m m . )

2n = 4x = 48

+

2n = 6 2n = 4

+ +

2n = 34 2n = 14

+ +

2n = 16 2n = 16 2n = 16

-

Solanaceae Lycopersicon esculentum MILL. 2n Nicotiana plumbaginifolia Viv. 2n Nicotiana sylvestris SPE~. & COMES 2n Petunia × hybrida HORT. ex 2n

= = = =

+

+

VILM.

Solanum tuberosum L. Asteraceae Crepis capillaris (L.) WALLR. Haplopappus gracilis (NuTT.)

this p a p e r m

A. GRAY

Helianthus annuus L. Leontodon hispidus L. Alliaceae Allium cepa L. A. chevsuricum TSCHOLOK. A. fistulosum L.

m

m

m

this p a p e r Cox & al. (1993), this p a p e r this p a p e r this p a p e r this p a p e r this p a p e r this p a p e r

J. FucHs & al.:

230 Table 1 (continued) Species

A. globosum BIEB. A. sativum L. Orchidaceae Paphiopedilum insigne (WALL.)

Chromosome number

Terminal signals

Nonterminal signals

References

2n = 16 2n = 16

-

-

this paper this paper

2n = 26+2B

+

+

Cox & al. (1993)

2n = 12

+

-

this paper

2n = 12

+

+

Cox & al. (1993)

2n = 6x = 22

+

+

Cox & al. (1993)

2n = 12

+

-

this paper

PFITZ.

Yuncaceae Luzula luzuloides (LAM.)D. ~¢ WILM.

Commelinaceae Gibasis pulchella (KuNTH) RAFIN.

Tradescantia commelinoides S CHULTESfil.

T. paludosa ANDERS. & WOODS Poaceae Aegilops markgrafii (GREUTER) HAMMERvar. markgrafii Dactylis glomerata L. Festuca arundinacea SCHREB. Hordeum vulgate L.

2n = 14

+

-

this paper

2n = 4x = 28 2n = 6x = 42 2n = 14

+ + +

-

Lolium multiflorum LAM. Milium vernale B~EB. Oryza sativa L. subsp, japonica

2n = 2x = 14 2n = 8 2 n = 24

+ + +

m

Secale cereale L.

2n = 14

+

+

Triticum aestivum L.

2n = 6x = 42

+

this paper this paper SCHWARZACHER• HESLOPHARRISON(1991); WANG& al. (1991); Cox & al. (1993) this paper Cox & al. 1993 OnMIDO& FUKUI(pets. COmlTl.) SCHWARZACHER& I-IEsLOPHARRISON(1991); T. ScrrvVARZACHZR& J. S. HFSLoP-HAR~SON(pers. COnllTI.) WF~NZR& al. (1992)

W h e r e a s the o c c u r r e n c e o f d i f f e r e n t t y p e s o f t e l o m e r i c s e q u e n c e s is o f interest w i t h r e g a r d to the e v o l u t i o n a r y r e l a t i o n s h i p b e t w e e n p h y l a , the distribution o f telomeric sequences within chromosome complements may elucidate mechanisms of k a r y o t y p e e v o l u t i o n - e s p e c i a l l y the alteration o f the d i p l o i d c h r o m o s o m e n u m b e r - b e t w e e n m o r e c l o s e l y r e l a t e d taxa. T h i s p a p e r c o m p i l e s the data c o n c e r n i n g the o c c u r r e n c e a n d l o c a t i o n o f t e l o m e r i c s e q u e n c e s in c h r o m o s o m e s o f 44 p l a n t s p e c i e s b e l o n g i n g to 14 f a m i l i e s (see T a b l e 1). F o r 23 s p e c i e s a n d s e v e n f a m i l i e s (Alliaceae, Apiaceae, Cycadaceae, Juncaceae, Pinaceae, Pelliaceae a n d Rosaceae) this is the first report. In six s p e cies (including f i v e species o f the Alliaceae) no t e l o m e r i c s e q u e n c e s o f the Arabidopsis-type w e r e d e t e c t a b l e . N i n e s p e c i e s r e v e a l e d , in addition, at least o c c a s i o n ally interstitial loci.

Telomere sequence localization in higher plants

231

Material and methods Chromosome preparation. Preparations were made from root tips of seedlings grown on wet filter paper or from cuttings (Tradescantia) of flowering plants. After treatment for 2 to 3 h in 0.05% colchicine (Picea abies and Pinus sylvestris for 15 h in 1% colchicine), fixation in 3 : 1 ethanol : acetic acid and digestion in 1% pectinase (Sigma) and 1% cellulase (Onozuka R10) for 30 rain at 37 °C, meristems were squashed in 45% acetic acid. Slides were used immediately for fluorescent in situ hybridization (FISH) or stored at 4 °C in glycerol. Thalli of Pellia epiphylla with antheridia were fixed as described above. Antheridia were prepared under a stereomicroscope and squashed in 45% acetic acid. Probe labelling, fluorescent in situ hybridization and signal detection. Heptameres (49 bp) of the Arabidopsis-type telomeric repeat (5'-TTTAGGG-3') were synthesized both in sense and antisense orientation and either end-labelled with Bio-11-dUTP (Sigma) by terminal transferase (GIBCO/Bethesda Research Laboratories) or labelled by incorporation of Bio-16-dUTP (Boehringer) via PCR according to Imo & al. (1991 b). In situ hybridization was performed as described in detail by Ft:cHs & SCHUBERt(1995). Between 20 and 100 ng biotinylated DNA were used per slide. Signals were detected with streptavidin-FITC (Vector Laboratories) and one or two rounds of amplification using biotinylated antistreptavidin (Vector Laboratories). Chromosomes were counterstained with propidium iodide. Photographs were taken using a Zeiss MC100 camera system and Kodak Ektachrom 400 colour side films or, for weak hybridization signals, using a computer-assisted cooled CCD camera (Photometrics). In the latter case, each fluorochrome was captured separately and images were pseudocoloured (Gene Join, Yale University) and merged (Adobe Photoshop 3.0). The complete images were printed on a Tektronix Phaser IISD.

i+1

a

Fig. 1. Evolutionary alterations of the metacentric Viciafaba chromosome I. a Scheme of reversible fusion/fission of chromosomes, b Giemsa-banded metacentric chromosome I and two telocentric chromosomes obtained after a spontaneous fission of the metacentric one (ScHuBERT & RIGGER 1990). Bars indicate the centromeric dot-like Giemsa-bands between which fission had occurred, c Metacentric chromosome I of the reconstructed karyotype ACB with signals after in situ hybridization with a biotinylated (TTTAGGG)nprobe. The signals at the centromeric position may indicate the origin of the metacentric chromosome by a remote fusion of two telocentrics (ScHuBERT1992)

232

J. FvcHs & al.:

1

11

Fig. 2. Scheme of possible events explaining the observed substitution of a metacentric by two acrocentric chromosomes in Tradescantia paludosa after pollination with X-irradiated pollen (C)STERGREN& (3STERG~EN1983). A translocation between homologous chromosomes or chromatids resulting in a metadicentric chromosome is followed by breakagefusion-bridge-cycles and eventual stabilization of the centric products

Results The species chosen for these experiments were selected firstly because of the occurrence of karyotype rearrangements, mostly resulting in altered diploid chrom o s o m e numbers within or between related species, secondly for comparing these with species of the same family which have already been investigated, and thirdly for searching for the Arabidopsis-type of telomeric sequences in hitherto untested families. Fabaceae. Six species of this family have already been investigated by FISH for the chromosomal localization of telomeric repeats (see Table 1, NAGL 1991, RAWLINS & al. 1991, SCat:BERa" 1992, SCt~tJBE~r & al. 1992, Cox & al. 1993). Except for Vicia faba, signals were found exclusively at the termini of metaphase or polytene (Phaseolus coccineus, NAGL 1991) chromosomes. We were able to confirm (Vicia sativa) and extend (V. narbonensis) these data (Fig. 3 d, e). In V.faba a regular interstitial locus of telomeric repeats was observed within the short arm of c h r o m o s o m e II and a polymorphic one within the centromeric region of the metacentric satellite c h r o m o s o m e I (ScauBERX 1992). The latter indicates that this chrom o s o m e m a y have originated by a remote c h r o m o s o m e fusion (Fig. 1). This is

Fig. 3. Fluorescent in situ hybridization with biotinylated Arabidopsis-type telomeric sequences to chromosomes of various plant species after counterstaining with propidium iodide. Pictures were either taken directly on a colour slide film (c-C) or captured using a cooled CCD-camera and pseudocoloured on a computer ( a, b ). a P ellia epiphylla, b Zamia furfuracea, c Pinus sylvestris, d Vicia narbonensis, e Vicia sativa, fFISH with biotinylated rDNA to metaphase chromosomes of V. sativa revealed a major and a minor locus of rDNA-genes on two pairs of chromosomes. Bar: 10 ~m


C

233

234

J. FucHs & al.:

supported by the recent finding of an individual showing a stable homozygous centric fission of this chromosome (ScHuBERT& RIEaER 1990) with both new centric termini possessing telomeric sequences (ScHuBERT& al. 1992). V. sativa (2n = 12) does not show interstitial telomeric sequences at centromere positions. Therefore, alterations of the chromosome number in this group of species (2n = 10/12/14, HANELT& METTIN 1966) may have evolved by meiotic missegregation in individuals heterozygous for two translocations involving three chromosomes (ScHuBERT& RIEGER 1985) rather than by direct chromosome fusion or fission. (Additionally, we were able to demonstrate, using FISH with biotinylated rDNA, that not one, but two chromosome pairs of this species carry nucleolus organizers; see Fig. 3 f.) Asteraceae. Haplopappus gracilis represents one of the few species with 2n = 4, the lowest chromosome number in plants. Although its karyotype structure is certainly derived, no clear interstitial telomere sequence loci indicative for direct chromosome fusions were found (Cox & al. 1993). This result is confirmed by our studies (Fig. 4 f). A similar situation occurs within the genus Crepis (BABCOCK& STEBBINS1938). Crepis capillaris (2n = 6), as well as Helianthus annuus (2n = 36) and Leontodon hispidus (2n = 14) revealed only terminal signals after FISH with telomeric sequences (Fig. 4 d, e, g, h). Solanaceae. For Nicotiana plumbaginifolia, N. sylvestris (PAROKONNY& al. 1992, Cox & al. 1993) and Lycopersicon esculentum (GANAL8~; al. 1991) Arabidopsis-type telomeres were found at terminal positions only. The same was true for Solanum tuberosum (2n = 4x = 48) in our investigations (Fig. 4 c). Apiaceae. Daucus carota subsp, sativa ( 2 n = 18) and D. montevidensis (2n = 22) are the first members of this family investigated for telomeric sequences. Signals were detectable only at terminal chromosome positions in both species (Fig. 4 a, b). Commelinaceae. Two species of this family were investigated by Cox & al. (1993), the hexaploid Tradescantia cornmelinoides and Gibasis pulchella with karyotypes resulting from Robertsonian fusions and harbouring complex interchanges, respectively. Both species revealed additional signals within some heterochromatic regions but not at the centromeres (Cox & al. 1993). For Tradescantia paludosa, 13Sa'ERCR~N& 0STERGREN(1983) observed a substitution of a submetacentric by two acrocentric chromosomes (2n = 12 ~ 2n = 14) in a progeny obtained after pollination with X-irradiated pollen. This was probably due to stabilization of centric chromosome fragments resulting from postmeiotic breakagefusion-bridge-cycles of a homodicentric chromosome (Fig. 2). The chromosomes of the tested individuals of Tradescantia paludosa did not show detectable signals for telomeric sequences at other than terminal positions (Fig. 4 k). AUiaceae. This family has not yet been tested for the presence of Arabidopsistype telomere sequences. In Allium cepa and A. fistulosum the heterochromatic satellites are dispensible and become, therefore, frequently deleted (ScHuBERT 1984). This raises the question whether terminal stabilization of nucleolus organizers is due to pre-existing telomeric sequences or to de novo formation of telomeres. However, it has not yet been possible in several independent experiments (with successful controls) to find any hybridization signals with telomeric


235

sequences on chromosomes of A. cepa, A. chevsuricum, A. fistulosum, A. globosum and A. sativum, although other monocotyledonous species showed these sequences at least at the ends of their chromosomes (Table 1). Juncaceae. Species of the genus Luzula exhibit deviating chromosome numbers. Luzula luzuloides (2n = 12) represents a species of this genus with a low number of comparatively large chromosomes revealing a polycentric constitution (BRASELTON1972). Therefore, these derived karyotypes might have originated by chromosome fusion. FISH with telomeric sequences resulted in terminal signals of varying intensity on the chromosomes of L. luzuloides. Internal signals indicating direct chromosome fusions were not regularly observed (Fig. 4 i, j). Rosaceae. The so far only investigated member of the family, Malus domestica (2n = 34), showed at least one clear signal at the ends of most chromosomes after FISH with telomeric sequences. Signals at interstitial positions were not detectable (Fig. 5 a, b). Poaceae. Except for Secale cereale, all hitherto tested species of this family showed telomeric sequences only at terminal positions. The same is true for all species checked in our investigations [Aegilops markgrafii (Fig. 5 c), Dactylis glomerata (Fig. 5 d), Festuca arundinacea (Fig. 5 d) and Lolium multiflorum (Fig. 5 e)]. Pinaceae. We were interested in including gymnosperm species into our investigations, since only angiosperms have previously been tested for the presence of Arabidopsis-type telomeric sequences. Most conifers possess karyotypes very similar in number and morphology of their chromosomes (2n = 24). Pinus sylvestris chromosomes showed signals at their ends as well as at some interstitial loci, including centromeric regions (Fig. 3 c), but no signals were detectable on the chromosomes of Picea abies. Cycadaceae. This family is considered to represent a primitive group of gymnosperms. The species Zamiafurfuracea (2n = 18) revealed only terminal signals (Fig. 3 b). Pelliaceae. Although chromosome preparations of antheridia of the liverwort Pellia epiphylla (n = 8) were covered by cytoplasm - a fact which prevented a good resolution of the hybridization signals - the chromosome ends showed signals (Fig. 3 a). This shows that the Arabidopsis-type of telomeric sequences is already present in bryophytes and has, therefore, been highly conserved during evolution of plants.

Discussion Occurrence of the Arabidopsis-type of telomeric sequences in higher plants. The compilation of data obtained by in situ hybridization revealed telomeres formed by TTTAGGG-sequences in 38 species belonging to 13 families of monoand dictoyledonous angiosperms, gymnosperms and bryophytes in addition to Arabidopsis thaliana. Sequencing of tomato telomeres revealed the same sequence (GANAL • al. 1991)and hybridization of the vertebrate telomere sequence TTAGGG to V.faba chromosomes did not yield signals when hybridized under comparable stringency (ScHuBERT& al. 1992). Therefore, it is inferred that this repeat, which is very similar to the TTTTAGGG repeat found in the alga

236

J. FucHs & al.:

a

d

C

f

%

h

g


a

b

237

c

d Fig. 5. Propidium iodide-stained metaphase chromosomes of various plant species after FISH with the biotinylated (TTTAGGG)n-probe. a-c were captured using a cooled CCDcamera and pseudocoloured on a computer; d, e were directly photographed, a, b Malus domestica; c Aegilops markgrafii; d Festuca arundinacea × Dactylis glomerata (2n = 35, MATZK1981). e Lolium multiflorum. Bar: 10 gm

C h l a m y d o m o n a s reinhardtii (PETRACgK & a l . 1990), is the basic telomeric sequence of all phyla of higher plants. Its redundancy may vary not only between species, but even between individual chromosome termini of one metaphase plate (see Figs. 3 and 4, GANAL & al. 1991, SCHWARZACHER• HESLOP-HARRISON 1991, WANG & al. 1991, Cox & al. 1993). The only family of which several species did not reveal any hybridization signals with the plant telomeric sequence after FISH was the Alliaceae. A possible

Fig. 4. FISH of Arabidopsis-type telomeric sequences to propidium iodide-counterstained chromosomes of different plant species, a, c, f, h, i, k were photographed directly, b, d, e, g, j were pseudocoloured on a computer, a, b Daucus montevidensis, c Solanum tuberosum. d, e Crepis capillaris, d incomplete metaphase, showing clear signals on all three types of chromosomes, f Haplopappus gracilis; g Helianthus annuus; h Leontodon hispidus; i, j Luzula luzuloides; k Tradescantia paludosa. Bar: 10 ~m

238

J. FucHs & al.:

explanation could be a secondarily evolved mechanism for the stabilization of chromosome termini comparable to that observed in Drosophila and possibly other Diptera lacking TTAGG-sequences at their chromosome ends. It is tempting to speculate that a large tandemly arranged repetitive sequence described for the terminal heterochromatin of Allium cepa (BARNES& al. 1985) stabilizes the chromosome termini by sequence and locus specific recombination events. Possibly, the previous observation of mobility of nucleolus organizers which occurs between chromosome ends after deletion of heterochromatic satellites in A. cepa × A. fistulosum hybrids (ScHuBERT& WOBUS 1985) is related to such a process. It is therefore of interest to test related liliaceous species for the occurrence of terminally located TTTAGGG-sequences. Chromosomal distribution of telomerie sequences. In addition to the terminal positions of the telomeric sequences in the majority of the investigated plant species, interstitial loci of these sequences were observed, at least occasionally, in nine species. In general, interstitial locations of telomeric sequences seem to be less frequent in plant than in vertebrate chromosomes. Further, in contrast to the predominant location of the interstitial loci of vertebrates at the centromeric regions, only Vicia faba, Pinus elliottii (A. KAMM& J. S. HESLOP-HARRrSON,pers. comm.) and Pinus sylvestris showed telomeric sequences in these chromosomal areas. Interstitially positioned telomeric sequences raise the question as to the cause(s) and consequence(s) of their internal location. According to BIESSMANN (1994) interstitial telomeric repeats could have developed either as random short sequence arrays which may have become extended by slippage during replication, or by attachment via telomerase or recombination to extrachromosomal linear DNA fragments which may then integrate into the genome, or by chromosome rearrangements such as fusions or inversions. Indications for interstitial telomeric sequences as remnants of chromosome fusion were obtained, e.g., from chromosomes of tissue-cultured cells of Sigmodon mascotensis (MEYNE& al. 1990), from chromosomes of canine tumor cells (REIMANN• al. 1994), from human (IJDO& al. 1991 a), Indian muntjac (LEE & al. 1993), Gonatodes taniae, a gekkonid lizard (Sci~Mm & al. 1994), and Vicia faba chromosomes (SchuBERT 1992). Interstitial telomeric sequences seem to be free of function and therefore dispensable. The centromeric locus of these repeats in the metacentric chromosome of Viciafaba is not present in all cultivars, which indicates spontaneous loss, possibly due to unequal recombination (ScHuBERT1992). A persistence of internal telomeric sequences after chromosome fusions should ensure the reversibility of these events, provided that essential parts, such as the centromere, have not been lost, or may be gained from elsewhere. Further, interstitial telomeric repeats may result in stabilization of centric fragments, if their positions become involved in chromosome breakage. However, a stabilization of chromosome breaks can also be mediated either by de novo addition of telomeric sequences via telomerase activity (WmKIE & al. 1990, MoRIN 1991), or by recombination with telomeric sequences or chromosome fragments containing telomeres, or by transposition of retroposons as in Drosophila (BIEsSMANN& MASON 1992). In some species, e.g., Paramecium primaurelia (KATINKA & BOURGAIN 1992) and Armenian hamster


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(ASHLEY • WARD 1993), interstitial telomeric sequences proved to be recombination hot spots which might increase genetic variation and contribute to genomic rearrangements. We thank Prof. G. WANNER,Munich, Dr J. L. S. KEESING,Kew, UK, and Prof. K. HAMMER, Genebank Gatersleben, for providing plant and seed material; Dr R. FRITSCH,OTTO AURICH, CARSTENORTEL and BARBARAHILDEBRANDT,Gatersleben, for determinations and preparations; Dr O. SCHRADER,Quedlinburg, Dr V. SCHUBERT,Halle and Dr M. SCHUSTER, Dresden-Pillnitz, for chromosome preparations of Daucus, Helianthus, Aegilops and MaIus, Dr R HANELT,Gatersleben, for critical reading of the manuscript and HANNALOETS, Heidelberg, for improving the English. This work was supported by a grant of the Ministerium ftir Wissenschaft und Technik of the Land Sachsen-Anhalt (LSA 623A09012). References

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Address of the authors: JORG Fucns, ANDREABRANDES*,and INoo SCHUBERT,Institut ftir Pflanzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Federal Republic of Germany. * Present address: John Innes Centre, Norwich Research Park Colney, Norwich NR4 7UH, UK. Accepted January 20, 1995 by F. EHRENDORFER

Verleger: Springer-Verlag KG, Sachsenplatz 4-6, A-120l Wien. - - Herausgeber: Prof. Dr. Friedrich Ehrendorfer, Institut ftir Botanik und Botanischer Garten der Universit~it Wien, Rennweg 14, A-1030 Wien. Redaktion: Rennweg 14, A-1030 Wien. - Hersteller: Adolf Holzhausens Nfg., Kandlgasse 19-21, A-1070 Wien, Verlagsort: Wien. - - Herstellungsort: Wien. - - Printed in Austria.