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Cloning and Characterization of Leishmania donovani Telomeres
Experimental Parasitology 94, 248–258 (2000) doi:10.1006/expr.2000.4499, available online at http://www.idealibrary.com on
Cloning and Characterization of Leishmania donovani Telomeres1
Miguel A. Chiurillo,* Anita E. Beck,†,2 Theo Devos,‡,3 Peter J. Myler,‡,§ Ken Stuart,‡,§ and Jose Luis Ramirez*,4 *Instituto de Biologı´a Experimental, Universidad Central de Venezuela, Caracas, Venezuela; †Washington University School of Medicine, Saint Louis, Missouri 63110, U.S.A.; ‡Seattle Biomedical Research Institute, Seattle, Washington 98109-1635, U.S.A.; and §Department of Pathobiology, University of Washington, Seattle, Washington 98195, U.S.A.
Chiurillo, M. A., Beck, A. E., Devos, T., Myler, P. J., Stuart, K., and Ramirez, J. L. 2000. Cloning and characterization of Leishmania donovani telomeres. Experimental Parasitology 94, 248–258. We describe here the cloning and sequence characterization of the absolute termini of several telomeres from the human parasite Leishmania donovani using a vector-adapter protocol. The 38 protruding strand of L. donovani telomeres terminates with the sequence 58-GGTTAGGGTOH 38. This single-stranded sequence is adjacent to tandemly repeated blocks of double-stranded sequence consisting of variable numbers of the hexameric repeat 58-TAGGGT-38, variable numbers of an octameric repeat 58-TGGTCATG-38, and a single 62-bp sequence, in that order. A number of additional, more chromosome-internal, nonrepeated sequences were found adjacent to the telomere sequences. Hybridization analyses indicated that some of these telomere adjacent sequences are found on all L. donovani chromosomes, some are more abundant on certain subsets of chromosomes, and some are unique to individual chromosomes. q 2000 Academic Press Index Descriptors and Abbreviations: vector-adapter telomeric cloning; telomere terminus.
INTRODUCTION
Knowledge of telomere sequence organization is critical to the understanding of telomere function. The sequence of the telomere terminus can provide useful information for the study of telomere function and to determine the binding sites for specific proteins (Gottschling and Zakian 1986; Price 1990). Information about the intact telomere is limited by the technical difficulty of cloning the absolute terminus; the 38OH overhang must be modified to allow cloning (Blackburn and Challoner 1984; Van der Ploeg et al. 1984; De Lange et al. 1990). The alternative of using half-YAC complementation (Cheng et al. 1989) is adequate for cloning subtelomeric sequences, but yeast telomeric sequences are added on the original termini. Thus, it is not surprising that few sequences of telomeric termini are known (Klobutcher et al. 1981; Henderson and Blackburn 1989). Recently, a PCR-based protocol was used to clone Leishmania subtelomeric regions (Fu and Barker 1998), confirming the presence of a hexameric repeat (58-CCCTAA-38 or 58-TTAGGG-38) and identifying a conserved 100-bp telomere-associated sequence. However, the terminal telomere sequence and organization is still unknown. In this study, we have characterized the telomeric ends of the human parasite Leishmania donovani, cloned by a recently developed vector-adapter method (Beck 1997; Chiurillo et al. 1999) which
1
The DNA sequences in this work have been submitted to GenBank and assigned Accession Nos. AFO84819-27, AFO95759, AFO95761, and AF95766. 2 Current address: University of California, San Francisco, San Francisco, CA 94143, U.S.A. 3 Current address: Icogen Corporation, Seattle, Washington 98103, U.S.A. 4 To whom correspondence should be addressed at IBE-UCV Apdo. 47525, Caracas 1041 A, Venezuela. Fax 58-2-753.5897. E-mail: [email protected].
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0014-4894/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
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complements the natural 38 overhang to preserve the telomere terminus. L. donovani telomeres have a 38 overhanging terminal sequence 58-GGTTAGGGT-OH 38 and contain hexameric, octameric, and 62-bp repeats. Several telomereadjacent sequences were found; some of them were present in all L. donovani chromosomes and others were more abundant in specific subsets of chromosomes or unique to individual chromosomes.
METHODS Parasites and DNA isolation. The following parasite species and strains were used in this study: L. donovani LSB-52.1 (MHOM/SD/ 00/1S clone cl2D); L. donovani LSB-51.1 (MHOM/SD/00/Khartoum); L. major LSB-132.1 (MHOM/IL/81/Friedlin); L. braziliensis (MHOM/ BR/75/M2903); L. amazonensis (MHOM/BR/73/M2269); Trypanosoma cruzi (CL-Brener), and Crithidia fasciculata (HS6). Telomere sequences were cloned from L. donovani LSB-52.1, which contains a multicopy small linear chromosome representing an amplification of the LD1 region (Tripp et al. 1991). Cells were grown in axenic culture as promastigotes and DNA isolated following a published procedure (Barker and Butcher 1983). Cloning of L. donovani telomeric sequences. L. donovani telomeres were cloned using a vector-adapter method (Beck 1997) outlined in Fig. 1. The 38 telomeric overhang is partially complemented with a nine-nucleotide single-stranded sequence (58-ACCCTAACC-OH 38), which is part of an adapter made from two overlapping oligonucleotides (see Table I). This particular 9-nt sequence was selected from the six possible permutations of the hexameric telomere repeat (CCCTAA) because it preferentially ligated to undigested L. donovani DNA (see Results). The adapter was ligated (via the 58-AGCT overhang) to the HindIII site of HindIII/BamHI-digested pBluescript KS2, and the vector-adapter fragment was gel-purified. It was subsequently ligated (via the 38-CCAATCCCA adapter and 38 telomere overhang) to Sau3AI-digested genomic DNA from L. donovani LSB-52.1 and the ligation mix used to transform Escherichia coli DH10B electrocompetent cells (Gibco BRL). After plating on LB plates supplemented with ampicillin, IPTG, and X-gal, white colonies were selected and those containing telomeric sequences identified by hybridization with an oligonucleotide containing three copies of the hexameric telomere repeat sequence (Hexamer probe, Table I). Plasmid DNA was isolated from these clones by alkaline SDS lysis (Sambrook et al. 1989) and sequenced with T3 and T7 primers using a Taq dye terminator cycle sequencing kit and a Model 373A DNA sequencer (Perkin–Elmer/ Applied Biosystems Inc.). Computer analysis of nucleotide sequences was performed using Lasergene software (DNASTAR). Separation of L. donovani chromosomes. Chromosomal-size DNA was prepared in agarose blocks containing 107 cells/ml as previously described (Scholler et al. 1986) and separated by clamped homogeneous electric field (CHEF) gel electrophoresis using a Bio-Rad CHEF-DR III System, in 1% DNA typing grade agarose (Gibco BRL) gels and 0.53TBE buffer. All electrophoresis were done at 148C, with an electrode angle of 1208 and a voltage gradient of 6 V/cm, and three different switching parameters were used: 14 h with 60-s pulses, 9 h with 90s pulses, and 5 h with 120-s pulses for separating small, intermediate,
and large chromosomes, respectively. Gels were stained with ethidium bromide and transferred to nylon filters (Hybond N, Amersham) by capillary action (Sambrook et al. 1989). Probes. Oligonucleotide probes were labeled with [g-32P] ATP, 3000 Ci/mmol (Amersham), using T4 polynucleotide kinase (Sambrook et al. 1989) or with dUTP-digoxigenin using terminal transferase according to the manufacturer’s protocol (Boehringer Mannheim). Probes for telomere-adjacent sequences were labeled by PCR using [a-32P]dCTP, 3000 Ci/mmol (Amersham) (Mertz and Rashtchian 1994). The primers and templates are listed in Table I. The 26S rRNA probe (clone Ldtel221, containing part of the large-subunit ribosomal gene) was labeled by incorporating [a-32P]dCTP 3000 Ci/mmol (Amersham) using the Megaprime DNA labeling system (Amersham). Hybridization conditions. For double-stranded radiolabeled probes, hybridizations were carried out in 0.5 M Na–phosphate, pH 7.2, 7% SDS, 1 mM EDTA, 0.1 mg/ml E. coli tRNA at 658C for 18 h. After hybridization, filters were washed twice at room temperature with 40 mM Na–phosphate, 0.1% SDS, for 15 min and twice with the same buffer at 658C for 15 min. For oligonucleotide probes, the same hybridization solutions were used, but the hybridization temperatures were 588C (Hexamer probe), 548C (Octamer probe), or 658C (62-bp probe). After hybridization, the filters were washed as before, with the last wash being done at the same temperatures used for hybridization. In the case of digoxigenin-labeled Hexamer probe, hybridization was carried out in 5 3 SSC, 0.1% sodium–N-laurylsarcosinate, 0.02% SDS, 1% blocking reagent (Boehringer Mannheim) at 588C for 3 h. After hybridization, filters were washed at room temperature in 6 3 SSC, 0.1% SDS for 5 min, then washed with 2 3 SSC, 0.1% SDS at room temperature for 15 min, and finally washed at 588C for 15 min. Hybridized probe was detected by chemiluminescense using Lumiphos 530 according to the manufacturers (Boehringer Mannheim). Bal 31 digestions. High-molecular-weight genomic DNA from L. donovani was incubated with 20 units of Bal31 exonuclease (Gibco BRL) at 308C. The reaction was stopped at 0, 2, 5, 15, 30, 40, and 60 min by the addition of EGTA to 10 mM. DNA was extracted with phenol–chloroform, ethanol precipitated, and then digested with HindIII and size fractionated in 0.8% agarose gel for Southern blot analysis (Brown et al. 1990).
RESULTS
Cloning by the Vector-Adapter Protocol Previous experiments with Trypanosoma cruzi, another member of the Trypanosomatidae family, indicated that when a mixture of the six adapters was used for telomeric cloning, the resultant telomeric recombinants contained only one adapter (Chiurillo et al. 1999). Thus, it appeared that
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FIG. 1. The vector-adapter method for cloning telomere termini in L. donovani.
all T. cruzi telomeres terminate in a single sequence (58GGGTAGGG-OH 38). To determine whether a similar situation applies for L. donovani, we annealed undigested genomic DNA with radiolabeled oligonucleotide adapters containing a 9-nt 38 overhang representing the six different permutations of the telomeric hexameric repeat (CCCTAA) and added T4 ligase. After ligation, the DNA was heated at 658C to release annealed but nonligated adapters and separated by agarose gel electrophoresis and the location of the adapters was determined by autoradiography. One adapter (containing the 38 terminal overhang 58-ACCCTAACC-OH
38) showed substantially more ligation to L. donovani telomeres than the others, as indicated by its comigration with high-molecular-weight DNA after ligation (Beck 1997). Although the adapters could potentially ligate to overhangs shorter than nine nucleotides, we found that no ligation occurred (nor were recombinants obtained, see below) unless Adapter No. 1 (the upper oligonucleotide in Fig. 1) was 58 phosphorylated (Chiurillo et al. 1999). Therefore the 38 overhang of L. donovani telomeres is at least 9 nt and terminates with the sequence 58-GGTTAGGGT-0H 38. In order to confirm these results and to characterize the
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TABLE I Oligonucleotides Used in These Experiments Oligonucleotide Adapter No. 1 Adapter No. 2 Hexamer probe Octamer probe 62-mer probe J forward primer J reverse primer K forward primer K reverse primer L forward primer L reverse primer M forward primer M reverse primer O forward primer O reverse primer P forward primer P reverse primer Q forward primer Q reverse primer R forward primer R reverse primer
TABLE II Sequence Organization of Telomeric Clones
Sequence
Clone
Size (kbp)
58P-GGCCGGGGAGGCCA-OH 38 58P-AGCTTGGCCTCCCCGGCCACCCTAACC-OH 38 58-CCCTAACCCTAACCCTAA-38 58-ACCAGTACACCAGTAC-38 58-TCACGCCCCCGTCCTGTTGGAGAGGGT-38 58-TCGATAGTGGCGGTCGCCCTGAA-38 58-GAGCTCATAATCCTCCCACACCGGTG-38 58-CAGCGAAGTTGAGCACGG-38 58-ATCATTCGCGCGCGGGCA-38 58-GCTTTGACGCGAGTCTGG-38 58-TGTTTGCTTGCCCGCGGA-38 58-CGTTGCGTTGTCTGTCACA-38 58-CGCATTGGGTTACGTGCCA-38 58-GCTCCTCTGCTCTCATGAC-38 58-AGGTTGGGGAGAATAGGTG-38 58-CCGGTACACTCGAGCATCT-38 58-TCCCTCTTGATGCTCCTAC-38 58-ACCCTGTCGTTCAATGCA-38 58-GATGTACAGCTGGTGAACAC-38 58-GCCCACGGCAAACACCA-38 58-TGTTTGTCGTCGTCGCGCC-38
200 201 202 204 205 206 207 208 209 210 211 212
1.2 3.0 0.9 0.8 1.4 2.2 1.9 0.4 2.8 0.8 3.2 2.2
213 215 217 218 219 221 223 226 228 230 233 235 237 238 B2 C1 C3 D1 D2 D3 E1 E2 E3 F2 F3 G1 H2
1.2 1.5 1.4 2.6 3.1 4.4 1.2 0.2 1.7 2.2 0.7 2.2 0.5 0.6 0.7 4.0 1.9 2.5 1.2 3.2 1.7 1.8 3.4 4.1 1.0 1.0 2.2
sequences adjacent to the telomeric termini, we used this adapter to clone L. donovani telomeres (see Methods and Fig. 1). Of the 1085 white colonies selected, 65 hybridized with the Hexamer probe. We selected 45 of these recombinants for further restriction and sequence analysis. Four recombinants were false positives (they did not contain the hexameric repeat or the adapter) and two had cloning artifacts (the HindIII cloning site was absent and the adapter sequence was truncated). The remainder had inserts ranging from 0.2 to 4 kbp with the sequence of selected adapter at their T3 end (see Table II). Several clones were found to contain one or more Sau3AI sites, identified by restriction digestion and/or sequence analysis. Since these could result from either partial digestion or ligation of multiple inserts into the cloning vector, subsequent analysis of these clones considered only the sequence between the adapter and the first Sau3AI site. Immediately adjacent to the adapter sequence, the recombinants contained a variable number of hexameric repeats (58-TAGGGT-38), the first one and a half copies of which represent the 38 terminal telomere overhang that annealed with the adapter sequence. Eight clones (200, 204, 226, 233, B2, C1, D2, and F3) contained nonrepeated Leishmania
Sequence organization I H27 J......H.13 [S*O2H4] [SO2H4] [SO2H11] J H12 [SO10H22] J H30 ......H.18 [SO2H8] ......H.32 ......H.72 a H4 [S*O2H11] [SO6H2] [SO2H1] ......H.8 [SO2H36] K [S*O2H.8]......H.56 L......H.8 [SO3H30] K [SO6bH.3]......H.5 [S*O2H7] [SO2H2] [SO2H8] M H19 [S*O1H1] ......H.9c [SO2H6] [SO3dH13e] [SO3dH11] ......H.21f ......H.5 L......H.9 ......H.6 [SO2H5] [S*O2H3] [SO2H11] ......H.63f N H15 O H.22......H.39 T H29g [S*O6H18] P H11 ......H.21 [SO1H23] N H20 [SO11H9] h [SO2H35] J H9 Q H8 ......H.9 [SO2H18] ......H.4 [SO2H21] Q H11 ......H.40f ......H.21f R......[SO2H5] [SO2H5] [SO2H11] ......H.24 R......H.14 Q H16 R......H.22 [S O2H15] ......H.27 [S*O2H12] [SO2H8]
Note. ......, Uncharacterized sequence; *, sequence variant of 62-bp repeat. I, J, K, L, M, N, O, P, Q, R, S, and T are non-repeated subtelomeric sequences (LSTS). The reading of the sequences are on the C-rich strand with the terminal at the right side a No Sau3AI site at the T7 end. b Second and fourth octameric repeats have sequence GTACTGGTGGT. c Fourth hexameric repeats have sequence GAGGGT. d Octameric repeats have sequence ATACTGGT/GCACTGCT/ GTACTGGA. e Second hexameric repeat has sequence GAGGGT. f Hybridized to 62-bp probe. g First 10 copies have sequence GAGGGT. h 62-bp repeat truncated at the T7 end.
252 subtelomeric sequences(LSTS, Table III) immediately adjacent to the hexameric repeats (Table II). One clone (228) contained LSTS adjacent to hexameric repeats at its T7 end and only hexameric repeats at its T3 end, but it was too long (1.7 kbp) to sequence in its entirety and so may contain other sequences. A further 10 clones (206, 207, 217, 218, 219, 223, D3, E1, E3, and F2) contained only hexameric repeats at their T3 ends. While some of these contained LSTS at their T7 ends, the junction between these sequences was not elucidated, since they were not completely sequenced. In contrast, 20 clones (Table II), contained variable numbers of octameric repeats (58-GTACTGGT-38) and a 62-bp sequence (Fig. 2) immediately adjacent to the hexameric repeats at their T3 ends. These sequences formed a repeating block with the general form, [SOyHx]n , where H, O, and S represent the hexameric, octameric, and 62-bp repeats respectively; and x, y, and n represent variable numbers of copies of each repeat unit. In the sample analyzed here, x ranged from 1 to 72, y from 1 to 11, and n from 1 to 4. These three sequences are clearly related, since they contain the same sequence (GGT) at their 38 ends. Clones 208 and 213 have a single copy of the hexameric repeat at the terminus, immediately followed by one or two copies of the octameric repeat. Thus, the first three nucleotides of the first octamer were part of the 38 telomeric overhang. Four clones (202, 213, 230, and 237) contained LSTS adjacent to hexameric repeats following the SOyHx blocks. Two clones (210 and 212) contain LSTS K immediately adjacent to the 62bp sequence at their T7 ends, although the sequence at the 38 end of LSTS K (58- GAGGGTTACGTT-38) resembles two degenerate copies of the hexameric repeat. Both clones also contain an unsequenced region between their T3 and T7 sequences. Another clone (238) contains a single SOyHx block, with a truncated 62-bp sequence, while one more (208), is truncated within the fourth SOyHx block. In the remaining 12 clones, the junction between the SOyHx blocks and the LSTS remains uncharacterized. In addition, hybridization analysis using the 62-bp probe indicates that four
FIG. 2. Alignment of telomere-associated sequences from different Leishmania species. The consensus sequence of a typical block of 62bp, octameric, and hexameric repeats is shown for each species, with only a single copy of the hexameric repeat included. The L. donovani sequence is from this paper; the L. major, L. mexicana, L. braziliensis, and L. lainsonsi sequences are from Fu and Barker (1998). The conserved sequence boxes (CSB1 and CSB2) identified by Fu and Barker (1998) are indicated by underlining, CSB2 light, and CSB1 dark. Dots represent nucleotide matches; dashes are blank spaces to make alignment possible.
CHIURILLO ET AL.
CLONING AND CHARACTERIZATION OF L. donovani TELOMERES
additional clones (217, 223, D3, and E1) contain a 62-bp sequence within their unsequenced regions. The location of the telomere-associated sequences was confirmed by Bal31 exonuclease sensitivity experiments. High-molecular-weight DNA from L. donovani was digested with Bal31 for a range of time periods, followed by HindIII digestion and Southern hybridization with oligonucleotide probes for the hexameric repeats, 62-bp sequence, and LSTS Q (Figs. 3A–3C). All three probes hybridize to a range of fragments in DNA without Bal31 treatment, with the strongest signal being seen to fragments $22 kbp. This signal was largely eliminated after 40 min of digestion with Bal31. Since a similar-size fragment detected by a probe for the chromosomal-internal 26S rRNA was not effected by Bal31
253 digestion of up to 60 min (Fig. 3D), this supports a telomeric location for these sequences. To determine the chromosomal distribution of the telomere-associated sequences, we probed blots of CHEF gelseparated chromosomal DNA from a number of Leishmania species, C. fasciculata, and T. cruzi with oligonucleotide probes for the hexameric, octameric, and 62-bp repeats (Fig. 4). As expected, the hexameric repeat probe hybridized to all chromosomal bands in all organisms (Fig. 4B), although the intensity of hybridization varied between chromosomes. This presumably reflects differences in the number of hexameric repeats on each chromosome, as well as a difference in the number of comigrating chromosomes in each band. In contrast, the octameric repeat (Fig. 4C) and 62-bp (Fig.
FIG. 3. Bal31 exonuclease analysis of telomere-associated sequences. L. donovani LSB-52.1 genomic DNA was digested with Bal31 exonuclease, for the times indicated, digested with HindIII, and transferred to nylon membranes as described under Methods. The membranes were then hybridized with radiolabeled oligonucleotide probes for the hexameric (A) and 62-bp (B) repeats; a PCR-labeled probe for telomere-adjacent sequence Q (C); and a mixed hexamer-labeled probe for 26S rRNA (D).
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FIG. 4. Chromosomal localization of telomere-associated sequences. Chromosome-sized DNA from Saccharomyces cerevisiae (lane 1), L. donovani LSB-52.1 (lane 2), L. donovani LSB-51.1 (lane 3), L. major LSB-132.1 (lane 4), L. amazonensis M2269 (lane 5), L. braziliensis M2903 (lane 6), Crithidia fasciculata (HS6) (lane 7), and Trypanosoma cruzi (CL Brener) (lane 8) was separated by PFG electrophoresis as described under Methods. Gels were stained with ethidium bromide (A), transferred to nylon filters, and probed with radiolabeled oligonucleotide probes for hexameric (B), octameric (C), and 62-bp (D) repeats. Arrows indicate chromosomal bands with different hybridization signals between L. donovani LSB-52.1 and LSB-51.1.
4D) probes hybridized only to chromosomes from L. donovani, probably because of sequence divergence between species (see Fig. 2). Both probes hybridized to all L. donovani chromosomes, but there were obvious differences in signal intensities between L. donovani LSB-52.1 and LSB-51.1 for some specific chromosomal bands. In particular, some chromosomes (see arrows in Fig. 4D) appeared to hybridize more strongly with the 62-bp probe than the octameric repeat probe, suggesting that the telomeres on these chromosomes may contain fewer copies of the octameric repeat. These telomeres may be represented by clones 213 and 235 (Table II), which contain only single copies of the octameric repeat. Hybridization to CHEF gel-separated chromosomal DNA with LSTS probes indicated that most (but not all) of the LSTS appear to be present in all chromosomes of L. donovani and most appear to be present in all Leishmania species. The chromosomal locations of selected LSTS are shown in Fig. 5. The probe for LSTS R (Fig. 5B) hybridized with all (or most) L. donovani chromosomes, whereas probes for P and L (Figs. 5C and 5D, respectively) recognized unique
bands in the low- and high-molecular-weight range, respectively. Several LSTS (J, K, L, N, Q, and R) were found in more than one clone (see Table III). Small sequence differences were sometimes found between different clones containing the same LSTS (e.g., Q), suggesting that they may be derived from different telomeres.
DISCUSSION In this study, we report the sequence of the 38 overhang of a Leishmania telomere terminus. In L. donovani, the majority of (perhaps all) telomeres terminate with the sequence 58GGTTAGGGT-OH 38. The same result was obtained for L. major telomeres (Chiurillo, unpublished data). In contrast, the telomeres of the related kinetoplastids T. brucei (Beck 1997) and T. cruzi (Chiurillo et al. 1999) terminate with the sequence 58-GGGTTAGGG-OH 38. Thus, although these organisms have the same telomeric hexameric
CLONING AND CHARACTERIZATION OF L. donovani TELOMERES
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FIG. 5. Chromosomal distribution of nonrepeated telomere-adjacent sequences (LSTS). Chromosome-size DNA from Saccharomyces cerevisiae (lane 1), L. donovani LSB-52.1 (lane 2), and L. donovani LSB-51.1 (lane 3) was separated by PFG electrophoresis as described under Methods. Gels were stained with ethidium bromide (A), transferred to nylon filters, and probed with radiolabeled PCR probes for LSTS R (B), P (C), and L (D).
repeat sequence, the register for the 38 overhang is different. The significance of this fact is not clear, but in a recent study in T. brucei telomerase (Cano et al. 1999) it was suggested that the minimal 9-nt templating domain of the telomerase RNA was 38OH-CCCAAUCCC-58, that is, exactly complementary to the termini reported for T. brucei (Beck 1997) and T. cruzi (Chiurillo et al. 1999). If these results are confirmed, then we can suggest that the minimal 9-nt template domain of Leishmania telomerase RNA template is 38OH-CCAAUCCCA-58. This fact is compatible with the mechanistic model of telomerase involving primer
annealing, primer elongation, and translocation (Greider and Blackburn 1989) for the synthesis of 58-TAGGGT-38 hexameric repeats. Since the telomeric terminus in other organisms is the target for the binding of important proteins (Gottschling and Zakian 1986; Greider and Blackburn 1989; Price 1990; Shippen et al. 1994; Horvath et al. 1998), this knowledge should be important for future biological studies on telomere function in these parasites, as well as to the synthesis and maintenance of telomeric DNA. The sequence structure of L. donovani telomeres may be summarized as 58-LSTS-Hz[SOyHx]n , where S, O, and H
256 represent 62-bp, octameric, and hexameric repeats, respectively; x and z are $1 (often .10), y is $1 (usually 2), and n is $0 (we counted up to 4). The 62-bp sequence was first reported in L. donovani by Ellis and Crampton (1988) in the opposite orientation relative to the hexameric repeats to the one reported here. Although this has been attributed to a cloning artifact (Fu and Barker 1998), telomeric sequences with inverted orientation have been also reported in T. brucei (Weiden et al. 1991) and T. cruzi (Chiurillo et al. 1999). Some sequence variation is seen within these telomere-associated repeats (see Tables II and III). Sequence variants of the hexameric and octameric repeats tend to occur within clusters, suggesting that they may have arisen and spread by the “slippage” mechanism proposed by Fu and Barker (1998). However, sequence variations within the 62-bp sequence tend not to be clustered in the same recombinant (see Table II), which may argue against the model as originally proposed. The block organization of telomeric sequences observed for L. donovani is also consistent with the telomeric sequences reported for L. major and L. mexicana (Fu and Barker 1998). The 100-bp LCTAS sequence in these organisms is the equivalent of the octameric and 62-bp repeats reported here (see Fig. 2). The L. major 62-bp sequence contains seven nucleotide differences from that of L. donovani. Five of these differences are in the region of the oligonucleotide probe used in Fig. 4D, explaining its lack of hybridization to L. major. The octameric sequence, which typically occurs as two identical repeats in L. donovani, is more frequently found as four divergent copies in L. major and L. mexicana (Fig. 2). The canonical telomeric sequence organization described above is less well-conserved in L. braziliensis and L. lainsoni (see Fig. 2). The 62-bp sequence is moderately conserved, especially in the region of one of the conserved sequence boxes (CSB2) described by Fu and Barker (1998), but appears to be somewhat longer in L. braziliensis and L. lainsoni. CSB1 is not conserved with in the 62-bp sequence of L. donovani, L. major, and L. mexicana, but is similar to part of the octameric sequence (see Fig. 2). The octameric sequence has also diverged in L. braziliensis and L. lainsoni and appears commonly as a single copy between the 62-bp and hexameric repeats. Interestingly, the 14-bp tandem repeat described within L. braziliensis telomeres by Fu and Barker (1998) is composed of alternating hexameric and octameric repeats. The origin and function of the particular organization of these telomereassociated sequences are not clear. The sequence similarity at the 38 end of the hexameric, octameric, and 62-bp repeats suggests that the last two may have been derived from the first.
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We found that 51% (20/39, Table II) of the telomeric clones analyzed contained variable numbers of [SOyHx]n blocks. Although 49% (19/39, Table II) lacked the octameric and 62-bp repeats based on sequence analysis, at least 10% (4/39, Table II) contained the 62-bp sequence based on hybridization. However, at least some clones contained only hexameric repeats followed by LSTS. This distribution of telomere-associated sequences is similar to that found by Fu and Barker (1988) in clones from L. braziliensis (50%:50%) and L. major (41%:58%) using a PCR-based protocol. If this distribution is a genuine representation of the telomere population, this suggests that only about half of the telomeres contain the [SOyHx]n block organization. We have ruled out the possibility that just one telomere on each chromosome contains [SOyHx]n blocks using bidimensional CHEF gels and RARE endonuclease digestion (Ferrin and Camerini-Otero 1991)(data not shown). Thus, it appears that the [SOyHx]n blocks are randomly distributed in all chromosomes. Alternatively, the block organization may be present or absent at particular times during the cell cycle. Since the DNA for cloning was isolated from nonsynchronized cells, this may explain the mixed population of telomeres with and without the [SOyHx]n block structure. It is interesting that the number and structure of the [SOyHx]n blocks vary between clones which contain the same LSTS, even in the case when these sequences appear to be specific for a single chromosome (clones 211 and 219). Similar results were also obtained in the study of L. braziliensis and L. major (Fu and Barker 1998), suggesting that individual telomere ends are polymorphic within Leishmania nuclei. Certainly, evidence from L. major Friedlin, indicates that the size of the telomeric repeats can vary by .1 kbp between chromosome homologs (Myler et al. 1999). Perhaps surprisingly, most of the LSTS appear to be present in all chromosomes and most appear to be present in all Leishmania species (as determined by hybridization). However, only one (Q) showed sequence homology with L. major or L. braziliensis telomere-associated sequences (Accession Number AF031202). Since these sequence were not found together in individual telomere clones, this suggests that the order in which they occur varies between chromosomes. Sequence rearrangements in the subtelomeric and telomeric regions have been documented for other protozoa (see Lanzer et al. 1995 for a review). Several hypotheses have been raised regarding their role in generating chromosome polymorphism and antigenic variation. The significance of telomeric and subtelomeric sequence heterogeneity in L. donovani remains to be determined.
CLONING AND CHARACTERIZATION OF L. donovani TELOMERES
ACKNOWLEDGMENTS
We thank Maynard Olson for his guidance in telomeric cloning and Ellen Sisk for the DNA sequencing. This work was supported by Grants S1-95000524 from CONICIT-Venezuela to JLR, as well as TDR/WHO T23/181/1 ID:940509 and NIH Grant AI17375 to KS.
REFERENCES
Barker, D., and Butcher, J.1983. The use of DNA probes in the identification of Leishmanias: Discrimination between isolates of Leishmania mexicana and L. braziliensis complex. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 285–297. Beck, A. E. 1997. “Telomeres: Requirements for Creation of a New Telomere in Vivo and Molecular Characterization and Cloning of Telomeric Ends by a Novel Telomere Adapter Scheme.” Ph.D. Thesis, Washington University, Division of Biology and Biomedical Sciences, Department of Genetics, Saint Louis, MO. Blackburn, E. H., and Challoner, P. B. 1984. Identification of a telomeric DNA sequence in Trypanosoma brucei. Cell 36, 447–457. Brown, W. R. A., MacKinnon, P. J., Villasante, A., Spurr, N., Buckle, V. J., and Dobson, M. J. 1990. Structure and polymorphism of human telomere-associated DNA. Cell 63, 119–132. Cano, M. I. N., Dungan, J. M., Agabian, N., and Blackburn, E. H. 1999. Telomerase in kinetoplastid parasitic protozoa. Proceedings of the National Academy of Sciences USA 96, 3616–3621. Cheng, J. F., Smith, C. L., and Cantor, C. R. 1989. Isolation and Characterization of a human telomere. Nucleic Acids Research 17, 6109–6127. Chiurillo, M. A., Cano, M. I., Franco Da Silveira, J., and Ramirez, J. L. 1999. Organization of telomeric and sub-telomeric regions of chromosomes from the protozoan parasite Trypanosoma cruzi. Molecular and Biochemical Parasitology 100, 173–183.
257 Fu, G., and Barker, D. C. 1998. Characterisation of Leishmania telomeres reveals unusual telomeric repeats and conserved telomeric sequence. Nucleic Acids Research 29, 2161–2167. Gottschling, D. E., and Zakian, V. A. 1986. Telomere proteins: Specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell 47, 195–205. Greider, C. W., and Blackburn, E. H.1989. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomeric repeat synthesis. Nature 337, 331–337. Henderson, E. R., and Blackburn, E. H. 1989. An overhanging 38 terminus is a conserved feature of telomeres. Molecular and Cellular Biology 9, 345–348. Horvath, M. P., Schweiker, V. L., Bevilacqua, J. M., Ruggles, J. A., and Schultz, S. C.1998. Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA. Cell 95, 963–974. Klobutcher, L. A., Swanton, M. T., Donini, P., and Prescott, D. M. 1981. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 38 terminus. Proceedings of the National Academy of Sciences USA 78, 3015–3019. Lanzer, M., Fischer, K., and Le Blancq, S. M. 1995. Parasitism and chromosome dynamics in protozoan parasites: Is there a connection? Molecular and Biochemical Parasitology 70, 1–8. Mertz, L. M., and Rashtchian, A. 1994. PCR radioactive labeling system: A rapid method for synthesis of high specific activity DNA probes. FOCUS (Published for the Life Scientist by Life Technologies, Inc.) 16(2): 45–48. Myler, P. J., Audleman, L., Devos, T., Hixson, G., Kiser, P., Lemley, C., Magness, C., Rickel, E., Sisk, E., Sunkin, S., Swartzell, S., Westlake, T., Bartien, P., Fu G., Ivens, A. and Stuart, K. 1999. Leishmania major Friedlin chromosome 1 has an unusual distribution of protein-coding genes. Proceedings of the National Academy of Sciences USA 96, 2902–2906. Pluta, A. F., Klaine, B. P., and Spear, B. B. 1982. The terminal organization of macromolecular DNA in Oxytricha fallax. Nucleic Acids Research 10, 8145–8154. Price, C. M. 1990. Telomere structure in Euplotes crassus: Characterization of DNA–protein interactions and isolation of telomere-binding protein. Molecular and Cellular Biology 10, 3421–3431.
De Lange, T., Shiue, L., Myers, R. M., Cox, D. R., Naylor, S. L., Killery, A. M., and Varmus, H. E. 1990. Structure and variability of human chromosome ends. Molecular and Cellular Biology 10, 518–527.
Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Ellis, J., and Crampton, J. 1988. Characterization of a simple, highly repetitive DNA from the parasite Leishmania donovani. Molecular and Biochemical Parasitology 29, 9–18.
Scholler, J. K., Reed, S. G., and Stuart, K. 1986. Molecular Karyotype of species and subspecies of Leishmania. Molecular and Biochemical Parasitology 20, 279–293.
Feng, J., Funk, W. D., Wang, S., Weinrich, S. L., Avilion, A. A., Chiu, C., Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., Le, S., West, M. D., Harley, C., Andrews, W., Greider, C. W., and Villeponteau, B. 1995. The RNA Component of human telomerase. Science 269, 1236–1241.
Shippen, D. E., Blackburn, E. H., and Price, C. M. 1994. DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases. Proceedings of the National Academy of Sciences USA 91, 405–409.
Ferrin, L. J., and Camerini-Otero, R. D. 1991. Selection cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage. Science 254, 1425–1556.
Shippen-Lentz, D., and Blackburn, E. H. 1990. Functional evidence for a RNA template in telomerase. Science 247, 546–552. Tripp, C. A., Myler, P. J., and Suart, K. 1991. A DNA sequence
258 (LD1) which occurs in several genomic organizations in Leishmania. Molecular and Biochemical Parasitology 47, 151–160. Van Der Ploeg, L. H. T., Liu, A. Y. C., and Borst, P. 1984. Structure of the growing telomeres of Trypanosomes. Cell 36, 459–468. Weiden, M., Osheim, Y. N., Beyer, A. L., and Van Der Ploeg. 1991.
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Chromosome structure: DNA nucleotide sequence elements of subset of the minichromosomes of the protozoan Trypanosoma brucei. Molecular and Cellular Biology 11, 3823–3834. Received 8 November 1999, accepted with revision 23 February 2000
Cloning and Characterization of Leishmania donovani Telomeres1
Miguel A. Chiurillo,* Anita E. Beck,†,2 Theo Devos,‡,3 Peter J. Myler,‡,§ Ken Stuart,‡,§ and Jose Luis Ramirez*,4 *Instituto de Biologı´a Experimental, Universidad Central de Venezuela, Caracas, Venezuela; †Washington University School of Medicine, Saint Louis, Missouri 63110, U.S.A.; ‡Seattle Biomedical Research Institute, Seattle, Washington 98109-1635, U.S.A.; and §Department of Pathobiology, University of Washington, Seattle, Washington 98195, U.S.A.
Chiurillo, M. A., Beck, A. E., Devos, T., Myler, P. J., Stuart, K., and Ramirez, J. L. 2000. Cloning and characterization of Leishmania donovani telomeres. Experimental Parasitology 94, 248–258. We describe here the cloning and sequence characterization of the absolute termini of several telomeres from the human parasite Leishmania donovani using a vector-adapter protocol. The 38 protruding strand of L. donovani telomeres terminates with the sequence 58-GGTTAGGGTOH 38. This single-stranded sequence is adjacent to tandemly repeated blocks of double-stranded sequence consisting of variable numbers of the hexameric repeat 58-TAGGGT-38, variable numbers of an octameric repeat 58-TGGTCATG-38, and a single 62-bp sequence, in that order. A number of additional, more chromosome-internal, nonrepeated sequences were found adjacent to the telomere sequences. Hybridization analyses indicated that some of these telomere adjacent sequences are found on all L. donovani chromosomes, some are more abundant on certain subsets of chromosomes, and some are unique to individual chromosomes. q 2000 Academic Press Index Descriptors and Abbreviations: vector-adapter telomeric cloning; telomere terminus.
INTRODUCTION
Knowledge of telomere sequence organization is critical to the understanding of telomere function. The sequence of the telomere terminus can provide useful information for the study of telomere function and to determine the binding sites for specific proteins (Gottschling and Zakian 1986; Price 1990). Information about the intact telomere is limited by the technical difficulty of cloning the absolute terminus; the 38OH overhang must be modified to allow cloning (Blackburn and Challoner 1984; Van der Ploeg et al. 1984; De Lange et al. 1990). The alternative of using half-YAC complementation (Cheng et al. 1989) is adequate for cloning subtelomeric sequences, but yeast telomeric sequences are added on the original termini. Thus, it is not surprising that few sequences of telomeric termini are known (Klobutcher et al. 1981; Henderson and Blackburn 1989). Recently, a PCR-based protocol was used to clone Leishmania subtelomeric regions (Fu and Barker 1998), confirming the presence of a hexameric repeat (58-CCCTAA-38 or 58-TTAGGG-38) and identifying a conserved 100-bp telomere-associated sequence. However, the terminal telomere sequence and organization is still unknown. In this study, we have characterized the telomeric ends of the human parasite Leishmania donovani, cloned by a recently developed vector-adapter method (Beck 1997; Chiurillo et al. 1999) which
1
The DNA sequences in this work have been submitted to GenBank and assigned Accession Nos. AFO84819-27, AFO95759, AFO95761, and AF95766. 2 Current address: University of California, San Francisco, San Francisco, CA 94143, U.S.A. 3 Current address: Icogen Corporation, Seattle, Washington 98103, U.S.A. 4 To whom correspondence should be addressed at IBE-UCV Apdo. 47525, Caracas 1041 A, Venezuela. Fax 58-2-753.5897. E-mail: [email protected].
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0014-4894/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
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complements the natural 38 overhang to preserve the telomere terminus. L. donovani telomeres have a 38 overhanging terminal sequence 58-GGTTAGGGT-OH 38 and contain hexameric, octameric, and 62-bp repeats. Several telomereadjacent sequences were found; some of them were present in all L. donovani chromosomes and others were more abundant in specific subsets of chromosomes or unique to individual chromosomes.
METHODS Parasites and DNA isolation. The following parasite species and strains were used in this study: L. donovani LSB-52.1 (MHOM/SD/ 00/1S clone cl2D); L. donovani LSB-51.1 (MHOM/SD/00/Khartoum); L. major LSB-132.1 (MHOM/IL/81/Friedlin); L. braziliensis (MHOM/ BR/75/M2903); L. amazonensis (MHOM/BR/73/M2269); Trypanosoma cruzi (CL-Brener), and Crithidia fasciculata (HS6). Telomere sequences were cloned from L. donovani LSB-52.1, which contains a multicopy small linear chromosome representing an amplification of the LD1 region (Tripp et al. 1991). Cells were grown in axenic culture as promastigotes and DNA isolated following a published procedure (Barker and Butcher 1983). Cloning of L. donovani telomeric sequences. L. donovani telomeres were cloned using a vector-adapter method (Beck 1997) outlined in Fig. 1. The 38 telomeric overhang is partially complemented with a nine-nucleotide single-stranded sequence (58-ACCCTAACC-OH 38), which is part of an adapter made from two overlapping oligonucleotides (see Table I). This particular 9-nt sequence was selected from the six possible permutations of the hexameric telomere repeat (CCCTAA) because it preferentially ligated to undigested L. donovani DNA (see Results). The adapter was ligated (via the 58-AGCT overhang) to the HindIII site of HindIII/BamHI-digested pBluescript KS2, and the vector-adapter fragment was gel-purified. It was subsequently ligated (via the 38-CCAATCCCA adapter and 38 telomere overhang) to Sau3AI-digested genomic DNA from L. donovani LSB-52.1 and the ligation mix used to transform Escherichia coli DH10B electrocompetent cells (Gibco BRL). After plating on LB plates supplemented with ampicillin, IPTG, and X-gal, white colonies were selected and those containing telomeric sequences identified by hybridization with an oligonucleotide containing three copies of the hexameric telomere repeat sequence (Hexamer probe, Table I). Plasmid DNA was isolated from these clones by alkaline SDS lysis (Sambrook et al. 1989) and sequenced with T3 and T7 primers using a Taq dye terminator cycle sequencing kit and a Model 373A DNA sequencer (Perkin–Elmer/ Applied Biosystems Inc.). Computer analysis of nucleotide sequences was performed using Lasergene software (DNASTAR). Separation of L. donovani chromosomes. Chromosomal-size DNA was prepared in agarose blocks containing 107 cells/ml as previously described (Scholler et al. 1986) and separated by clamped homogeneous electric field (CHEF) gel electrophoresis using a Bio-Rad CHEF-DR III System, in 1% DNA typing grade agarose (Gibco BRL) gels and 0.53TBE buffer. All electrophoresis were done at 148C, with an electrode angle of 1208 and a voltage gradient of 6 V/cm, and three different switching parameters were used: 14 h with 60-s pulses, 9 h with 90s pulses, and 5 h with 120-s pulses for separating small, intermediate,
and large chromosomes, respectively. Gels were stained with ethidium bromide and transferred to nylon filters (Hybond N, Amersham) by capillary action (Sambrook et al. 1989). Probes. Oligonucleotide probes were labeled with [g-32P] ATP, 3000 Ci/mmol (Amersham), using T4 polynucleotide kinase (Sambrook et al. 1989) or with dUTP-digoxigenin using terminal transferase according to the manufacturer’s protocol (Boehringer Mannheim). Probes for telomere-adjacent sequences were labeled by PCR using [a-32P]dCTP, 3000 Ci/mmol (Amersham) (Mertz and Rashtchian 1994). The primers and templates are listed in Table I. The 26S rRNA probe (clone Ldtel221, containing part of the large-subunit ribosomal gene) was labeled by incorporating [a-32P]dCTP 3000 Ci/mmol (Amersham) using the Megaprime DNA labeling system (Amersham). Hybridization conditions. For double-stranded radiolabeled probes, hybridizations were carried out in 0.5 M Na–phosphate, pH 7.2, 7% SDS, 1 mM EDTA, 0.1 mg/ml E. coli tRNA at 658C for 18 h. After hybridization, filters were washed twice at room temperature with 40 mM Na–phosphate, 0.1% SDS, for 15 min and twice with the same buffer at 658C for 15 min. For oligonucleotide probes, the same hybridization solutions were used, but the hybridization temperatures were 588C (Hexamer probe), 548C (Octamer probe), or 658C (62-bp probe). After hybridization, the filters were washed as before, with the last wash being done at the same temperatures used for hybridization. In the case of digoxigenin-labeled Hexamer probe, hybridization was carried out in 5 3 SSC, 0.1% sodium–N-laurylsarcosinate, 0.02% SDS, 1% blocking reagent (Boehringer Mannheim) at 588C for 3 h. After hybridization, filters were washed at room temperature in 6 3 SSC, 0.1% SDS for 5 min, then washed with 2 3 SSC, 0.1% SDS at room temperature for 15 min, and finally washed at 588C for 15 min. Hybridized probe was detected by chemiluminescense using Lumiphos 530 according to the manufacturers (Boehringer Mannheim). Bal 31 digestions. High-molecular-weight genomic DNA from L. donovani was incubated with 20 units of Bal31 exonuclease (Gibco BRL) at 308C. The reaction was stopped at 0, 2, 5, 15, 30, 40, and 60 min by the addition of EGTA to 10 mM. DNA was extracted with phenol–chloroform, ethanol precipitated, and then digested with HindIII and size fractionated in 0.8% agarose gel for Southern blot analysis (Brown et al. 1990).
RESULTS
Cloning by the Vector-Adapter Protocol Previous experiments with Trypanosoma cruzi, another member of the Trypanosomatidae family, indicated that when a mixture of the six adapters was used for telomeric cloning, the resultant telomeric recombinants contained only one adapter (Chiurillo et al. 1999). Thus, it appeared that
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FIG. 1. The vector-adapter method for cloning telomere termini in L. donovani.
all T. cruzi telomeres terminate in a single sequence (58GGGTAGGG-OH 38). To determine whether a similar situation applies for L. donovani, we annealed undigested genomic DNA with radiolabeled oligonucleotide adapters containing a 9-nt 38 overhang representing the six different permutations of the telomeric hexameric repeat (CCCTAA) and added T4 ligase. After ligation, the DNA was heated at 658C to release annealed but nonligated adapters and separated by agarose gel electrophoresis and the location of the adapters was determined by autoradiography. One adapter (containing the 38 terminal overhang 58-ACCCTAACC-OH
38) showed substantially more ligation to L. donovani telomeres than the others, as indicated by its comigration with high-molecular-weight DNA after ligation (Beck 1997). Although the adapters could potentially ligate to overhangs shorter than nine nucleotides, we found that no ligation occurred (nor were recombinants obtained, see below) unless Adapter No. 1 (the upper oligonucleotide in Fig. 1) was 58 phosphorylated (Chiurillo et al. 1999). Therefore the 38 overhang of L. donovani telomeres is at least 9 nt and terminates with the sequence 58-GGTTAGGGT-0H 38. In order to confirm these results and to characterize the
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TABLE I Oligonucleotides Used in These Experiments Oligonucleotide Adapter No. 1 Adapter No. 2 Hexamer probe Octamer probe 62-mer probe J forward primer J reverse primer K forward primer K reverse primer L forward primer L reverse primer M forward primer M reverse primer O forward primer O reverse primer P forward primer P reverse primer Q forward primer Q reverse primer R forward primer R reverse primer
TABLE II Sequence Organization of Telomeric Clones
Sequence
Clone
Size (kbp)
58P-GGCCGGGGAGGCCA-OH 38 58P-AGCTTGGCCTCCCCGGCCACCCTAACC-OH 38 58-CCCTAACCCTAACCCTAA-38 58-ACCAGTACACCAGTAC-38 58-TCACGCCCCCGTCCTGTTGGAGAGGGT-38 58-TCGATAGTGGCGGTCGCCCTGAA-38 58-GAGCTCATAATCCTCCCACACCGGTG-38 58-CAGCGAAGTTGAGCACGG-38 58-ATCATTCGCGCGCGGGCA-38 58-GCTTTGACGCGAGTCTGG-38 58-TGTTTGCTTGCCCGCGGA-38 58-CGTTGCGTTGTCTGTCACA-38 58-CGCATTGGGTTACGTGCCA-38 58-GCTCCTCTGCTCTCATGAC-38 58-AGGTTGGGGAGAATAGGTG-38 58-CCGGTACACTCGAGCATCT-38 58-TCCCTCTTGATGCTCCTAC-38 58-ACCCTGTCGTTCAATGCA-38 58-GATGTACAGCTGGTGAACAC-38 58-GCCCACGGCAAACACCA-38 58-TGTTTGTCGTCGTCGCGCC-38
200 201 202 204 205 206 207 208 209 210 211 212
1.2 3.0 0.9 0.8 1.4 2.2 1.9 0.4 2.8 0.8 3.2 2.2
213 215 217 218 219 221 223 226 228 230 233 235 237 238 B2 C1 C3 D1 D2 D3 E1 E2 E3 F2 F3 G1 H2
1.2 1.5 1.4 2.6 3.1 4.4 1.2 0.2 1.7 2.2 0.7 2.2 0.5 0.6 0.7 4.0 1.9 2.5 1.2 3.2 1.7 1.8 3.4 4.1 1.0 1.0 2.2
sequences adjacent to the telomeric termini, we used this adapter to clone L. donovani telomeres (see Methods and Fig. 1). Of the 1085 white colonies selected, 65 hybridized with the Hexamer probe. We selected 45 of these recombinants for further restriction and sequence analysis. Four recombinants were false positives (they did not contain the hexameric repeat or the adapter) and two had cloning artifacts (the HindIII cloning site was absent and the adapter sequence was truncated). The remainder had inserts ranging from 0.2 to 4 kbp with the sequence of selected adapter at their T3 end (see Table II). Several clones were found to contain one or more Sau3AI sites, identified by restriction digestion and/or sequence analysis. Since these could result from either partial digestion or ligation of multiple inserts into the cloning vector, subsequent analysis of these clones considered only the sequence between the adapter and the first Sau3AI site. Immediately adjacent to the adapter sequence, the recombinants contained a variable number of hexameric repeats (58-TAGGGT-38), the first one and a half copies of which represent the 38 terminal telomere overhang that annealed with the adapter sequence. Eight clones (200, 204, 226, 233, B2, C1, D2, and F3) contained nonrepeated Leishmania
Sequence organization I H27 J......H.13 [S*O2H4] [SO2H4] [SO2H11] J H12 [SO10H22] J H30 ......H.18 [SO2H8] ......H.32 ......H.72 a H4 [S*O2H11] [SO6H2] [SO2H1] ......H.8 [SO2H36] K [S*O2H.8]......H.56 L......H.8 [SO3H30] K [SO6bH.3]......H.5 [S*O2H7] [SO2H2] [SO2H8] M H19 [S*O1H1] ......H.9c [SO2H6] [SO3dH13e] [SO3dH11] ......H.21f ......H.5 L......H.9 ......H.6 [SO2H5] [S*O2H3] [SO2H11] ......H.63f N H15 O H.22......H.39 T H29g [S*O6H18] P H11 ......H.21 [SO1H23] N H20 [SO11H9] h [SO2H35] J H9 Q H8 ......H.9 [SO2H18] ......H.4 [SO2H21] Q H11 ......H.40f ......H.21f R......[SO2H5] [SO2H5] [SO2H11] ......H.24 R......H.14 Q H16 R......H.22 [S O2H15] ......H.27 [S*O2H12] [SO2H8]
Note. ......, Uncharacterized sequence; *, sequence variant of 62-bp repeat. I, J, K, L, M, N, O, P, Q, R, S, and T are non-repeated subtelomeric sequences (LSTS). The reading of the sequences are on the C-rich strand with the terminal at the right side a No Sau3AI site at the T7 end. b Second and fourth octameric repeats have sequence GTACTGGTGGT. c Fourth hexameric repeats have sequence GAGGGT. d Octameric repeats have sequence ATACTGGT/GCACTGCT/ GTACTGGA. e Second hexameric repeat has sequence GAGGGT. f Hybridized to 62-bp probe. g First 10 copies have sequence GAGGGT. h 62-bp repeat truncated at the T7 end.
252 subtelomeric sequences(LSTS, Table III) immediately adjacent to the hexameric repeats (Table II). One clone (228) contained LSTS adjacent to hexameric repeats at its T7 end and only hexameric repeats at its T3 end, but it was too long (1.7 kbp) to sequence in its entirety and so may contain other sequences. A further 10 clones (206, 207, 217, 218, 219, 223, D3, E1, E3, and F2) contained only hexameric repeats at their T3 ends. While some of these contained LSTS at their T7 ends, the junction between these sequences was not elucidated, since they were not completely sequenced. In contrast, 20 clones (Table II), contained variable numbers of octameric repeats (58-GTACTGGT-38) and a 62-bp sequence (Fig. 2) immediately adjacent to the hexameric repeats at their T3 ends. These sequences formed a repeating block with the general form, [SOyHx]n , where H, O, and S represent the hexameric, octameric, and 62-bp repeats respectively; and x, y, and n represent variable numbers of copies of each repeat unit. In the sample analyzed here, x ranged from 1 to 72, y from 1 to 11, and n from 1 to 4. These three sequences are clearly related, since they contain the same sequence (GGT) at their 38 ends. Clones 208 and 213 have a single copy of the hexameric repeat at the terminus, immediately followed by one or two copies of the octameric repeat. Thus, the first three nucleotides of the first octamer were part of the 38 telomeric overhang. Four clones (202, 213, 230, and 237) contained LSTS adjacent to hexameric repeats following the SOyHx blocks. Two clones (210 and 212) contain LSTS K immediately adjacent to the 62bp sequence at their T7 ends, although the sequence at the 38 end of LSTS K (58- GAGGGTTACGTT-38) resembles two degenerate copies of the hexameric repeat. Both clones also contain an unsequenced region between their T3 and T7 sequences. Another clone (238) contains a single SOyHx block, with a truncated 62-bp sequence, while one more (208), is truncated within the fourth SOyHx block. In the remaining 12 clones, the junction between the SOyHx blocks and the LSTS remains uncharacterized. In addition, hybridization analysis using the 62-bp probe indicates that four
FIG. 2. Alignment of telomere-associated sequences from different Leishmania species. The consensus sequence of a typical block of 62bp, octameric, and hexameric repeats is shown for each species, with only a single copy of the hexameric repeat included. The L. donovani sequence is from this paper; the L. major, L. mexicana, L. braziliensis, and L. lainsonsi sequences are from Fu and Barker (1998). The conserved sequence boxes (CSB1 and CSB2) identified by Fu and Barker (1998) are indicated by underlining, CSB2 light, and CSB1 dark. Dots represent nucleotide matches; dashes are blank spaces to make alignment possible.
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CLONING AND CHARACTERIZATION OF L. donovani TELOMERES
additional clones (217, 223, D3, and E1) contain a 62-bp sequence within their unsequenced regions. The location of the telomere-associated sequences was confirmed by Bal31 exonuclease sensitivity experiments. High-molecular-weight DNA from L. donovani was digested with Bal31 for a range of time periods, followed by HindIII digestion and Southern hybridization with oligonucleotide probes for the hexameric repeats, 62-bp sequence, and LSTS Q (Figs. 3A–3C). All three probes hybridize to a range of fragments in DNA without Bal31 treatment, with the strongest signal being seen to fragments $22 kbp. This signal was largely eliminated after 40 min of digestion with Bal31. Since a similar-size fragment detected by a probe for the chromosomal-internal 26S rRNA was not effected by Bal31
253 digestion of up to 60 min (Fig. 3D), this supports a telomeric location for these sequences. To determine the chromosomal distribution of the telomere-associated sequences, we probed blots of CHEF gelseparated chromosomal DNA from a number of Leishmania species, C. fasciculata, and T. cruzi with oligonucleotide probes for the hexameric, octameric, and 62-bp repeats (Fig. 4). As expected, the hexameric repeat probe hybridized to all chromosomal bands in all organisms (Fig. 4B), although the intensity of hybridization varied between chromosomes. This presumably reflects differences in the number of hexameric repeats on each chromosome, as well as a difference in the number of comigrating chromosomes in each band. In contrast, the octameric repeat (Fig. 4C) and 62-bp (Fig.
FIG. 3. Bal31 exonuclease analysis of telomere-associated sequences. L. donovani LSB-52.1 genomic DNA was digested with Bal31 exonuclease, for the times indicated, digested with HindIII, and transferred to nylon membranes as described under Methods. The membranes were then hybridized with radiolabeled oligonucleotide probes for the hexameric (A) and 62-bp (B) repeats; a PCR-labeled probe for telomere-adjacent sequence Q (C); and a mixed hexamer-labeled probe for 26S rRNA (D).
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FIG. 4. Chromosomal localization of telomere-associated sequences. Chromosome-sized DNA from Saccharomyces cerevisiae (lane 1), L. donovani LSB-52.1 (lane 2), L. donovani LSB-51.1 (lane 3), L. major LSB-132.1 (lane 4), L. amazonensis M2269 (lane 5), L. braziliensis M2903 (lane 6), Crithidia fasciculata (HS6) (lane 7), and Trypanosoma cruzi (CL Brener) (lane 8) was separated by PFG electrophoresis as described under Methods. Gels were stained with ethidium bromide (A), transferred to nylon filters, and probed with radiolabeled oligonucleotide probes for hexameric (B), octameric (C), and 62-bp (D) repeats. Arrows indicate chromosomal bands with different hybridization signals between L. donovani LSB-52.1 and LSB-51.1.
4D) probes hybridized only to chromosomes from L. donovani, probably because of sequence divergence between species (see Fig. 2). Both probes hybridized to all L. donovani chromosomes, but there were obvious differences in signal intensities between L. donovani LSB-52.1 and LSB-51.1 for some specific chromosomal bands. In particular, some chromosomes (see arrows in Fig. 4D) appeared to hybridize more strongly with the 62-bp probe than the octameric repeat probe, suggesting that the telomeres on these chromosomes may contain fewer copies of the octameric repeat. These telomeres may be represented by clones 213 and 235 (Table II), which contain only single copies of the octameric repeat. Hybridization to CHEF gel-separated chromosomal DNA with LSTS probes indicated that most (but not all) of the LSTS appear to be present in all chromosomes of L. donovani and most appear to be present in all Leishmania species. The chromosomal locations of selected LSTS are shown in Fig. 5. The probe for LSTS R (Fig. 5B) hybridized with all (or most) L. donovani chromosomes, whereas probes for P and L (Figs. 5C and 5D, respectively) recognized unique
bands in the low- and high-molecular-weight range, respectively. Several LSTS (J, K, L, N, Q, and R) were found in more than one clone (see Table III). Small sequence differences were sometimes found between different clones containing the same LSTS (e.g., Q), suggesting that they may be derived from different telomeres.
DISCUSSION In this study, we report the sequence of the 38 overhang of a Leishmania telomere terminus. In L. donovani, the majority of (perhaps all) telomeres terminate with the sequence 58GGTTAGGGT-OH 38. The same result was obtained for L. major telomeres (Chiurillo, unpublished data). In contrast, the telomeres of the related kinetoplastids T. brucei (Beck 1997) and T. cruzi (Chiurillo et al. 1999) terminate with the sequence 58-GGGTTAGGG-OH 38. Thus, although these organisms have the same telomeric hexameric
CLONING AND CHARACTERIZATION OF L. donovani TELOMERES
255
FIG. 5. Chromosomal distribution of nonrepeated telomere-adjacent sequences (LSTS). Chromosome-size DNA from Saccharomyces cerevisiae (lane 1), L. donovani LSB-52.1 (lane 2), and L. donovani LSB-51.1 (lane 3) was separated by PFG electrophoresis as described under Methods. Gels were stained with ethidium bromide (A), transferred to nylon filters, and probed with radiolabeled PCR probes for LSTS R (B), P (C), and L (D).
repeat sequence, the register for the 38 overhang is different. The significance of this fact is not clear, but in a recent study in T. brucei telomerase (Cano et al. 1999) it was suggested that the minimal 9-nt templating domain of the telomerase RNA was 38OH-CCCAAUCCC-58, that is, exactly complementary to the termini reported for T. brucei (Beck 1997) and T. cruzi (Chiurillo et al. 1999). If these results are confirmed, then we can suggest that the minimal 9-nt template domain of Leishmania telomerase RNA template is 38OH-CCAAUCCCA-58. This fact is compatible with the mechanistic model of telomerase involving primer
annealing, primer elongation, and translocation (Greider and Blackburn 1989) for the synthesis of 58-TAGGGT-38 hexameric repeats. Since the telomeric terminus in other organisms is the target for the binding of important proteins (Gottschling and Zakian 1986; Greider and Blackburn 1989; Price 1990; Shippen et al. 1994; Horvath et al. 1998), this knowledge should be important for future biological studies on telomere function in these parasites, as well as to the synthesis and maintenance of telomeric DNA. The sequence structure of L. donovani telomeres may be summarized as 58-LSTS-Hz[SOyHx]n , where S, O, and H
256 represent 62-bp, octameric, and hexameric repeats, respectively; x and z are $1 (often .10), y is $1 (usually 2), and n is $0 (we counted up to 4). The 62-bp sequence was first reported in L. donovani by Ellis and Crampton (1988) in the opposite orientation relative to the hexameric repeats to the one reported here. Although this has been attributed to a cloning artifact (Fu and Barker 1998), telomeric sequences with inverted orientation have been also reported in T. brucei (Weiden et al. 1991) and T. cruzi (Chiurillo et al. 1999). Some sequence variation is seen within these telomere-associated repeats (see Tables II and III). Sequence variants of the hexameric and octameric repeats tend to occur within clusters, suggesting that they may have arisen and spread by the “slippage” mechanism proposed by Fu and Barker (1998). However, sequence variations within the 62-bp sequence tend not to be clustered in the same recombinant (see Table II), which may argue against the model as originally proposed. The block organization of telomeric sequences observed for L. donovani is also consistent with the telomeric sequences reported for L. major and L. mexicana (Fu and Barker 1998). The 100-bp LCTAS sequence in these organisms is the equivalent of the octameric and 62-bp repeats reported here (see Fig. 2). The L. major 62-bp sequence contains seven nucleotide differences from that of L. donovani. Five of these differences are in the region of the oligonucleotide probe used in Fig. 4D, explaining its lack of hybridization to L. major. The octameric sequence, which typically occurs as two identical repeats in L. donovani, is more frequently found as four divergent copies in L. major and L. mexicana (Fig. 2). The canonical telomeric sequence organization described above is less well-conserved in L. braziliensis and L. lainsoni (see Fig. 2). The 62-bp sequence is moderately conserved, especially in the region of one of the conserved sequence boxes (CSB2) described by Fu and Barker (1998), but appears to be somewhat longer in L. braziliensis and L. lainsoni. CSB1 is not conserved with in the 62-bp sequence of L. donovani, L. major, and L. mexicana, but is similar to part of the octameric sequence (see Fig. 2). The octameric sequence has also diverged in L. braziliensis and L. lainsoni and appears commonly as a single copy between the 62-bp and hexameric repeats. Interestingly, the 14-bp tandem repeat described within L. braziliensis telomeres by Fu and Barker (1998) is composed of alternating hexameric and octameric repeats. The origin and function of the particular organization of these telomereassociated sequences are not clear. The sequence similarity at the 38 end of the hexameric, octameric, and 62-bp repeats suggests that the last two may have been derived from the first.
CHIURILLO ET AL.
We found that 51% (20/39, Table II) of the telomeric clones analyzed contained variable numbers of [SOyHx]n blocks. Although 49% (19/39, Table II) lacked the octameric and 62-bp repeats based on sequence analysis, at least 10% (4/39, Table II) contained the 62-bp sequence based on hybridization. However, at least some clones contained only hexameric repeats followed by LSTS. This distribution of telomere-associated sequences is similar to that found by Fu and Barker (1988) in clones from L. braziliensis (50%:50%) and L. major (41%:58%) using a PCR-based protocol. If this distribution is a genuine representation of the telomere population, this suggests that only about half of the telomeres contain the [SOyHx]n block organization. We have ruled out the possibility that just one telomere on each chromosome contains [SOyHx]n blocks using bidimensional CHEF gels and RARE endonuclease digestion (Ferrin and Camerini-Otero 1991)(data not shown). Thus, it appears that the [SOyHx]n blocks are randomly distributed in all chromosomes. Alternatively, the block organization may be present or absent at particular times during the cell cycle. Since the DNA for cloning was isolated from nonsynchronized cells, this may explain the mixed population of telomeres with and without the [SOyHx]n block structure. It is interesting that the number and structure of the [SOyHx]n blocks vary between clones which contain the same LSTS, even in the case when these sequences appear to be specific for a single chromosome (clones 211 and 219). Similar results were also obtained in the study of L. braziliensis and L. major (Fu and Barker 1998), suggesting that individual telomere ends are polymorphic within Leishmania nuclei. Certainly, evidence from L. major Friedlin, indicates that the size of the telomeric repeats can vary by .1 kbp between chromosome homologs (Myler et al. 1999). Perhaps surprisingly, most of the LSTS appear to be present in all chromosomes and most appear to be present in all Leishmania species (as determined by hybridization). However, only one (Q) showed sequence homology with L. major or L. braziliensis telomere-associated sequences (Accession Number AF031202). Since these sequence were not found together in individual telomere clones, this suggests that the order in which they occur varies between chromosomes. Sequence rearrangements in the subtelomeric and telomeric regions have been documented for other protozoa (see Lanzer et al. 1995 for a review). Several hypotheses have been raised regarding their role in generating chromosome polymorphism and antigenic variation. The significance of telomeric and subtelomeric sequence heterogeneity in L. donovani remains to be determined.
CLONING AND CHARACTERIZATION OF L. donovani TELOMERES
ACKNOWLEDGMENTS
We thank Maynard Olson for his guidance in telomeric cloning and Ellen Sisk for the DNA sequencing. This work was supported by Grants S1-95000524 from CONICIT-Venezuela to JLR, as well as TDR/WHO T23/181/1 ID:940509 and NIH Grant AI17375 to KS.
REFERENCES
Barker, D., and Butcher, J.1983. The use of DNA probes in the identification of Leishmanias: Discrimination between isolates of Leishmania mexicana and L. braziliensis complex. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 285–297. Beck, A. E. 1997. “Telomeres: Requirements for Creation of a New Telomere in Vivo and Molecular Characterization and Cloning of Telomeric Ends by a Novel Telomere Adapter Scheme.” Ph.D. Thesis, Washington University, Division of Biology and Biomedical Sciences, Department of Genetics, Saint Louis, MO. Blackburn, E. H., and Challoner, P. B. 1984. Identification of a telomeric DNA sequence in Trypanosoma brucei. Cell 36, 447–457. Brown, W. R. A., MacKinnon, P. J., Villasante, A., Spurr, N., Buckle, V. J., and Dobson, M. J. 1990. Structure and polymorphism of human telomere-associated DNA. Cell 63, 119–132. Cano, M. I. N., Dungan, J. M., Agabian, N., and Blackburn, E. H. 1999. Telomerase in kinetoplastid parasitic protozoa. Proceedings of the National Academy of Sciences USA 96, 3616–3621. Cheng, J. F., Smith, C. L., and Cantor, C. R. 1989. Isolation and Characterization of a human telomere. Nucleic Acids Research 17, 6109–6127. Chiurillo, M. A., Cano, M. I., Franco Da Silveira, J., and Ramirez, J. L. 1999. Organization of telomeric and sub-telomeric regions of chromosomes from the protozoan parasite Trypanosoma cruzi. Molecular and Biochemical Parasitology 100, 173–183.
257 Fu, G., and Barker, D. C. 1998. Characterisation of Leishmania telomeres reveals unusual telomeric repeats and conserved telomeric sequence. Nucleic Acids Research 29, 2161–2167. Gottschling, D. E., and Zakian, V. A. 1986. Telomere proteins: Specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell 47, 195–205. Greider, C. W., and Blackburn, E. H.1989. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomeric repeat synthesis. Nature 337, 331–337. Henderson, E. R., and Blackburn, E. H. 1989. An overhanging 38 terminus is a conserved feature of telomeres. Molecular and Cellular Biology 9, 345–348. Horvath, M. P., Schweiker, V. L., Bevilacqua, J. M., Ruggles, J. A., and Schultz, S. C.1998. Crystal structure of the Oxytricha nova telomere end binding protein complexed with single strand DNA. Cell 95, 963–974. Klobutcher, L. A., Swanton, M. T., Donini, P., and Prescott, D. M. 1981. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 38 terminus. Proceedings of the National Academy of Sciences USA 78, 3015–3019. Lanzer, M., Fischer, K., and Le Blancq, S. M. 1995. Parasitism and chromosome dynamics in protozoan parasites: Is there a connection? Molecular and Biochemical Parasitology 70, 1–8. Mertz, L. M., and Rashtchian, A. 1994. PCR radioactive labeling system: A rapid method for synthesis of high specific activity DNA probes. FOCUS (Published for the Life Scientist by Life Technologies, Inc.) 16(2): 45–48. Myler, P. J., Audleman, L., Devos, T., Hixson, G., Kiser, P., Lemley, C., Magness, C., Rickel, E., Sisk, E., Sunkin, S., Swartzell, S., Westlake, T., Bartien, P., Fu G., Ivens, A. and Stuart, K. 1999. Leishmania major Friedlin chromosome 1 has an unusual distribution of protein-coding genes. Proceedings of the National Academy of Sciences USA 96, 2902–2906. Pluta, A. F., Klaine, B. P., and Spear, B. B. 1982. The terminal organization of macromolecular DNA in Oxytricha fallax. Nucleic Acids Research 10, 8145–8154. Price, C. M. 1990. Telomere structure in Euplotes crassus: Characterization of DNA–protein interactions and isolation of telomere-binding protein. Molecular and Cellular Biology 10, 3421–3431.
De Lange, T., Shiue, L., Myers, R. M., Cox, D. R., Naylor, S. L., Killery, A. M., and Varmus, H. E. 1990. Structure and variability of human chromosome ends. Molecular and Cellular Biology 10, 518–527.
Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Ellis, J., and Crampton, J. 1988. Characterization of a simple, highly repetitive DNA from the parasite Leishmania donovani. Molecular and Biochemical Parasitology 29, 9–18.
Scholler, J. K., Reed, S. G., and Stuart, K. 1986. Molecular Karyotype of species and subspecies of Leishmania. Molecular and Biochemical Parasitology 20, 279–293.
Feng, J., Funk, W. D., Wang, S., Weinrich, S. L., Avilion, A. A., Chiu, C., Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., Le, S., West, M. D., Harley, C., Andrews, W., Greider, C. W., and Villeponteau, B. 1995. The RNA Component of human telomerase. Science 269, 1236–1241.
Shippen, D. E., Blackburn, E. H., and Price, C. M. 1994. DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases. Proceedings of the National Academy of Sciences USA 91, 405–409.
Ferrin, L. J., and Camerini-Otero, R. D. 1991. Selection cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage. Science 254, 1425–1556.
Shippen-Lentz, D., and Blackburn, E. H. 1990. Functional evidence for a RNA template in telomerase. Science 247, 546–552. Tripp, C. A., Myler, P. J., and Suart, K. 1991. A DNA sequence
258 (LD1) which occurs in several genomic organizations in Leishmania. Molecular and Biochemical Parasitology 47, 151–160. Van Der Ploeg, L. H. T., Liu, A. Y. C., and Borst, P. 1984. Structure of the growing telomeres of Trypanosomes. Cell 36, 459–468. Weiden, M., Osheim, Y. N., Beyer, A. L., and Van Der Ploeg. 1991.
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Chromosome structure: DNA nucleotide sequence elements of subset of the minichromosomes of the protozoan Trypanosoma brucei. Molecular and Cellular Biology 11, 3823–3834. Received 8 November 1999, accepted with revision 23 February 2000