The Journal of Heredity 2002:93(4)
© 2002 The American Genetic Association 93:282-285
Brief Communication |
Conservation of (TTAGG)n Telomeric Sequences Among Ants (Hymenoptera, Formicidae)
From the Departamento de Biología Experimental, área de Genética, Universidad de Jaén, 23071 Jaén, Spain.
Address correspondence to T. Palomeque at the address above, or e-mail: tpalome{at}ujaen.es.
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Abstract |
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To determine the telomere sequence in Tapinoma nigerrimum, we carried out in situ hybridization using TTAGGG and TTAGG repeat polymerase chain reaction (PCR)-generated probes. No hybridization signals were found when TTAGGG was used as a probe. However, strong signals were observed at the end of the chromosomes with the TTAGG probe. Southern blot analysis carried out on genomic DNA using TTAGG as a probe showed a strong hybridization signal even under highly stringent conditions. Similar results were obtained in Southern blot analysis carried out on genomic DNA of 19 species of ants belonging to three different subfamilies. In accordance with all the results shown in this article, the TTAGG repeat seems to be the major component of the telomere sequence in the majority of ant species.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Telomeres are at the end of eukaryotic chromosomes and consist of many tandemly repeated copies of basic short sequences, which are synthesized by the reverse transcriptase activity of telomerase (Blackburn 1991). Telomere sequences are very similar among species, consisting of tandem arrays of simple sequence 3' G-rich, according to the (T)nA(G)n pattern (reviewed in Henderson 1995; Zakian 1995).
In the insect species for which telomere organization has been studied, three different types of organization have been described: a pentanucleotide sequence repeat (TTAGG)n from the silkworm (Bombyx mori) (Okazaki et al. 1993), HeT-A and TART transposable elements in Drosophila melanogaster (Diptera) (reviewed in Mason and Biessmann 1995), and long complex and regular tandem repeats in chironomids (Diptera) (Rosén and Edström 2000; Zhang et al. 1994). The (TTAGG)n repeat appears to be a widespread, though not the only telomere DNA motif in insects and other arthropods according to Southern hybridization and fluorescence in situ hybridization (FISH) data (Okazaki et al. 1993; Sahara et al. 1999).
Recently telomerase activity of insects has been detected using a modified telomeric repeat amplification protocol (TRAP) (Sasaki and Fujiwara 2000). This telomerase required dATP, dGTP, and dTTP, but not dCTP, as a substrate and sequence analyses of the products of TRAP revealed that the (TTAGG)n repeats are synthesized by telomerase. However, in some species which have (TTAGG)n, such as B. mori, the absence or very weak activity of telomerase has been reported (Sasaki and Fujiwara 2000).
The telomeric organization of the order Hymenoptera has been studied in only a few species. (TTAGG)n-containing telomeres were found in the honeybee (Apis mellifera) (Hymenoptera, Apidae) and in the ant Manica yessensis (Hymenoptera, Formicidae) (Okazaki et al. 1993; Sahara et al. 1999). However, telomeric hybridization with the TTAGG oligomers and with the putative vertebrate telomere sequence, TTAGGG, has also been reported in several species of ants belonging to the genus Myrmecia (Meyne et al. 1995).
In this article we analyze the telomeric sequences of the ant Tapinoma nigerrimum by Southern and in situ hybridization using synthetic oligonucleotides with the sequences (TTAGGG)n and (TTAGG)n. In addition, we used Southern blotting to determine the presence of the TTAGG and TTAGGG repeats in 19 species of ants included in three subfamilies: Dolichoderinae, Formicinae, and Myrmicinae.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Ant Material and DNA Isolation
The ant species examined are indicated in the following figures. Ants were collected in Spain. Adult workers were used for DNA extraction according the technique described by Heinze et al. (1994). Drosophila melanogaster and spleens from mice were also used for DNA isolation using the same technique.
DNA Probes Labeling for Southern Analysis
Synthetic oligonucleotides with the sequence (TTAGGG)5 or (TTAGG)6 were directly DIG-labeled using terminal transferase (Roche) according to the instructions of the supplier. Labeled probes were dissolved in hybridization solution (5x SSC, 0.1% N-laurosylsarcosine, 0.02% SDS, 1% blocking reagent) to a final concentration of 2 pmol/ml.
Southern Hybridization
DNA samples (2 µg) digested with EcoRI were separated by electrophoresis on a 0.8% agarose gel and blotted to a nitrocellulose filter. Hybridization with oligonucleotides (TTAGGG)5 and (TTAGG)6 was performed overnight at 55°C. Posthybridization washes were carried out in different steps with four different stringency conditions: (1) 2x SSC, (2) 2x + 1x SSC, (3) 2x + 1x + 0.5x SSC, and (4) 2x + 1x + 0.5x + 0.1x SSC, according to the procedure of Okazaki et al. (1993). Each wash was conducted at 55°C for 20 min. Hybridization signals were detected using the alkaline phosphatase DIG-detection kit (Roche).
DNA Probes Labeling for In Situ Hybridization
Telomeric probes were generated by polymerase chain reaction (PCR) using (TTAGG)6 and (TAACC)6 as primers, which when mixed, annealed to two stable double-stranded DNA forms with two base 3' and three base 5' protruding ends, respectively. PCR was performed in 100 µl using 100 pmol of each primer and 2.5 U of Taq polymerase in the absence of a template. Amplification consisted in 30 cycles, each 1 min at 95°C, 1 min at 50°C, 1 min at 72°C, and a final step of 10 min at 72°C. PCR-generated fragments between 200 bp and 1 kb were labeled with biotin using the biotin labeling kit from Roche. A similar procedure was used to obtain a mammal telomeric probe, using the oligonucleotides (TTAGGG)5 and (TAACCC)5 as primers.
Chromosome Preparations and In Situ Hybridization
Metaphase chromosome preparations from T. nigerrimum were obtained as described (Palomeque et al. 1988). Slides were denatured with the hybridization solution (20 ng probe/µl 50% formamide) for 2 min at 80°C and incubated in a moist chamber at 37°C overnight. After hybridization they were rinsed three times in 50% formamide at 37°C. Hybridization signals were detected using an avidin-alkaline phosphatase/biotinilated antiavidin system. Three rounds of amplification were performed using 100 µl of avidin-alkaline phosphatase (5 µg/ml) or biotinilated antiavidin (5 µg/ml) and finally an alkaline phosphatase DIG-detection kit (Roche). The chromosomes were subsequently stained with Giemsa.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Southern hybridization was used to detect telomeric sequences ((TTAGG)6 and (TTAGGG)5) in genomic DNA from T. nigerrimum, D. melanogaster, and the mouse (Figure 1). Using the (TTAGGG)5 oligonucleotide as probe, under conditions of low stringency (final wash in 2x SSC at 55°C), strong hybridization could be observed in mouse and T. nigerrimum, while weak hybridization was observed in D. melanogaster (Figure 1a). However, under high stringency (0.5x SSC or 0.1x SSC at 55°C), hybridization disappeared from D. melanogaster and T. nigerrimum, whereas in mouse the intense signal remained (Figure 1b). When (TTAGG)6 was used as probe, hybridization signal was observed only in T. nigerrimum (Figure 1c).
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The chromosome number of T. nigerrimum is n = 9 in males and 2n = 18 in females and workers, and the standard haploid karyotype formula is 5m + 2sm + 2st (Palomeque et al. 1988). In situ hybridization to metaphase chromosomes was performed using TTAGGG- or TTAGG-repeat PCR-generated probes. No hybridization signals were found when TTAGGG repeat was used (data not shown). However, with the TTAGG repeat, strong signals were observed at the telomeres of the chromosomes (Figure 2). As can be seen in Figures 2b and c, there were some differences in hybridization intensities among different chromosomes and between homologous telomeres; probably due to technical artifacts, since these differences were not consistently observed when several metaphases were analyzed.
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To determinate if other ant species contain (TTAGG)n in telomeric regions, DNAs from several species were digested with EcoRI and hybridized with the (TTAGG)6 and the (TTAGGG)5 probes under highly stringent conditions (0.5x SSC at 55°C). Figure 3a shows that all species tested presented intense hybridization signals when (TTAGG)6 was used as a probe. However, when (TTAGGG)5 was used as a probe, no clear hybridization signals were observed in any species, although a very faint band was noted in almost all species (Figure 3b).
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The few species of Hymenoptera for which telomere organization has been studied have shown two types of patterns. Okazaki et al. (1993) performed Southern hybridization with two oligonucleotides, (TTAGG)6 and (TTAGGG)5, in the ant M. yessensis, and only with the TTAGG repeat was hybridization detected. Similar results were obtained with the honeybee (A. mellifera) (Sahara et al. 1999). However, Meyne et al. (1995) observed that telomeres of several species of ants (genus Myrmecia) hybridized with the putative insect telomere repeat sequence (TTAGG) and also with the putative vertebrate telomere sequences (TTAGGG)n.
The presence of telomeric repeats in nontelomeric locations has been detected in a variety of species, generally associated with fusion-fission processes (Go et al. 2000; Slijepcevic 1998). We did not find any hybridization signals in nontelomeric locations. However, previous articles have reported that Robertsonian processes could play an important role in karyotypic evolution in the genus Tapinoma (Lorite et al. 1998b; Palomeque et al. 1988), as well as in other genera of the family Formicidae (Imai et al. 1984; Lorite et al. 1998a, 2000). The lack of nontelomeric hybridization could be due to processes of partial or total elimination after Robertsonian fission/fusion, resulting in no or very low copies of telomeric repeats in nontelomeric regions. The loss of telomeric repeats during Robertsonian processes has also been observed in other species (Silva and Yonenaga-Yassuda 1997).
The results obtained showed that the major telomere sequence of ants is TTAGG. In spite of this, TTAGGG repeats as a minor component in ant telomeric and subtelomeric regions could not be totally excluded. We did not detect positive signals by in situ hybridization using the (TTAGGG)n probe, although faint bands were observed by Southern hybridization. It is possible that the TTAGGG repeats are present in very low copy numbers and are consequently difficult to detect by in situ hybridization or Southern blotting techniques.
In summary, the presence of the pentanucleotide repeat TTAGG has now been found in species belonging to two families of Hymenoptera, the honeybee (A. mellifera, family Apidae) (Sahara et al. 1999), and in several species of ants (family Formicidae) belonging to the subfamilies Myrmeciinae (Meyne et al. 1995), Myrmicinae (Okazaki et al. 1993; present study), Dolichoderinae, and Formicinae (present study). Approximately 80% of all ant species belong to these four subfamilies (Bolton 1995), thus the TTAGG repeat may be a widespread telomere motif among the vast majority of ant species. However, the Hymenoptera (ants, bees, and wasps) is one of the most species-rich insect orders, with more than 200,000 described species (Bolton 1995), and further studies will be needed to determinate the organization of telomeres in this insect order.
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Acknowledgments |
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We are very grateful to Dr. A. Tinaut from the University of Granada for taxonomic identification of the material studied. This work was supported by PGCDGESIC PB98–0293 and PAI CECJA CVI 220.
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Footnotes |
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Corresponding Editor: William S. Modi
Received July 26, 2001
Accepted May 17, 2002
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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