The Journal of Heredity 2002:93(4)
© 2002 The American Genetic Association 93:279-282
Brief Communication |
rDNA (18S–28S and 5S) Colocalization and Linkage Between Ribosomal Genes and (TTAGGG)n Telomeric Sequence in the Earthworm, Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), Revealed by Single- and Double-Color FISH
From the Dipartimento di Biologia Animale, Università di Palermo, Palermo, Italy (Vitturi); Facoltà di Scienze Ambientali, Università di Urbino, Urbino (PU), Italy (Colomba); Dipartimento di Biologia Cellulare e dello Sviluppo ‘A. Monroy', Università di Palermo, Palermo, Italy (Pirrone); and Dipartimento di Biologia Animale, Università di Modena e Reggio Emilia, Modena, Italy (Mandrioli).
Address correspondence to Roberto Vitturi, Dipartimento di Biologia Animale, Via Archirafi 18, 90123 Palermo, Italy, or e-mail: zuvitcol{at}unipa.it.
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Abstract |
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Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes—5S rDNA, 18S–26S rDNA, and (TTAGGG)n. Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S–26S rDNA as probes. Simultaneous (18S–26S)-5S and (18S–26S)-(TTAGGG)n FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.
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Introduction |
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The genes coding for rRNA in eukaryotes are arranged into two different rDNA multicopy families, that is, major (18S, 5.8S, and 28S rDNA) and minor (5S rDNA) ribosomal genes. The two rDNAs, detectable as chromosomal hybridized labels, were found to be located at different chromosome pairs as the most frequent configuration in vertebrates (Lucchini et al. 1993; Suzuki et al. 1996). However, 5S units have also been found to be associated with repeated copies of major rDNA (Drouin and Moniz de Sá 1995; Mandrioli et al. 2000; Pendás et al. 1994) or histone genes (Andrews et al. 1987; Barzotti et al. 2000).
Fluorescent in situ hybridization (FISH) with ribosomal sequences as probes allowed the mapping of 18S–28S and 5S rDNA genes on the chromosomes of several vertebrate species. In contrast, invertebrate taxa remained poorly characterized in this respect. For example, in the class Oligochaeta, major rDNA genes have been mapped using single-color FISH to spermatocyte chromosomes of two lumbricid species, Eisenia foetida and Octodrilus complanatus (Vitturi et al. 2000a), whereas to the authors' knowledge, nothing is known about the 5S rDNA localization among these organisms.
In the present work we mapped for the first time in Oligochaeta the 5S rDNA to the chromosomes of Octodrilus complanatus using single-color FISH. Moreover, since next to nothing or very little is known about the mapping of repetitive DNAs in the oligochete earthworms, we used, in addition to single-color FISH, double-color FISH to examine the physical relationship between 18S–28S rDNA, 5S rDNA, and (TTAGGG)ntelomeric repeats.
O. complanatus is a lumbricid earthworm of the class Oligochaeta. Its chromosome complement (2n = 36) consists of seven metacentric/submetacentric pairs (M/SM) and 11 subtelocentric pairs (ST) homogeneously fluorescent after CMA3 and DAPI staining. FISH using a sea urchin (Paracentrotus lividus) 18S–26S ribosomal probe mapped major rDNA genes on one small subtelocentric chromosome pair (Vitturi et al. 2000a). In the same study the (TTAGGG)nFISH showed that the hexanucleotide hybridized to the ends of the chromosome of E. foetida and O. complanatus.
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Materials and Methods |
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A total of 20 O. complanatus (Dugés 1828) specimens were collected at the Botanic Garden of Palermo (Sicily). Chromosomes were prepared by means of the air-drying technique (Vitturi et al. 1991) from dissected testes of sexually mature specimens previously soaked in a freshwater colchicine solution (0.1%) at room temperature.
FISH was performed on fixed spermatocyte chromosomes of five specimens as described by Vitturi et al. (2000a) using three different probes: a 4.3 kb sea urchin (Paracentrotus lividus) rDNA probe (prR14) consisting of sequences from the 3' end of the 18S to the 3' end of the 26S rDNA (Cantone et al. 1993); a PCR-obtained 5S rDNA probe using as primers F (5'-TGCACGTAGTGTTCCCAAGC) and R (5'-ACGACCATACCACGTTGAATAC), deduced from the 5S coding sequence of insects available in GenBank, according to the protocol described by Mandrioli et al. (2000); and a telomeric hexanucleotide (TTAGGG)ngenerated by PCR (PCR DIG-probe Synthesis Kit, Roche) in the absence of template (Ijdo et al. 1991) using (TTAGGG)5 and (CCCTAA)5 as primers. For single-color FISH, nick translation labeling with digoxigenin of 18S–26S rDNA was performed according to the manufacturer's (Boehringer Mannheim) instructions, while the remaining two probes were DIG-labeled following the Roche protocol.
Slides treated with single-color FISH were mounted in an antifade solution containing propidium iodide (5 µm/ml) and viewed under a Leica I3 filter set (BP 450–490, LP 515). For multiple FISH the 18S–26S rDNA probe was labeled with biotin (Biotin-Nick Translation Mix, Roche), whereas both 5S rDNA and (TTAGGG)nprobes were the same as those used in single-color experiments.
Chromosomes were denatured for 4 min in 70% formamide/2x SSC at 72°C. The mixed probe solution (4 ng/µl of each probe) was denatured for 5 min at 80°C. Hybridization was allowed to proceed in a moist plastic chamber at 37°C overnight. Slides were washed twice in 50% formamide/2x SSC at 37–38°C (5 min each), twice in 2x SSC at 37–38°C (5 min each), once (5 min) in 4x SSC/0.1% Tween at RT, and finally, once (5 min) in PBS/0.1% Tween/0.5% skim milk powder at RT. Probe hybridization sites were detected using both anti-DIG-fluorescein, Fab fragments, and streptavidin-Texas Red conjugate, according to the manufacturer's (Boehringer Mannheim, molecular probes) instructions. Slides were mounted in an antifade solution containing DAPI (3 µg/ml) and viewed under a Leica three-color filter set (B/G/R filter set, BP 400/20, BP 465/20) which allowed the simultaneous visualization of fluorescein- and Texas Red-labeled hybridization sites (green and red, respectively) and chromosomal DNA (blue). Chromosomes were observed with a Leica microscope and photographed on with Kodak Ektacolor 1000 ASA film.
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Results |
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As was reported in a previous article (Vitturi et al. 2000a), in this study as well, single-color 18S–26S rDNA FISH showed that in O. complanatus, two hybridization sites per spread occurred at spermatogonial metaphase (Figure 1a). Two positive sites per spread were also obtained using single-color FISH with the 5S rDNA as a probe (Figure 1b). Both major (18S–28S rDNA) (Figure 1a) and minor (5S rDNA) (Figure 1b) ribosomal genes were located at the terminal region of two small sized chromosomes.
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After single-color FISH with either the 18S–26S (Figure 1c) or 5S (Figure 1d) rDNA as a probe, a single metaphase I bivalent showed ribosomal sites, thus demonstrating that repeated units of major and minor rDNA were located at homologous chromosomes. Simultaneous double-color FISH was performed on diakinetic chromosomes of O. complanatus using the biotin-labeled 18S–26S rDNA and the digoxigenin-labeled (TTAGGG)nas probes. Telomeric signals, probably due to their small dimension, were not clearly distinguishable in all bivalents. In spite of this it was possible to establish that ribosomal (red spot) and telomeric (green spot) sequences were located adjacently in the rDNA-bearing bivalent (Figure 1e).
After simultaneous double-color FISH using the biotin-labeled 18S–26S rDNA and the digoxigenin-labeled 5S rDNA as probes, two hybridized chromosomes were obtained at spermatogonial metaphase (Figure 1f) and one hybridized bivalent at metaphase I (Figure 1g). Hybridization sites showed a mixed-color appearance. Analysis of these sites using a spot software diagnostic instrument for decomposition of colors revealed that each hybridization spot (Figure 1g) was the result of the overlapping of two distinct colors, namely red (Figure 1h) and green (Figure 1i).
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Discussion |
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Cytological studies in the class Oligochaeta are predominantly restricted to chromosome counts of about 160 species and to gross karyotype description of some of them (Christensen 1980 and references therein). Conventional banding techniques, such as silver staining (Ag-NOR) and C-banding, have been attempted on spermatogonial chromosomes of the lumbricid Eisenia foetida (Vitturi et al. 1991). Recently single-color FISH has been used to map major rDNA genes (18S–28S) and to test the presence of the (TTAGGG)ntelomeric repeats in the lumbricid species E. foetida and O. complanatus (Vitturi et al. 2000a).
In the present article, the single-color FISH has been used for the first time to map the 5S rDNA on the chromosomes of O. complanatus. In addition, simultaneous double-color FISH in combination with DAPI counterstaining has been used to investigate the relationship between repeated units of three multigene families, namely 18S–28S rDNA, 5S rDNA, and (TTAGGG)ntelomeric sequences.
The results obtained with single-color FISH demonstrate that in O. complanatus, the 5S and 18S–26S rDNA hybridized to the short arms of one small-size chromosome pair. However, this technique did not allow establishment of whether the hybridized pair was the same in the two experiments. Double-color FISH conclusively demonstrated that in O. complanatus, the 5S and 18S–28S sites were overlapped. This assumption was deduced from two main arguments. One argument is that the use of two different probes in the same experiment gave a single hybridization signal. A second argument is that the analysis of hybridization sites using a spot software for decomposition of colors showed that each signal was the result of the overlapping of two spots, red (18S–28S rDNA) and green (5S rDNA).
Potentially functional 5S rRNA-encoding genes were found interspersed with the rDNA repeats in the nematode Meloidogyne arenaria (Vahidi et al. 1991) and with histone genes in the crustacean Artemia spp. (Andrews et al. 1987) and Asellus aquaticus (Barzotti et al. 2000). Moreover, the existence of a colocalization of 5S rDNA and 18S–28S rDNA has been reported in 7 of 11 calanoid copepod species analyzed thus far (Drouin and Moniz de Sá 1995; Drouin et al. 1987, 1992), in the cenogastropod Melarhaphe neritoides (Mollusca: Prosobranchia) (Colomba et al. 2002), and in four fish species (Mandrioli et al. 2000; Morán et al. 1996; Pendás et al. 1994).
The double-color FISH was also used to investigate the linkage of the (TTAGGG)ntelomeric sequence and 18S–28S ribosomal repeats. The results revealed that in the NOR-bearing chromosomes of O. complanatus, the telomeric signal was closely associated with major ribosomal sequences. Therefore, as recently found in M. neritoides (Colomba et al. 2002), and in the earthworm as well, three multicopy families are linked on the same chromosome pair.
By combining the FISH and silver-staining techniques, linkage or interspersion between the (TTAGGG)nsequences and NORs was found in some vertebrate species including the Eastern American mole (Scalopus aquaticus), the wood lemming (Myopus schisticolor), two Atlantic eels, and four salmonid species (Abuin et al. 1996; Liu and Fredga 1999; Meyne et al. 1990; Salvadori et al. 1995). The hexanucleotide (TTAGGG)nwas also found at NORs in nonvertebrate species such as Neurospora, Giardia, and Tetrachymena (Adam et al. 1991; Butler 1992; Yu and Blackburn 1991).
The significance of such an association and/or interspersion involving repeated units of different multigene families is still unclear. However, available literature provides two possible interpretations. One interpretation is that this close association might have a functional role in nucleolus organization, as demonstrated for a human nonribosomal tandemly repeated block of a pentamer sequence located near the NORs (Kaplan et al. 1993).
A second possibility is that the colocalization or interspersion of repeated units of different multigene families might result in unequal crossing-over with consequent heteromorphism which, according to some authors (Dover 1986; Liu and Fredga 1999), is important in the maintenance of a conserved and multiple array. Taking into account that in most vertebrate species a colocalization of 18S–28S and 5S rDNAs is not the rule, but rather the exception (Mandrioli et al. 2000; Sola et al. 2000), this possibility may be true only for some species.
As far as invertebrates are concerned, where a high degree of NOR polymorphism has been described (Sella et al. 1995 and references therein; Vitturi et al. 2000b), the FISH technique to investigate the relationship between repeated units of multigene families has rarely been used. However, in disagreement with data reported for vertebrates, where an adjacent disposition between NORs and telomeres is unusual (Liu and Fredga 1999), in invertebrate species results obtained using silver staining seem to indicate that major rDNA clusters are closely associated with telomeric sequences. In fact, in most studied species, Ag-NORs were found to be terminal or subterminal.
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Acknowledgments |
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The authors thank R. Barbieri for kindly providing the 18S–26S rDNA probe. This work was supported by a MURST grant (to R.V. and M.Z.).
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Footnotes |
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Corresponding Editor: Martin Tracey
Received March 18, 2002
Accepted May 17, 2002
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References |
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