Springer 2005
Genetica (2005) 123: 245–253
Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, Taeniura lymma and Raja montagui (Chondrichthyes, Batoidea) L. Rocco1, D. Costagliola1, M. Fiorillo1, F. Tinti2 & V. Stingo1 1
Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 – Caserta, Italy; (Phone: +39-0823-274555; Fax: +39-0823-274571; E-mail:
[email protected]); 2Molecular Genetics for Environmental & Fishery Resources Laboratory, Interdept. Center for Research in Environmental Sciences, via Tombesi dall’Ova 55, Ravenna, Italy Received 15 July 2004 Accepted 18 August 2004
Key words: 5S rDNA, cartilaginous fish, chromosome markers, FISH, nucleolar organizer region, ribosomal genes Abbreviations: bp – base pair(s); CMA3 – chromomycin A3; FISH – fluorescent in situ hybridization; NOR – nucleolar organizer region; NTS – non transcribed sequences; PBS – phosphate buffer saline; PCR – polymerase chain reaction
Abstract We used silver nitrate staining, CMA3 and FISH to study the chromosomal localization of both the major ribosomal genes and the nucleolar organizer regions as well as that of the minor ribosomal genes (5S rDNA) in two species of Batoidea, Taeniura lymma (Dasyatidae) and Raja montagui (Rajidae). In both species, all the metaphases examined showed the presence of multiple NOR-bearing sites, while the gene for 5S rRNA proved to be localized on two chromosome pairs. Furthermore, one of the two 5S rDNA sites in T. lymma was shown to be co-localized with the major ribosomal cluster. The presence of multiple nucleolar organizer regions in the two species might be interpreted as being the result of intraspecific polymorphisms, or as a phenomenon of the amplified transposition of mobile elements of the genome. We also determined the nucleotide sequence of the 5S rRNA gene, consisting of 564 bp in R. montagui and 612 bp in T. lymma. We also found TATA-like and (TGC)n trinucleotides, (CA)n dinucleotides and (GTGA)n tetranucleotides, which probably influence gene regulation.
Introduction Ribosomal RNA is by far the most abundant product of transcription and accounts for between 80 and 90% of the total mass of cellular RNA, both in prokaryotes and in eukaryotes. In addition to the major class, which consists of the genes for 18S, 28S and 5.8S rRNA, the genes for the ribosomal RNA of the eukaryotes are represented by a class of genes that code for 5S RNA (5S rDNA), a component of the major subunit of ribosomes. In the majority of higher eukaryotes, the genes for 5S rRNA are separated in the genome from the major
cluster (Fedoroff, 1979a, b) and are present as tandem repetitions, separated by non-transcribed spacer regions, with a ‘‘head-to-tail’’ arrangement (Long & David, 1980). Chondrichthyes are probably the class of vertebrates least studied from the standpoint of systematic evolution and genome organization. Even though karyological studies of fish increased considerably in recent times, the selachians remain among the least researched in this regard. In the last few years, different approaches have been used to shed some light upon as yet unresolved questions about the taxonomic evolution of these fish.
246 Although the literature now includes extensive data on genome composition in terms of DNA at varying degrees of repetitivity, information on base composition, the distribution of the heterochromatin fraction and the characterization of highly repeated DNAs (Olmo et al., 1982; Stingo, Rocco & Improta, 1989; Rocco, Stingo & Bellitti, 1996; Rocco et al., 2002a), no final conclusions can yet be made about the karyologic evolution of these vertebrates. One of the most effective methods of studying the karyologic characteristics of these fish is to combine the classic techniques of chromosome banding with molecular approaches such as in situ hybridization and the characterization of specific repeated gene sequences. In this study, we used FISH to analyze the number and the position of both active and non-active nucleolar organizer regions and the chromosomal localization of major and minor rDNA clusters in two species, belonging to different orders of Batoidea, Raja montagui (Rajidae) and Taeniura lymma (Dasyatidae). (For detailed systematic implications, see Stingo & Rocco, 2001). Moreover, we determined the nucleotide sequence of the gene for 5S rDNA in both species.
Materials and methods Specimens The Raja montagui specimens (three males and four females) were collected in the waters of the Gulf of Pozzuoli in Southern Italy. The specimens of Taeniura lymma (one male and two females) came from the Indian Ocean (Bali). DNA extraction and PCR amplification Genomic DNAs were isolated according to Rocco, Stingo & Bellitti (1996) and amplified by PCR in a PE Biosystems 2400 thermal cycler, using the primers A and B indicated by Pendas et al. (1994) for 5S rDNA and M1 (5¢GGCTTCTGGTTG ATTCTGCCAGT-3¢) and M2 (5¢-TACGCCCGATCTCGTCCGATC-3¢) taken from 18S rDNA fish sequences available in the GenBank database. The amplifications were performed under the conditions reported in Rocco et al. (1999).
Cloning and nucleotide sequences of 5S rDNA The amplified DNA products were visualized after staining with ethidium bromide on 1.4% agarose gel. Cloning and nucleotide sequences were carried out according to Rocco et al. (1999). Chromosome preparation Metaphase plates were obtained from testis and spleen according to Stingo et al. (1995). Fluorescent in situ hybridization (FISH) and banding procedures Chromosome preparations were stored for at least one week at room temperature or for 12 h at 60 C before hybridization. Fluorescent in situ hybridization was performed using 18S rDNA amplification products and/or 5S rDNA clones as probes. 5S rDNA Dig-probe labeling and detection were carried out according to Rocco et al. (2002a). For double FISH the 18S rDNA probe was biotin-labeled by nick translation using the Biotin-Nick Translation Mix (ROCHE). Double FISH with biotin plus digoxigenin was performed and simultaneous signals detected. Probe hybridization sites were detected using both anti-Dig-fluorescein, Fab fragments (green signals) and avidin–rhodamine conjugates (red signals) according to the manufacturer’s (ROCHE) instructions. The probes were hybridized in situ on the same metaphase plates (double FISH) in order to assess the probable co-localization of the two ribosomal clusters. We also performed detection with two or three layers of FITC conjugated antibody to visualize the weaker signals of 5S rDNA clusters. NOR locations were identified by CMA3/methyl green and Ag-staining, carried out according to Rocco et al. (2002a). For sequential staining, the same plates were first treated with CMA3/methyl green, then, after rinsed the slides in McIlvaine buffer (pH 7.0), we performed the double color FISH. After one rinse in 1· PBS the metaphase plates were finally stained with silver nitrate according to the above mentioned procedure.
247
Figure 1. Nucleotide sequences of the 5S rDNA clones studied in the two species (orientation 5¢–3¢). The TATA-like sequences are shown in grey; (*) corresponds to identical bases; ()) corresponds to insertions; the short repeated sequences are reported in italics. Rmon ¼ Raja montagui (GenBank accession number AY278250), Tlym ¼ Taeniura lymma (GenBank accession number AY278251).
Results The amplification of the gene for 5S rRNA from the genomic DNA of R. montagui and T. lymma showed the presence of two bands, one with 300 bp, the other with about 600 bp. The latter was cloned and sequenced (Figure 1). The cloned fragments consisted of 564 bp in R. montagui (GenBank accession number AY278250) and 612 bp in T. lymma (GenBank accession number AY278251). Moreover, both contained a coding region of 120 bp, which showed 100% identity with that of other fish present in the GenBank database. The NTS region included some TATA-like and (TGC)n trinucleotides, (CA)n dinucleotides and (GTGA)n tetranucleotides, as illustrated in Figure 1. In T. lymma we also observed an insertion of the tetranucleotides (GTGA)11. We examined more than 300 metaphase plates for R. montagui and more than 200 for T. lymma. The karyotype of R. montagui is made up of 48 chromosome pairs (9 m/sm, 8 st, 31 t; FN ¼ 114) and microchromosomes are present (Rocco et al., 2002b). All metaphase plates examined revealed at
least 10 chromosomes positive to silver staining and located at telomere and centromere level in acrocentric elements (Figure 2(a)). Furthermore, at least 20% of the plates examined revealed one or two additional sites. The bands revealed after staining with CMA3 were located on six chromosome pairs. Sites sensitive to fluorochrome were found at telomere level in one pair of acrocentric elements, at telomere and centromere levels in two pairs of medium-sized acrocentrics and at centromeric level in one bi-armed and two uni-armed pairs (Figure 2(b)). FISH with 18S rDNA (Figure 2(c)) showed fluorescent spots coinciding with those revealed by CMA3 staining, while FISH with 5S rDNA revealed the presence of bands on the telomeres of two acrocentric pairs, one medium-sized, the other small (Figure 2(d)). Unfortunately the double color FISH on R. montagui chromosomes did not provide satisfactory results. We previously reported the karyotype of T. lymma (2n ¼ 64) as having 19 bi-armed and 13 uni-armed pairs (Rocco et al., 2002a). All the metaphases examined evidenced at least three chromosomes positive to silver staining
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Figure 2. Ribosomal genes localization on R. montagui chromosomes after silver (a) and CMA3 (b) staining, FISH with 18S (c) and 5S (d) probes. In (e) are schematically represented the chromosome pairs bearing the ribosomal clusters.
(Figure 3(a)). Two sites were located at the terminal end of the short arms of a small submetacentric pair. The third site was found at paracentromeric level on the long arms of a large submetacentric element. Moreover, in at least 25% of the plates examined, the other homolog chromosome was also labeled (Figure 3(b)).
Staining with CMA3/methyl green, revealed the presence of CMA3-positive multiple loci (Figure 3(c)). In fact, in a great number of plates, very brilliant labeling was revealed on two chromosome pairs, in particular on the telomere portion of the short arms of a small submetacentric pair and at paracentromere level on the long arms of a large
249
Figure 3. Ribosomal genes localization on T. lymma chromosomes after silver (a, b) and CMA3 (c, d) staining, FISH with 18S and 5S probes (e). In (e), the green spots (dark arrows) represent the 5S rDNA hybridization signal, the red spots (white arrows) indicate the bands that correspond to 18S rDNA, and the orange (asterisks) are the co-located signals of 5S and 18S rDNA. In (F) are schematically represented the chromosome pairs bearing the ribosomal clusters.
250 submetacentric one. There was also a small percentage of metaphases (about 8%) that showed two other CMA3-sensitive sites (Figure 3(d)). The data obtained for the 18S rDNA probe revealed the presence of strong fluorescent signals at the terminal end of the short arms of a small submetacentric pair in all the plates (Figure 3(e)). We also found two small spots on the same Agsensitive and CMA3-sensitive chromosome pair. This latter site showed strong fluorescence after FISH with the 5S rDNA probe (Figure 3(e)). This minor ribosomal gene is also localized at telomeric level in another small submetacentric pair. The size and fluorescent intensity of this signal were strictly correlated with the enhanced signals used in FISH (two or three layers of antibody FITC-conjugated) that were fundamental in visualizing these small clusters.
Discussion The Chondrichthyes are among the least studied vertebrates from a cytotaxonomic standpoint, both within their own class and in relation to other vertebrates. As regards the localization of nucleolar organizer regions in the two species that we studied, results indicate the presence of multiple NORbearing sites. The presence of multiple rDNA sites has been observed in various families of bony fish such as salmonids, for example, whose NOR organization has been studied in greater depth (Phillips, Zajicek & Ihssen, 1989; Zhuo, Reed & Phillips, 1995). Multiple Ag–NOR sites have also been revealed in other species of Raja and in the torpedoes T. ocellata and T. mormorata (Stingo et al., 1995; Rocco et al., 2002b). This would appear to be a typical characteristic of species belonging to the superorder of Batoidea. In many cases, the variability in the number and location of the minor NORs has been related to the polymorphism of the constitutive heterochromatin associated with the CMA3-positive NORs, rather than directly to the polymorphism of the rDNA sites. Such NOR-associated heterochromatin is thus considered responsible for the frequent rearrangements that appear to involve the NORs as well as the polymorphism of the NORs themselves (Oliveira et al., 1995; Almeida-Toledo et al., 1996). Transposition events brought about
by mobile elements of the genome, with subsequent amplification of the transposed sequences, might also give rise to the formation of new NOR sites (Eickbush & Eickbush, 1995; Almeida-Toledo et al., 1996). This hypothesis is supported by evidence of the terminal position of the NORs, which, according to Zhuo, Reed & Phillips (1995), makes transposition events more probable, and could also explain the detection of further AgNO3positive and CMA3-positive sites in the various metaphase plates examined in the two species. In both species, the chromosome sites of the major ribosomal genes revealed through CMA3 coincide with those detected when FISH was used with 18S rDNA, even though they are generally present in larger numbers than those detected by silver staining. Techniques like these have been widely used on bony fish, but have only been applied to Chondrichthyes for a couple of years (Rocco et al., 2002a). The differences in the number of nucleolar organizer regions revealed by CMA3 and silver staining in the two species we studied are thus no novelty in the superclass Pisces. In general, the number of Ag-positive nucleolar organizer regions is lower than those detected by CMA3, and this matches the results obtained in this study. FISH confirms the maximum number of Ag-NOR detected. As for the CMA3 extra-positive sites found in T. lymma, since these were not identified by FISH, they most likely correspond to heterochromatic GC-rich DNA regions, not related to ribosomal sequences. As far as 5S rDNA organization is concerned, the results of the amplification products are the same as those reported in the literature. The length of the bands obtained in the different species varies. This is undoubtedly due to the length of the NTS region, which can vary considerably, as several authors have observed (Little & Braaten, 1989; Srivastava & Schlessinger, 1991). The comparison of the rDNA 5S sequence of T. lymma and R. montagui with that of other fish in the database showed that the clones have a high homology of sequence with them. While the gene for 5S rDNA is highly conserved even among taxa that are not closely related, the non-transcribed spacer region can vary greatly in length, which might lead to a heightened dynamism of the gene itself (Williams & Strobeck, 1985). Though the NTS seem to have no specific function, it has recently been demonstrated in
251 different mammals that a TATA sequence localized in the NTS of the gene plays an important role in regulating its expression (Nederby-Nielsen et al., 1993; Suzuki, Sakurai & Matsuda, 1996). A TATA-like sequence has been observed upstream of the 5S gene of some Osteichthyes (Pendas et al., 1994; Rocco et al., 1999; Inafuku et al., 2000; Martins & Galetti, 2001a). Martins and Galetti (2001b) suggest that this sequence may have some influence on the transcription level of the genes for 5S rRNA, and deduce that other short sequences of the same type, as are present in the NTS, might be involved in the functions of gene expression and regulation. TATA-like trinucleotides are also present in the gene sequences that we determined (Figure 1), indicating that this characteristic may also be present in the gene organization of the 5S rDNA in Chondrichthyes. Moreover, the presence of (CA)n dinucleotides and tetranucleotides, most likely attributed to microsatellites, could have an involvement in the regulation of the gene (Martins & Galetti, 2001b). The sequences analyzed concern one of the two bands generated as amplification product by the primers used by Pendas et al. (1994). The other band is currently in the process of being studied in our laboratories. It has about 300 bp and we believe it is composed of a coding region of 120 bp, plus an NTS that is different in length and composition from the one that we examined. A dual tandem arrangement of the 5S rDNA has also been encountered in the Osteichthyes (Pendas et al., 1994; Sajdak, Reed & Phillips, 1998; Martins & Galetti, 2001b). We can thus deduce that such a characteristic can also be applied to the genome of the Chondrichthyes. As regards FISH with the gene for the 5S rDNA in R. montagui and T. lymma, the hybridization signal is present on two chromosome pairs in both species. One of the two 5S rDNA sites in T. lymma proved to be co-located with the major ribosomal cluster. It is not possible to make a comparison with the chromosomal localization of 5S rDNA in other species of Selachii, since there is no previous information in the literature. In most of the species of Osteichthyes examined (see Martins & Galetti, 2001b, for a review) and in one of the two species of Batoidea that we investigated, the genes for 5S rRNA are to be found at the interstitial level along the chromosome. This localization has also been observed in mammals
(Mellink et al., 1996; Frederiksen et al., 1997; Ma¨kinem et al., 1997) and in amphibians (Vitelli et al., 1982; Schmid, Vitelli & Batistoni, 1987; Lucchini et al., 1993), suggesting that this type of pattern is no accident. It probably provides some advantage related to the organization of these genes in the genome of vertebrates (Martins & Galetti, 2001b). Also to be taken into consideration is the number of chromosome sites on which the minor ribosomal cluster is located. In mammals, these genes are generally situated on one pair of chromosomes only, while in amphibians and fish, the genes for 5S rRNA can be localized on several chromosome pairs (Schmid, Vitelli & Batistoni, 1987; Fujiwara et al., 1998; Martins & Galetti, 1999). One explanation for the presence of multiple chromosome sites bearing the gene for 5S rRNA in Batoidea might be the extensive chromosomal rearrangements that took place during the evolution of cartilaginous fish. Indeed, cases of polyploidy could have taken place in the Elasmobranchs. This has been demonstrated by renaturation kinetics and is not evident in the chromosome arrangements because of phenomena of secondary diploidization of the karyotype (Olmo et al., 1982; Stingo & Rocco, 1991). Furthermore, the loci for rRNA 45S are transcribed by the enzyme RNA polymerase I, whereas the loci for 5S are transcribed outside the nucleolus by RNA polymerase III. Some authors believe that this functional divergence would require a different physical localization of the two gene cluster arrangements (Amarasinghe & Carlson, 1998). The chromosomal localization of 5S rDNA might represent further evidence to support the hypothesis that the karyotype of the Chondrichthyes evolved through fusion of acrocentric elements, forming metacentric or submetacentric ones. The chromosomes bearing the minor ribosomal clusters might in fact have been involved in these Robertsonian fusion phenomena that occurred in the karyological evolution of these fish, stemming from a progenitor with more primitive karyotype characteristics such as those of R. montagui. As the relationship between the type of tandem organization of 5S rDNA and the number of loci evidenced still needs to be better defined. It would be appropriate to extend investigations to other
252 species of Chondrichthyes in order to verify whether these different classes of 5S rDNA can be clustered in separate chromosomal sites. In conclusion, the study of the organization of the ribosomal genes and their chromosomal localization undoubtedly provides a range of useful information, especially if integrated with quantitative and qualitative evaluations using in situ hybridization and analysis of repeated sequences, such as the microsatellites, present in the NTS region. In this way it would be possible to obtain better insight into the molecular bases of the structure and the chromosomal function, thus the arrangements that contribute to the process of speciation.
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