5S ribosomal and U1 small nuclear RNA genes: A ...

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5S ribosomal and U1 small nuclear RNA genes: A new linkage type in the genome of a crustacean that has three different tandemly repeated units containing 5S ribosomal DNA sequences Genome Downloaded from www.nrcresearchpress.com by YALE MEDICAL LIBRARY on 11/19/12 For personal use only.

Franca Pelliccia, Rita Barzotti, Elisabetta Bucciarelli, and Angela Rocchi

Abstract: We investigated the 5S ribosomal RNA (rRNA) genes of the isopod crustacean Asellus aquaticus. Using PCR amplification, three different tandemly repeated units containing 5S rDNA were identified. Two of the three sequences were cloned and sequenced. One of them was 1842 bp and presented a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene. This type of linkage had never been observed before. The other repeat consisted of 477 bp and contained only an incomplete 5S rRNA gene lacking the first eight nucleotides and a spacer sequence. The third sequence was 6553 bp long and contained a 5S rRNA gene and the four core histone genes. The PCR products were used as probes in fluorescent in situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus. The possible evolutionary origin of the three repeated units is discussed. Key words: Asellus, isopoda, crustacea, 5S rDNA, U1 snDNA. Résumé : Les auteurs ont examiné les gènes codant pour les ARNr 5S chez le crustacé isopode Asellus aquaticus. Suite à l’amplification PCR, trois unités répétées en tandem et contenant un ADNr 5S ont été identifiées. Deux des trois séquences ont été clonées et séquencées. Une des deux mesure 1842 pb et comprend à la fois un ADNr 5S et un gène codant pour un ARNsn U1. C’est la première fois qu’une telle association est rapportée. L’autre unité répétée mesure 477 pb et ne contient qu’un ADNr 5S incomplet dépourvu des huit premiers nucléotides et d’un espaceur. La troisième séquence mesurait 6553 pb et contenait un ADNr 5S ainsi que les gènes qui codent pour les quatre histones du noyau octamérique. Les produits PCR ont été employés comme sondes lors d’expériences FISH pour les localiser sur les chromosomes du A. aquaticus. L’origine évolutive possible de ces trois unités répétées est discutée. Mots clés : Asellus, isopode, crustacé, ADNr 5S, ADNsn U1. [Traduit par la Rédaction]

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Introduction The 5S ribosomal RNA (rRNA) genes were investigated in a small number of crustacean species and, interestingly, several different genomic organization types were found. In particular, seven species belonging to the Calanoida, Thoracica, and Euphasiacea orders have their 5S rRNA genes linked to 18S, 5.8S, and 28S rDNA repeat units (Drouin et al. 1987, 1992). For two other species of calanoids and for a cladocer it was established that these genes are not linked to the rDNA repeats (Drouin et al. 1992). The 5S rRNA genes of the anostrac Artemia salina are linked to the tandem repeats of the histone genes (Andrews et al. 1987). Finally, in the genome of isopod Proasellus coxalis the 5S rRNA genes are tandemly arranged but not linked to other repeated genes (Pelliccia et al. Received November 6, 2000. Accepted January 25, 2001. Published on the NRC Research Press Web site April 20, 2001. Corresponding Editor: W. Traut. F. Pelliccia, R. Barzotti, E. Bucciarelli, and A. Rocchi.1 Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, p.le Aldo Moro 5, 00185 Roma, Italia. 1

Corresponding author (e-mail: [email protected]).

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1998). Recently, we found in the genome of the isopod Asellus aquaticus a 5S rRNA gene in a tandemly repeated unit containing the four core histone genes. The gene cluster was sequenced and mapped by fluorescent in situ hybridization (FISH); 5S rDNA clusters occurred in terminal positions on a variable number of chromosomes (from 6 to 12 per diploid cell) not associated with the telomeric heterochromatic areas (Barzotti et al. 2000). The linkage between 5S rRNA genes and histone genes has so far been observed in two crustacean species, A. salina and A. aquaticus, but has not been found in any other organisms (Drouin and Moniz de Sá 1995). In this work, we used PCR to identify two more tandemly repeated units containing 5S rDNA in the genome of A. aquaticus. The two repeats were cloned and sequenced. Interestingly, one of them presents a 5S rRNA gene and a U1 small nuclear RNA (snRNA) gene, a type of linkage never observed before, while the second repeat contains only a 5S rRNA gene lacking the first eighth nucleotides and a spacer sequence. The two repeated units have also been used as probes in FISH experiments to locate them on the chromosomes of A. aquaticus. The chromosomes of A. aquaticus (2n = 16) cannot be differentiated by G- or R-banding techniques, whereas an interindividual variable number of heterochromatic telomeric

DOI: 10.1139/gen-44-3-331

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regions is revealed by C-banding and chromomycin A3 (CMA) (Rocchi et al. 1980).

Materials and methods Specimens examined in this research were collected from a population of Asellus aquaticus in the Sarno river near Naples, Italy.

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DNA extraction Batches of 30 animals were homogenized in a buffer containing 100 mM EDTA, 100 mM Tris-HCl, pH 9.0. After treatment with 2% SDS and 0.8 mg/mL proteinase K at 37°C overnight, the solution was treated with 1 M potassium acetate and precipitated with isopropyl alcohol. DNA was purified by digestion with 100 µg/mL RNase A in TE buffer, pH 8.0, extracted with phenol–chloroform and precipitated by ethanol.

PCR amplification and blot analysis Primers used to amplify 5S rDNA fragments were C (5′GAAAGCACCACTTCTCGTCC-3′) and D (5′-AACGTGGTATGGCCGTTGAC-3′). These sequences correspond to the first 40 bp of the 5S rDNA sequence of P. coxalis (Pelliccia et al. 1998; Fig. 2). The primers E and F were designed to determine the 5S rDNA sequences corresponding to the heterologous primers in the 1842-bp fragment. The sequences of primers E and F were 5′AGTACTAACCAGACCCAACG-3′ and 5′-TGGATGGGAGACCGCCTGGG-3′, respectively. These sequences correspond to an internal tract of the 5S rDNA of A. aquaticus present in the 1842-bp repetitive unit, precisely at positions 79–60 and 80–99, respectively, (Fig. 1). Two more primers G and H were designed to determine the 5S rDNA sequence corresponding to the heterologous primers in the 477-bp fragment. The sequences of G and H primers were 5′-AACTGTATAGCTAACTTTCG-3′ and 5′-CATATAAGCGCTACACGTTTC-3′, respectively. These sequences are present in the spacer sequence of the 477-bp fragment at positions 402–383 and 403–423, respectively (Fig. 2). These three pairs of primers were designed as diverging contiguous sequences in such a way that only tandemly arranged units could yield amplification products. Two primers, I (5′-GTTGCTCTATGGTCATGCTA-3′) and L (5′-GAACGGAATTACAGAACGAAAA-3′), were designed to amplify a fragment of 1502 bp corresponding to the 1842-bp unit lacking the U1 snDNA sequence. The sequences of the primers I and L are present in the spacer sequences of the 1842-bp fragment at the positions 306–287 and 648–668, respectively (Fig. 1). The amplification mixture used for PCR contained 100 ng of genomic DNA, 15 µM of each primer, 500 µM dNTP and 2.5 U Taq polymerase (Expand Long Template PCR System, Roche Diagnostic). Thirty cycles of PCR amplification were performed with an annealing temperature of 50°C. Gel electrophoresis and Southern blots of the PCR products were performed. The probe for hybridization included two complete gene sequences of the somatic 5S rRNA gene from Xenopus borealis (Xbs5S) labelled with digoxigenin-11-dUTP by random priming (Roche Diagnostic).

DNA cloning and sequencing The 1842-bp PCR product obtained with primers C and D and the 477-bp PCR product obtained with primers E and F were cloned in pBluescript SK(–) (Stratagene). Two clones for each PCR product were sequenced by the dideoxysequencing method with a Sequenase Kit 2.0 (USB, Cleveland, Ohio). The 1842-bp sequence was sequenced by primer walking. The gene regions corresponding to the heterologous primers were sequenced from the PCR products without cloning.

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The FASTA3 package at the EMBL – European Bioinformatics Institute was used for sequence analyses. Chromosome preparations and in situ hybridization Chromosome preparations were obtained from squashes of testes in 45% acetic acid, 1.5 h after colchicine injection (conc. 0.05%). The slides were ethanol dehydrated and stored desiccated at –20°C for several days. Probes for FISH included: (i) a 2128-bp fragment obtained by digesting the 6553-bp sequence with BamHI and SacI restriction endonucleases. This fragment lacks the 5S rRNA gene and its flanking sequences (Barzotti et al. 2000; Fig. 1); (ii) the complete 1842-bp PCR product, or a 1730-bp fragment obtained by digesting the 1842-bp sequence with HindII restriction endonuclease. This removed almost completely the 5S rRNA gene (112 bp), or else the PCR fragment of 1502 bp, corresponding to the 1842-bp unit lacking the U1 snRNA gene (Fig. 3A); (iii) the entire 477-bp PCR product, or the 306-bp spacer sequence obtained by digestion of the 477-bp sequence with HindII and HaeII restriction endonucleases (Fig. 3B). The probes were labelled with biotin16-dUTP or digoxigenin-11-dUTP by random priming (Roche Diagnostic). FISH was carried out as previously described (Barzotti et al. 1996). Chromosome preparations were counterstained with DAPI (4′,6-diamidino-2-phenylindole). After observation, preparations were destained with methanol – acetic acid (3:1) and stained with CMA methyl green. The images were recorded using a Zeiss Axioscope epifluorescence microscope equipped with a charged coupled device (CCD) camera and then merged using Adobe Photoshop 4.0 software.

Results Genomic DNA of A. aquaticus was amplified by PCR using two primers (C and D) obtained from the first 40 bp of the 5S rDNA sequence of P. coxalis (Pelliccia et al. 1998). C and D primers and the EF and GH primer pairs used in this work were designed as diverging contiguous sequences in such a way that only tandemly arranged units could be amplified. Two products of amplification of about 6.5 and 1.8 kbp were obtained. We predicted that the first product was the 6553-bp sequence previously described. The fragment containing the four core histone genes (H2A, H2B, H3, H4) and a 5S rRNA gene had been previously obtained by PCR using two heterologous H4 histone gene primers (Barzotti et al. 2000). Restriction enzyme digestion and Southern blot analysis using histone DNA and 5S rDNA probes confirmed our hypothesis. The PCR product of about 1.8 kbp was cloned and sequenced, and was found to be 1842 bp. Sequence analysis revealed the presence of a 120-bp 5S rRNA gene and a 226-bp spacer sequence separating this gene from a 163-bp tract that presents a sequence identity of 72.4% with the U1 snRNA gene of Drosophila melanogaster (Mount and Steitz 1981). In the 5′ flanking region, this U1 snRNA gene lacks characteristic class II snRNA gene promoters containing a proximal sequence element (PSE) at a conserved location 40 to 65 bp upstream of the transcription start site and a distal sequence element located between positions –200 and –300. On the other hand, in the 3′ flanking region, this U1 snRNA gene has a highly conserved sequence, known as the 3′ box, which is required for 3′ end formation of mature snRNAs (e.g., Cuello et al. 1999 and © 2001 NRC Canada



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Fig. 1. Nucleotide sequence of the 1842-bp PCR fragment from A. aquaticus. The portion of the sequence corresponding to the 5S rRNA gene (120 bp) (type A) is in bold type. The U1 snRNA gene is underlined. The 3′ box is doubly underlined and HindII restriction sites are underdotted. EMBL accession AJ24001.

333 Fig. 3. Schematic representation of the PCR products and restriction fragments obtained from the 1842-bp unit (A) and from the 477-bp unit (B). H, HindII; Ha, HaeII; F, E, L, I: primers.

Fig. 4. Alignment of the 5S rDNA sequence of P. coxalis (Pc) with those of A. aquaticus (Aa).

Fig. 2. Nucleotide sequence of the 477-bp PCR fragment from A. aquaticus. The 5S rDNA (112 bp) (type C) is in bold type. The HindII and HaeII restriction sites are underdotted. EMBL accession AJ24002.

references therein). This sequence is located at position 523– 536. A spacer sequence of 1333 bp separates the U1 snRNA gene from the following 5S rRNA gene (Fig. 1). To determine the 5S rDNA sequences corresponding to the heterologous primers, PCR amplifications of genomic DNA were performed using two primers (E and F) obtained from an internal sequence of the 5S rDNA of A. aquaticus previously sequenced from the 1842-bp repetitive unit. In addition to the two bands of 6553 bp and 1842 bp, this type of amplification produced a third band of about 500 bp. This fragment was cloned and sequenced. It consisted of 477 bp and contained an incomplete 5S rRNA gene of 112 bp lacking the first 8 nucleotides and a spacer sequence of 365 bp (Fig. 2). The sequences of the incomplete 5S rRNA gene corresponding to the primers were determined by sequencing a PCR product that was obtained using two primers (G and H) derived from the spacer sequences of the 477 fragment. The incomplete gene sequence of 112 bp (type C) was identical to the corresponding nucleotide region (9–120) of the 5S rRNA gene present in the 1842-bp fragment (type A). These two sequences differed from the 5S rRNA gene present in the 6553 fragment (type B) at position 48 (A→G) (Fig. 4).

The three 5S rDNA sequences of A. aquaticus differ at two positions (30 and 117) from the 5S rRNA gene of P. coxalis; the type A sequence differs also at position 48 (Fig. 4). In this last position all the known 5S rRNA gene sequences of crustacean species have a G nucleotide, so only the type A sequence of A. aquaticus has an A nucleotide. The comparison between the three DNA sequences amplified from the A. aquaticus genome, excluding 5S rDNA sequences, revealed the existence, in the 3′ regions immediately flanking the 5S rDNA of the 6553-bp and 477-bp fragments, of two 248-bp segments with high sequence identity (92%). FISH experiments were performed to analyse the chromosome locations of the three PCR amplified sequences. After observation, the hybridized chromosome preparations were washed and then stained with CMA to reveal the heterochromatic areas. The 6553-bp sequence was digested with BamHI and SacI restriction enzymes, obtaining three fragments of 3136 bp, 2128 bp, and 1289 bp. The central 2128-bp fragment, lacking 5S rDNA and its 3′-flanking 248-bp sequence, was used as a probe for FISH. These experiments confirmed the results previously obtained in which the 6553-bp fragments were located on an interindividually variable number of chromosomes, from 6 to 12 per diploid cell, always in terminal position and never associated with the heterochromatic areas (Barzotti et al. 2000). The 1842-bp sequence was digested with HindII, which separates 112 bp of the 5S rRNA gene from the remaining fragment of 1730 bp (Figs. 1 and 3A). The complete 1842-bp sequence, the 1730-bp fragment, and a PCR fragment of 1502 bp, corresponding to the 1842 units lacking the U1 snDNA sequence, were used as probes for FISH. The hybridization pattern of the three probes displayed two signals located in subcentromeric position on one arm of two largesized chromosomes; a third subcentromeric signal was often observed on one middle-sized chromosome. Moreover, telo© 2001 NRC Canada



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Fig. 5. Spermatogonial metaphase of A. aquaticus after FISH simultaneously using the 2128-bp fragment (obtained after digestion of the 6553-bp fragment) (white) and the 1730-bp fragment (obtained after digestion of the 1842-bp fragment) (black) as probes. Bar = 10 µm. Fig. 6. Spermatogonial metaphase of A. aquaticus after FISH simultaneously using the 306-bp fragment (obtained after digestion of the 477-bp fragment) (white) and the 1730-bp fragment (obtained after digestion of the 1842-bp fragment) (black) as probes.

meric hybridization signals were present on a maximum number of four chromosomes. Some FISH experiments were performed simultaneously using the 2128-bp and the 1730-bp fragments labelled with biotin-16-dUTP and digoxigenin-11-dUTP, respectively. The telomeric hybridization sites of the 1730-bp probe co-maps with part of the hybridization sites of the 2128-bp probe that are more numerous (Fig. 5). The 477-bp sequence was digested with HindII and HaeII that separate 171 bp containing the 112- bp 5S rDNA from a spacer sequence of 306 bp (Figs. 2 and 3B). Both the entire 477-bp PCR product and the 306-bp spacer sequence were used for FISH. For both probes, the fluorescent hybridization signals observed on preparations obtained from 13 different individuals were always located in telomeric position. The chromosomes of four individuals showed that all telomeres were hybridized (32 per diploid cell). The chromosomes of the other nine individuals displayed a variable number of hybridized telomeres (from 10 to 20 per diploid cell). In some FISH experiments the differently labelled 306-bp and the 1730-bp fragments were simultaneously used (Fig. 6).

Discussion In higher eukaryotes the 5S rRNA genes are typically arranged in clusters of units that are tandemly repeated and (or) dispersed in the genome. However, these genes are also found to be linked to the repeated units of different genes, mainly the trans-spliced leader genes and the 18S, 5.8S, and 28S rRNA genes, in several lower and higher eukaryotic species (for review see Drouin and Moniz de Sá 1995). Moreover, the 5S rRNA genes have been found linked to the repeated units of the multigenic family of histone genes in the crustacean anostrac A. salina (Andrews et al. 1987) and in the crustacean isopod A. aquaticus (Barzotti et al. 2000).

In this work we demonstrate that in the genome of A. aquaticus the 5S rDNA is present in two more organization types: either linked to the U1 snRNA gene or not linked to any other sequences. A. aquaticus is the first organism in which the 5S rRNA genes have been found to be linked to U1 snDNA in the same tandemly arranged unit. We do not know if this U1 snDNA represents a functional gene or a pseudogene. If it is compared with the U1 snRNA gene of D. melanogaster, it has complete 5′ and 3′ ends and a high sequence identity (72.4%) (Mount and Steitz 1981). Moreover, in the 3′ flanking region it has the 3′ box, a conserved sequence required for proper 3′’ processing of transcript, but in the 5′ flanking regions it lacks characteristic class II snRNA gene promoter sequences (e.g., Cuello et al. 1999 and references therein). The U1 snRNA genes are transcribed by polymerase II whereas the 5S rRNA is transcribed by polymerase III, and so this type of linkage, like those described previously, is not justified by cotranscription. The U1 snRNA genes are tandemly arranged in the genomes of many organisms but also dispersed in the genomes of others (e.g., Zeller et al. 1984). The observation that the 5S rRNA genes are found linked to three different tandemly repeated multicopy genes, as well as not being linked to any other gene in the genome of the crustaceans, seems to indicate that the 5S genes have the feature of transposing frequently in the genome, as Drouin and Moniz de Sá (1995) have suggested. However, the complex case of A. aquaticus suggests a possible different evolutionary origin for the linkage between 5S rRNA and U1 snRNA genes, namely, that the U1 snDNA may have invaded the 5S rDNA unit. The type A 5S gene could represent the orthologous gene of the species (derived from speciation) and the type B and type C genes may be paralogous sequences (due to gene duplication). This hypothesis is based on a number of observations. © 2001 NRC Canada




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First, the sequence of the 5S rRNA gene of type A is the best conserved, differing in only two nucleotides, at positions 30 and 117, from the 5S rRNA gene of P. coxalis, the evolutionarily most closely related species. The G at position 48 (which corresponds to A nucleotide in the type B 5S rRNA gene) is shared by these two genes as well as by all the 5S rRNA crustacean genes described so far. The type A genes are located in interstitial positions on chromosomes of A. aquaticus as well as on some telomeres. An interstitial location of the 5S rDNA was observed by us in P. coxalis (Pelliccia et al. 1998). Second, the type B 5S rRNA gene is linked to the four core histone genes and has therefore probably invaded the large histone repeat by an RNA or DNA mediated transposition of the 5S rRNA type A gene. Third, the type C 5S rRNA gene differs from the type B gene in having the G nucleotide (nt) at position 48 and lacking the first eight nucleotides. However, the two genes share a sequence of 248 bp flanking their 3′ ends. This seems to suggest that the type C gene may result from the retrotransposition of a transcript of the type B gene that has included the 3′ flanking region before any base substitution (G→A) occured at position 48 in the type B gene. It has recently been demonstrated in the human genome that LINE-1 elements (a class of non-LTR retrotransposons, long interspersed elements), can transduce downstream sequences to new genomic locations, bypassing their termination sites during transcription (Moran et al. 1999). Moreover, insertions of non-LTR elements are often accompanied by 5′-end truncations. In any case, neither a DNA mediated transposition nor a common origin of the type B and type C genes from a 5S rDNA sequence dispersed in the genome can be excluded. All three 5S rDNA sequences identified in the A. aquaticus genome could be transcriptionally active; indeed, including the type C sequence lacking the first 8 nts. They have complete transcriptional control elements in the coding region. The internal control region (ICR) of the 5S rRNA genes lying between +50 and +90 would be sufficient to promote initiation of transcription by RNA polymerase III. The type C 5S gene could also be a pseudogene. The presence of multiple 5S rDNA sequences suggests the possible existence, in A. aquaticus of two classes of 5S rRNA gene, the somatic and the oocyte types, the expression of which is developmentally regulated as in the case of those observed in Xenopus (Brown 1994), chicken, lamprey, and in some fishes (Komiya et al. 1986; Sajdak et al. 1998). The interindividual variability of hybridization signal numbers observed for all three gene clusters is probably due to the different degrees of repetitivity of the repetitive units on various chromosome sites, responsible for a variable degree of efficiency of FISH detection of the different loci. Alternatively, polymorphism may exist with regard to the number of the loci and (or) the number of repetitions in the same loci in different organisms.

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Acknowledgements This work was supported by Ministero dell’Università e della Ricerca Scientifica (MURST), Italia. We thank the Cenci Bolognetti Foundation for supplying the 35S-labelled nucleotides.

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