A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes: Erythrinidae) is derived from 5S rDNA

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A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces
  A tandemly repetitive centromeric DNA sequence of the fish  Hopliasmalabaricus  (Characiformes: Erythrinidae) is derived from 5S rDNA Cesar Martins 1 , Irani Alves Ferreira 1 , Claudio Oliveira 1 , Fausto Foresti 1 & PedroManoel Galetti Jr 2 1 Departamento de Morfologia, UNESP-Universidade Estadual Paulista, Instituto de Biocie ˆ ncias, CEP 18618-000, Botucatu, SP, Brazil (E-mail: cmartins@ibb.unesp.br; Phone/Fax: +55-14-38116264);  2 Departamentode Gene´ tica e Evoluc¸a ˜ o, Universidade Federal de Sa ˜ o Carlos, Centro de Cie ˆ ncias Biolo´  gicas e da Sau´ de, CEP13565-905, Sa ˜ o Carlos, SP, Brazil  Received 15 June 2005; Accepted 31 August 2005 Key words:  5S rDNA, 5S rDNA variant, centromeric DNA, fish,  Hoplias malabaricus , satellite DNA Abstract A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites,minisatellites, microsatellites, and transposable elements. Although extensively studied for the past threedecades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes arestill discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs invertebrate genome, we searched for repetitive sequences in the genome of the fish species  Hoplias mala-baricus . A satellite sequence, named 5S Hind  III-DNA, which has a conspicuous similarity with 5S rRNAgenes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats wereclustered in the interstitial position of two chromosome pairs of   H. malabaricus , while the satellite5S Hind  III-DNA sequences were clustered in the centromeric position in nine chromosome pairs of thespecies. The presence of the 5S Hind  III-DNA sequences in the centromeres of several chromosomes indi-cates that this satellite family probably escaped from the selective pressure that maintains the structure andorganization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible toexplain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize thatit may be involved in some structural or functional role of the centromere organization. Introduction An interesting feature of eukaryote genome is thepresence of a substantial fraction of duplicatedDNA sequences, most of them composed by non-coding sequences that include satellite, minisatel-lite and microsatellite sequences, and transposableelements. Although studied extensively for the pastthree decades, the molecular forces that propagateand maintain repetitive DNAs in the genome arestill discussed. Among whole sequenced genomesthe repetitive areas remains as gaps because of thedifficulty in their correct positioning and array inthe genome. However, the role of these DNAs ingenome organization and evolution, and theirimpact on speciation has been frequently reported(Charlesworth, Snlegowski and Stephan, 1994).The variation in genome size of different eukary-otes is often reported to differences in the amountof repeated DNA sequences (Cavalier-Smith,1985; Brenner et al., 1993). Recently advances onstudies concerning non-coding repetitive DNAsequences have shown that such sequences areextremely important in the structural and func-tional organization of the genome.Among vertebrate species, studies about repeti-tive sequences have been mainly directed to theunderstanding of the structure and organization of satellite DNA and ribosomal DNA (rDNA)repeats. Satellite DNA families can correspond to Genetica (2006) 127:133–141    Springer 2006DOI 10.1007/s10709-005-2674-y  10–20% of some mammalian genomes (Beridze,1986)andare usually species-specific or found to beconserved within closely related species. They areuseful for molecular cytogenetic analysis, such astheidentificationofhomologous chromosomesandchromosomal abnormalities by  in situ  hybridiza-tion. The molecular organization, chromosomallocation, and possible functions of satellite DNAshave been studied in several groups of animals(Singer, 1982; Clabby et al., 1996). These studies have indicated that satellite-like repetitive DNAsequences may play an important role at thechromosomal and nuclear level (Singer, 1982;Larin, Fricker and Tyler-Smith, 1994; Sart et al.,1997).Although usually considered as a single biolog-ical species, the taxonomy of   Hoplias malabaricus  ispoorly understood. Growing evidence has pointedto the karyotypic diversity of   H. malabaricus ,showinginterpopulationaldifferencesinthediploidnumberandchromosomemorphology, aswellasinsex chromosome systems (Bertollo, Takahashi andMoreira-Filho, 1978, 1983; Dergam and Bertollo,1990; Bertollo, Moreira-Filho and Fontes, 1997a,Bertollo et al., 1997b; Lopes et al., 1998; Bertolloand Mestriner, 1998; Born and Bertollo, 2000). Specimens with a putative hybrid karyotype havenot been found when distinct chromosomal forms(cytotypes) are sympatric (Bertollo et al., 2000).Since the fish  H. malabaricus  has shown to be aninteresting model species for cytogenetic andevolutionarystudies,wehaveinvestigatedrepetitivesequences in the genome of this fish. A centromericsatellite DNA family srcinated from the 5S rDNArepeats was isolated and its composition and orga-nization in the genome of   H. malabaricus  studied. Material and methods Animals, DNA isolation, DNA digestion, PCRand cloning Genomic DNA of five individuals of   Hopliasmalabaricus  from the Araqua ´ river (Botucatu, SP,Brazil) was extracted according to standard phe-nol–chloroform procedures (Sambrook andRussel, 2001). Restriction enzyme digestions of thegenomic DNA were conducted with the endonuc-leases  Hind  III,  Msp I,  Pst I,  Hae III,  Eco RI,  Pvu II, Sca I,  Rsa I and  Spe I. The endonuclease  Hind  IIIgenerated a band of approximately 350 bp thatwas purified from agarose gel for cloning. PCRamplifications of 5S rDNA were performed usingprimers A (5 ¢ -TAC GCC CGA TCT CGT CCGATC-3 ¢ ) and B (5 ¢ -CAG GCT GGT ATG GCCGTA AGC-3 ¢ ) designed from the 5S rRNA se-quence of rainbow trout (Komiya and Takemura,1979) to amplify the 5S rRNA gene and the non-transcribed spacer (NTS) (hereafter refereed as 5SrDNA repeat). A standard PCR reaction wasperformed using 150 pmoles of each primer ,  20 ngof genomic DNA, 1x  Taq  buffer, 200  l M of eachdNTP and 2 U of   Taq  polymerase in a finalreaction volume of 50  l l. Cycling times were asfollows: 94  C of denaturation for 5 min, 35 cyclesof 95  C for 1 min, 63  C for 30 s and 72  C for1 min, with post-cycling extension at 72  C for7 min. The PCR-amplified products were visual-ized in 1% agarose gels. The  Hind  III-DNA frag-ments and the PCR-generated 5S rDNA repeatswere linked in the plasmids pUC18  Hind  III-BAP(Amersham Bioscience) and pGEM-T (Promega),respectively, and used to transform DH5 a  E. coli  competent cells (Invitrogen). Sequencing and sequence analysis The clones obtained from the 5S rDNA–PCRproducts (five clones) and the  Hind  III-DNA frag-ments(fiveclones)weresequencedonanABIPrism377 DNA sequencer (Perkin-Elmer) with the ABIPrism BigDye Terminator Cycle Sequencing ReadyReaction Kit(Perkin-Elmer).Nucleotide sequenceswere subjected to BLASTN (Altschul et al., 1990)searches at the National Center for BiotechnologyInformation (NCBI), web site (http://www.ncbi.nlm.nih.gov/blast), and the sequencealignment was performed using Clustal W(Thompson, Higgins and Gibson, 1994) andchecked by hands. UPGMA based phylogeneticanalyses and Kimura’s 2-parameter genetic dis-tances were determined using the software MEGA2.1 (Kumar et al . , 2001). Bootstrap resembling(Felsenstein,1985)wasappliedtoassesssupportforindividual nodes using 1000 replicates. Southern blot hybridization The genomic organization of the isolated sequenceswas investigated by Southern blot-hybridization.134  Genomic DNA (8  l g) was partially (10 and30 min) and overnight (14 h) digested with 45 U of the endonuclease  Hind  III, submitted to gel-elec-trophoresis in 1% agarose and Southern-trans-ferred to Hybon-N nylon membrane (Southern,1975). The enzyme  Hind  III was used since has onlyone cleaving site in both isolated sequence classes.Filters containing the immobilized DNA wereprobed with cloned monomeric units of thePCR-generated 5S rDNA and with  Hind  III-DNAsequences. To avoid cross-hybridization betweenthe 5S rDNA and the  Hind  III-DNA, the filterhybridization was carried out with the kit ECL-direct nucleic acid labeling and detection system(Amersham Bioscience) using high stringencyconditions. The probes were denatured at 95  C,covalently labeled with the enzyme horseradishperoxidase and hybridized for 14 h to filterimmobilized target DNA in a hybridization buffer(6 M urea/50% formamide/0.5 M NaCl) at 42  C.After the hybridization the filters were washed in6 M urea/0.4% SDS/0.5  SSC buffer at 42  C andthe hybridized DNA detected bychemiluminescence. Chromosome analyses Mitotic chromosomes were prepared from anteriorkidney cells with  in vivo  colchicine treatment(Bertollo, Takahashi and Moreira-Filho, 1978) andwere submitted to fluorescence  in situ  hybridization(FISH) (Pinkel et al., 1986) and C-banding(Sumner, 1972). The same probes employed in theSouthern hybridizations (5S rDNA repeat and Hind  III-DNA fragment) were used for FISH. Theprobes were labeled by nick translation with biotin-14-dATP (Bionick labelling system-Invitrogen).The hybridization was performed in three strin-gency conditions: 35, 50 or 65% of formamide. Themetaphase chromosome slides were incubated withRNase (40  l g/ml) for 1.5 h at 37  C. After dena-turationofchromosomalDNAin70%formamide/2  SSC for 4 min at 70  C, hybridization mixturescontaining 100 ng of denatured probe, 10 mg/mldextran sulfate, 2  SSC and 35, 50 or 65% of formamide were dropped on the slides and thehybridization was performed overnight at 37  C.Hybridization washes included 2  SSC and 35, 50or 65% of formamide at 37  C and 2  SSC and4  SSC at room temperature. Detection of hybrid-ized probes was carried out with avidin–FITCconjugate(Sigma)followedbytworoundsofsignal-amplification. After each amplification step, theslides were washed in the blocking buffer (1.26%NaHCO 3 , 0.018% sodium citrate, 0,0386% triton,1%non-fatdriedmilk)at42  C.Chromosomeswerecounterstained with propidium iodide, and theslides were mounted with antifade (Oncor). Results The cloning and sequencing of the repetitive5S Hind  III-350 bp band identified DNA fragmentsof 356–360 bp long with the presence of insertions,deletions and base substitutions among the clones(Figure 1). A search in the DDBJ-EMBL-Gen-Bank database showed DNA sequence similaritywith the 5S rRNA gene of several vertebrates,including fishes. To determine the relationshipbetween the repetitive  Hind  III-DNA sequence andthe 5S rRNA gene, PCR with specific primersdesigned to amplify monomeric repeat units of the5S rDNA from the genome of   H. malabaricus , wasperformed. The primers designed successfullyamplified the incomplete 5S rRNA gene (118 bp)and the complete NTS (Figure 1). The 5S rDNAconsensus sequence and the  Hind  III-repetitive se-quences (hereafter denominated  Hind  III-DNA)were aligned and the similarity between themdetermined (Figure 1). Analyses among the5S Hind  III-DNA sequences showed a mean geneticdistance of 0.049. The genetic distance between the5S rDNA and the 5S Hind  III-DNA sequencesranges from 0.173 (most similar sequences) to0.221 (most divergent sequences) (Table 1). Thephylogenetic analysis of the sequences discrimi-nated the 5S rDNA and the 5S Hind  III-DNA se-quences (Figure 2). The AT base content of 5SrDNA repeat units was 55.9% and the 5S Hind  III-DNA 65.48%. The main difference between 5SrDNA repeat units and the 5S Hind  III-DNA is thepresence of an expanded imperfect TAAA micro-satellite sequence and two short deletions.A GenBank search showed that the 5S rDNAand the 5S Hind  III-DNA sequences isolated in thepresent work had similarities with a satelliteDNA family previously identified in the genomeof   H. malabaricus  (Database accession numbersL11927 and L11928) (Haaf et al., 1993).135  However, no previous relationship was describedbetween that satellite sequence and the 5S rDNAsequences.Southern blot hybridization analyses wereconducted using DNA digested with the restrictionendonuclease  Hind  III, selected for its recognitionsite within the repetitive isolated sequences. Theenzyme  Hind  III has only one cleaving site in bothisolated sequences. Membrane hybridization usingthe 5S Hind  III-DNA and 5S rDNA isolated se-quences as probes showed that these repetitivesequences are tandemly arrayed in the genome of  H. malabaricus  (Figure 3).FISH was carried out using the 5S rDNA andthe 5S Hind  III-DNA sequences as probes. Underlow stringent conditions (35% of formamide) bothprobes hybridized in the centromeric region of 18chromosomesandnearthecentromeres intheshortarm of chromosome pairs 3 and 15 (Figure 4).Under high stringent conditions (50 and 65%formamide) the 5S Hind  III-DNA probe hybridizedto the centromeric region of 18 chromosomes and Figure 1.  Alignment of the 5S rDNA consensus sequence and 5S Hind  III-DNA repeat units (5S Hind  III-a to 5S Hind  III-e) identifiedin the genome of   H. malabaricus . The 5S rRNA gene sequence regions are in boldface type, the primers used to obtain the 5SrDNA sequences are underlined and the arrows indicate the direction of the PCR amplification. Hyphens represent gaps, dots baseidentity, and N non-defined nucleotides. The sequences are deposited in GenBank under the accession numbers AY624052-AY624061. 136  the 5S rDNA probe to the short arm of chromo-some pairs 3 and 15 (Figure 4). Dark C-band po-sitive segments were detected in the centromericregion of most chromosomes whereas faint C-bandpositive segments were evidenced in the terminalposition of several chromosomes as well as in thecentromeres of a few chromosomes (Figure 4). Discussion Ohno, Wolf and Atkin (1968) postulated that geneduplication was the main driving force of verte-brates evolution. Once a gene was duplicated, onecopy was no longer constrained by selection andany mutation that occurred in the duplicated copycould potentially lead to new expression patternsor altered function, leaving the original copy toprovide its required function. The genetic studies,including the human genome sequencing, over thelast years have identified that duplications of thegenome have led to the complexity of human geneswhen compared to flies and worms (Horvath et al.,2001). In the present paper a tandemly repetitivecentromeric DNA sequence (named 5S Hind  III-DNA) from the fish  H. malabaricus  that sharesequence similarities with repeat units of the 5SrDNA is reported. It seems probably that dupli-cated segments of the 5S rDNA gave srcin to the5S Hind  III-DNA satellite family.The presence of variant repeats of the5S Hind  III-DNA suggests that these sequenceshave an intense evolutionary rate in the genome.An evidence of the intense dynamism of the5S Hind  III-DNA sequences is the presence of theexpanded TAAA microsatellite. The organizationand evolution of tandem repetitive DNAs is gov-erned by particular patterns of evolution such asunequal exchange, transposition, RNA-mediatedtransposition and gene conversion (Dover, 1986).Drouin and Moniz de Sa ´ (1995) suggested the hypothesis that RNA-mediated transposition isthe mechanism responsible for the unusual linkageof 5S rRNA genes to other tandemly repeatedmultigene families. According to the authors, theRNA-mediated transposition could be responsiblefor the dispersion of single copies of 5S rDNArepeats whereas covalently closed circular DNA(cccDNA) molecules containing 5S rRNA geneswould be expected to sometimes lead to theinsertion of several 5S RNA gene copies withinothers sequences in the genome. Such cccDNA Table 1.  Genetic distances (Kimura’s 2-parameter) determined between 5S rDNA consensus sequence and 5S Hind  III-DNAsequences ( Hind  III-a to  Hind  III-e). Upper diagonal, standard error; lower diagonal, genetic distances5S rDNA 5S Hind  III-a 5S Hind  III-b 5S Hind  III-c 5S Hind  III-d 5S Hind  III-e5S rDNA – 0,025 0,025 0,027 0,027 0,0295S Hind  III-a 0,173 – 0,008 0,011 0,012 0,0145S Hind  III-b 0,177 0,026 – 0,010 0,010 0,1125S Hind  III-c 0,186 0,043 0,40 – 0,009 0,0155S Hind  III-d 0,194 0,62 0,41 0,029 – 0,0135S Hind  III-e 0,221 0,74 0,46 0,074 0,055 –   5SHindIII-a 5SHindIII-b 5SHindIII-c 5SHindIII-d 5SHindIII-e 5SrDNA 848377 0,02 Figure 2.  UPGMA three based on the 5S rDNA consensus and 5S Hind  III-DNA sequences (5S Hind  III-a to 5S Hind  III-e). Thenumbers at each node indicate the percentage recovery (>50%) of the particular node (1000 bootstrap replicates). 137
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