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• W2047423592 abstract "The hexamer repeat sequence (TTAGGG)n, found at the ends of all vertebrate chromosomes, was previously identified as the main building element of one member of a Hin dIII satellite DNA family characterized in the genome of the bivalve mollusc Donax trunculus. It was also found in 22 perfect tandem repeats in a cloned junction region juxtaposed to the proper satellite sequence, from which the DNA tract encompassing the clustered tandem copies was excised and subcloned. Here, the chromosomal distribution of (TTAGGG)n sequences in the Donax was studied by the sensitivity to Bal31 exonuclease digestion, fluorescence in situ hybridization (FISH) on metaphase chromosomes and rotating-field gel electrophoresis. To verify the occurrence of the hexamer repeat in the genomes of taxonomically related molluscs and other marine invertebrates, genomic DNA from the mussel Mytilus galloprovincialis and the echinoderm Holothuria tubulosa was also analyzed. The kinetics of Bal31 hydrolysis of high molecular mass DNA from the three marine invertebrates revealed a marked decrease over time of the hybridization with the cloned (TTAGGG)22 sequence, concomitantly with a progressive shortening of the positively reacting DNA fragments. This revealed a marked susceptibility to exonuclease consistent with terminal positioning on the respective chromosomal DNAs. In full agreement, FISH results with the (TTAGGG)22 probe showed that the repeat appears located in telomeric regions in all chromosomes of both bivalve molluscs. The presence of (TTAGGG)n repeat tracts in marine invertebrate telomeres points to its wider distribution among eukaryotic organisms and suggests an ancestry older than originally presumed from its vertebrate distinctiveness. The hexamer repeat sequence (TTAGGG)n, found at the ends of all vertebrate chromosomes, was previously identified as the main building element of one member of a Hin dIII satellite DNA family characterized in the genome of the bivalve mollusc Donax trunculus. It was also found in 22 perfect tandem repeats in a cloned junction region juxtaposed to the proper satellite sequence, from which the DNA tract encompassing the clustered tandem copies was excised and subcloned. Here, the chromosomal distribution of (TTAGGG)n sequences in the Donax was studied by the sensitivity to Bal31 exonuclease digestion, fluorescence in situ hybridization (FISH) on metaphase chromosomes and rotating-field gel electrophoresis. To verify the occurrence of the hexamer repeat in the genomes of taxonomically related molluscs and other marine invertebrates, genomic DNA from the mussel Mytilus galloprovincialis and the echinoderm Holothuria tubulosa was also analyzed. The kinetics of Bal31 hydrolysis of high molecular mass DNA from the three marine invertebrates revealed a marked decrease over time of the hybridization with the cloned (TTAGGG)22 sequence, concomitantly with a progressive shortening of the positively reacting DNA fragments. This revealed a marked susceptibility to exonuclease consistent with terminal positioning on the respective chromosomal DNAs. In full agreement, FISH results with the (TTAGGG)22 probe showed that the repeat appears located in telomeric regions in all chromosomes of both bivalve molluscs. The presence of (TTAGGG)n repeat tracts in marine invertebrate telomeres points to its wider distribution among eukaryotic organisms and suggests an ancestry older than originally presumed from its vertebrate distinctiveness. The ends of eukaryotic chromosomes are capped with functional nucleoprotein structures known as telomeres, which are required to complete the telomerase-dependent replication of the tips of the linear DNA molecules and to preserve the stability and integrity of chromosome arms, as well as for chromosome positioning and segregation (1Zakian V.A. Science. 1995; 270: 1601-1607Crossref PubMed Scopus (795) Google Scholar). Telomeres are structurally complex and contain several DNA components. Essentially, short double-stranded DNA repeats organized in tandem arrays at the tip regions and more complex satellite DNA sequences attached to the tandem repeats, which constitute the internal telomere-associated DNA and form the subtelomeric regions (2Biessman H. Mason J.M. Chromosoma (Berl.). 1994; 103: 154-161Crossref PubMed Scopus (63) Google Scholar). The former contain G-rich strands that are enzymatically elongated by the reverse transcriptase telomerase as single-stranded tails that extend beyond the complementary C-rich strand toward the chromosomal 3′ termini (3Collins K. Curr. Opin. Cell Biol. 2000; 12: 378-383Crossref PubMed Scopus (194) Google Scholar). The structural organization and function of telomeres are fairly conserved among widely divergent organisms from protozoa to vertebrates and higher plants (4Blackburn E.H. Cell. 1994; 77: 621-623Abstract Full Text PDF PubMed Scopus (308) Google Scholar). In contrast, telomeric DNA sequences appear to be variable between species and confined within large taxonomic groups, therefore telomeric repeats are considered group-specific (5Blackburn E.H. Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1772) Google Scholar). In this regard, vertebrates display a repeat motif, namely (TTAGGG)n, which is conserved in all species so far examined from mammals to fish (6Garrido-Ramos M.A. de la Herrán R. Rui Zrejón C. Rui Zrejón M. Cytogenet. Cell Genet. 1998; 83: 3-9Crossref PubMed Scopus (49) Google Scholar, 7Meyne J. Ratliff R.L. Moyzis R.K. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7049-7053Crossref PubMed Scopus (679) Google Scholar). Studies on telomeric DNA in invertebrates are less abundant and mainly restricted to insects (8Okazaki S. Tsuchida K. Maekawa H. Ishikawa H. Fujiwara H. Mol. Cell. Biol. 1993; 13: 1424-1432Crossref PubMed Scopus (194) Google Scholar, 9Saiga H. Edstrom J.E. EMBO J. 1985; 4: 799-804Crossref PubMed Scopus (73) Google Scholar), some other arthropods (10Sahara K. Marec F. Traut W. Chromosome Res. 1999; 7: 449-460Crossref PubMed Scopus (219) Google Scholar), and a few flat and roundworms (11Chiurillo M.A. Cano I. da Silveira J.F. Ramı́rez J.L. Mol. Biochem. Parasitol. 1999; 100: 173-183Crossref PubMed Scopus (55) Google Scholar, 12Joffee B.I. Solovei I.V. Macgregor H.C. Chromosoma. 1998; 107: 173-183Crossref PubMed Scopus (26) Google Scholar, 13Muller F. Wicky C. Spicher A. Tobler H. Cell. 1991; 67: 815-822Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 14Teschke C. Solleder G. Moritz K.B. Nucleic Acids Res. 1991; 19: 2677-2684Crossref PubMed Scopus (31) Google Scholar). Invertebrate telomeric repeats differ from those found in vertebrates in several respects. They exhibit a certain degree of heterogeneity in DNA sequence and repeat lengths (1Zakian V.A. Science. 1995; 270: 1601-1607Crossref PubMed Scopus (795) Google Scholar). In addition, most insects display the pentanucleotide (T2AG2)n as the telomeric repeat element (10Sahara K. Marec F. Traut W. Chromosome Res. 1999; 7: 449-460Crossref PubMed Scopus (219) Google Scholar). Moreover, synthetic oligonucleotides mimicking the pentanucleotide motif do not recognize sequences from vertebrate genomic DNA, nor do (TTAGGG)n oligomers hybridize with insect DNA (8Okazaki S. Tsuchida K. Maekawa H. Ishikawa H. Fujiwara H. Mol. Cell. Biol. 1993; 13: 1424-1432Crossref PubMed Scopus (194) Google Scholar). To date, studies on telomeric DNA in marine and freshwater invertebrates are rather scarce and fragmentary. For instance, (T2AG2)n -containing telomeres have been reported in a freshwater crustacean (10Sahara K. Marec F. Traut W. Chromosome Res. 1999; 7: 449-460Crossref PubMed Scopus (219) Google Scholar) but found absent in a holothuroid, whose genomic DNA yielded fuzzy hybridization signals with a (TTAGGG)n probe (8Okazaki S. Tsuchida K. Maekawa H. Ishikawa H. Fujiwara H. Mol. Cell. Biol. 1993; 13: 1424-1432Crossref PubMed Scopus (194) Google Scholar). A similar probe has been preliminarily reported to hybridize to some extent with genomic DNA from an echinoid (15Lejnine S. Makarov V.L. Langmore J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2393-2397Crossref PubMed Scopus (148) Google Scholar), two marine annelids (16Jha A.N. Dominguez I. Balajee A.S. Hutchinson T.H. Dixon D.R. Natarajan A.T. Chromosome Res. 1995; 3: 507-508Crossref PubMed Scopus (26) Google Scholar), a neogastropod (17Vitturi R. Colomba M.S. Gianguzza P. Pirrone A.M. Genetica. 2000; 108: 253-257Crossref PubMed Scopus (22) Google Scholar), and the bay scallop (18Estabrooks S.L. J. Shellfish Res. 1999; 18: 401-404Google Scholar). Some fluorescent in situ hybridizations to chromosomes of the pacific oyster (19Guo X. Allen S.K. J. Shellfish Res. 1997; 16: 87-89Google Scholar) and the freshwater snail Biwamelania habei (20Nomoto Y. Hirai M. Ueshima R. Zool. Sci. 2001; 18: 417-422Crossref Scopus (20) Google Scholar) have also been described. We recently characterized a family of Hin dIII satellite DNAs in the genome of the bivalve mollusc Donax trunculus (21Plohl M. Cornudella L. J. Mol. Evol. 1997; 44: 189-198Crossref PubMed Scopus (22) Google Scholar). Restriction endonuclease digestions of sperm DNA from the truncated wedgeshell with Hin dIII allowed detection of a DNA fragment the size of a satellite pentamer, which resisted endonuclease cleavage even under extensive digestion conditions. Cloning of the DNA in the corresponding band yielded a set of recombinants showing positive albeit weak reactivity toward one of the characterized Hin dIII satellites. The 836-bp cloned insert appeared to be of a heterogeneous nature, since it consisted of a satellite DNA sequence tract, preceded upstream by a segment 130 bp long made up of tandemly arrayed perfect copies of the hexanucleotide (TTAGGG). The 5′-end of the cloned insert consisted of a segment without any sequence elements resembling those of the characterized satellite structures. Studies of telomeric repeats and their modes of association with repetitive DNA sequences might provide insight into the structural organization and function of telomeric and subtelomeric regions. In this paper we report the molecular cloning of the clustered tandem copies of the hexanucleotide (TTAGGG) previously detected in the sperm DNA of D. trunculus. This cloned DNA segment was used as a probe to study the chromosomal localization of (TTAGGG)n sequences in the truncated wedgeshell clam by following the time course of digestion of DNA with the exonuclease Bal31. We also examined genomic DNA from the Mediterranean mussel Mytilus galloprovincialis and the sea cucumber Holothuria tubulosa. The results indicate that both molluscs as well as the echinoderm species contain clusters of the (TTAGGG) repeat and that the tandem arrays are preferentially located at their chromosome ends. In addition, FISH 1The abbreviations used are: FISHfluorescence in situ hybridizationRFGErotating-field gel electrophoresisFITCfluorescein isothiocyanate on metaphase chromosomes of both molluscs together with RFGE analyses supported the localization suggested by the exonuclease experiments. This work demonstrates the presence and location of the vertebrate-type hexamer repeat in telomeres of marine molluscs and echinoderms. fluorescence in situ hybridization rotating-field gel electrophoresis fluorescein isothiocyanate Adult specimens of the truncated wedgeshell (D. trunculus), the blue mussel (M. galloprovincialis), and the sea cucumber (H. tubulosa) were either obtained from commercial suppliers or collected on the Mediterranean or northwestern coast (Ria de Ribadeo and Balcobo beach) of Spain during the breeding season. Specimens of live H. tubulosa were moved to the laboratory and kept in cold seawater until use. Sperm fluid was obtained as described previously (22Drabent B. Kim J.S. Albig W. Prats E. Cornudella L. Doenecke D. J. Mol. Evol. 1999; 49: 645-655Crossref PubMed Scopus (14) Google Scholar). Briefly, mollusc shells were carefully opened with a scalpel, and the gills were removed to expose gonadal tissue. Sperm fluid was collected through a small incision with the aid of a Pasteur pipette. Male gonads from the echinoderm species were excised immediately before use, squeezed, and the resulting sperm fluid filtered through flannelette as detailed elsewhere (21Plohl M. Cornudella L. J. Mol. Evol. 1997; 44: 189-198Crossref PubMed Scopus (22) Google Scholar). High molecular mass DNA was isolated and purified from fresh sperm suspensions by standard phenol extraction with some modifications (23Sainz J. Cornudella L. Nucleic Acids Res. 1990; 18: 885-890Crossref PubMed Scopus (19) Google Scholar). To test for the chromosomal positioning of (TTAGGG) repeat tracts, purified sperm DNA samples were subjected to Bal31 exonucleolytic trimming with time. High molecular mass DNA (20 μg) in 20 mmTris-HCl (pH 8.0), 600 mm NaCl, 12.5 mmCaCl2, 12.5 mm MgCl2, 1 mm Na2EDTA was supplemented with λ-DNA Hin dIII fragments (0.5 μg) to monitor the extent of the digestion and treated with Bal31 nuclease (2 units) at 30 °C in a final volume of 150 μl. Aliquots of 3.3 μg of DNA (one-sixth of the bulk reaction) were taken at various times: time 0 (prior to enzyme addition), 10, 20, 30, 40, and 50 min, respectively. Reactions were halted by addition of Na2EDTA to 50 mm, inactivated at 75 °C for 10 min, and chilled on ice. Digested DNAs were then recovered by ethanol precipitation and finally dissolved in distilled water. A sixth part of the DNA digests (0.55 μg) was used to monitor λ-Hin dIII fragment trimming, whereas the remainder (2.75 μg) was further digested with AluI. All enzymatic digests were electrophoresed on 0.8% agarose gels and the resolved DNA fragments subsequently transferred to positively charged nylon membranes by alkaline blotting in 0.4 n NaOH after partial depurination (24Reed K.C. Mann D.A. Nucleic Acids Res. 1985; 13: 7207-7221Crossref PubMed Scopus (1283) Google Scholar). DNA probes were labeled with fluorescein-12-dUTP by random priming with the Klenow fragment of DNA polymerase I using the Ready-To-Go labeling beads (AmershamBiosciences). Hybridizations were carried out overnight at 42 °C in 50% formamide containing 0.25 mNa2HPO4 (pH 7.2), 7% SDS, 1 mmEDTA, and 50 μg/ml tRNA, followed by stringent washes in 0.1 × SSC (saline-sodium citrate), 1% SDS at 65 °C, except for the cloned histone H4 probe from H. tubulosa, which was hybridized at 35 °C, and the membrane washed at 57 °C. Stringency washes were followed by blocking with 0.2% casein, 0.5% SDS in phosphate-buffered saline and the filters finally reacted with an alkaline phosphatase-conjugated anti-fluorescein antibody (Tropix). Hybridization signals were visualized by chemiluminescence using the dioxetane CDP-Star (disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro) tricyclo[3.3.1.13,7]decan}-4-yl)-1-phenyl phosphate) (Roche Diagnostics) and recorded on x-ray film. The relative genomic abundance of the (TTAGGG) hexamer sequence was determined by dot-hybridization of graded amounts of both D. trunculus sperm DNA and the recombinant plasmid containing the 148-bp fragment encompassing the (TTAGGG)22 tandem repeat. DNA samples were spot-blotted onto nylon and the membrane subsequently probed with the repetitive insert released from the recombinant clone and 32P-labeled by random priming (25Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar). After exposure to film the intensities of the radioactive signals were quantified using a computer-assisted laser densitometer loaded with the ImageQuant program (Molecular Dynamics). Mean values were derived from two independent experiments. The genomic organization of (TTAGGG) tandem arrays was examined by RFGE. Aliquots of purified sperm suspensions from D. trunculus were embedded in 0.5% agarose plugs at a DNA concentration of 0.5 μg/μl as previously described (22Drabent B. Kim J.S. Albig W. Prats E. Cornudella L. Doenecke D. J. Mol. Evol. 1999; 49: 645-655Crossref PubMed Scopus (14) Google Scholar). Agarose plugs containing high molecular mass DNA larger than 400 kb were incubated with selected restriction endonucleases and the resulting large genomic fragments resolved on 1.2% agarose gels in 0.5 × TBE (Tris-borate-EDTA) at 11 °C. Electrophoresis was run at 100 V for 1 h, followed by successive pulses of 10 s for 15 h and 20 s for 20 h at 200 V with 120° reorientation angles. Gels were then visualized by ethidium bromide staining and the DNA fragments transferred to a nylon membrane. Subsequent hybridizations to the cloned (TTAGGG)22 probe were carried out as described above. Truncated wedgeshell and mussel specimens were continuously fed with Isochrisis galbana microalgae for 10 days in the laboratory. Before use, following treatment with 0.005% colchicine for 6–8 h, gills were dissected and metaphase spreads prepared as described previously (26González-Tizón A. Martı́nez-Lage A. Rego I. Ausio J. Méndez J. Genome. 2000; 43: 1-8PubMed Google Scholar). FISH was carried out with the (TTAGGG)22 cloned probe labeled with digoxigenin by a standard PCR procedure and denatured at 75 °C for 15 min. The hybridization was performed in a PTC-100 microscope slide thermal cycler (MJ Research), with a solution of 50% formamide, 10% dextran sulfate in 2 × SSC, containing sonicated salmon sperm DNA (0.3 mg/ml) and the denatured digoxigenin-labeled hexamer repeat (3.3 μg/ml). Post-hybridization washes were performed in 2 × SSC at 42 °C and then sequentially with 20% formamide in 0.2 × SSC, 0.1 × SSC, and 2 × SSC for 10 min each. For detection, slides were washed in 0.1 m Tris-HCl (pH 7.5), 0.15 m NaCl, 0.05% Tween 20 buffer and blocked in the same buffer containing 0.5% casein, but lacking detergent, at 37 °C for 30 min. The slides were then incubated with anti-digoxigenin mouse serum at a dilution of 1:200 in blocking buffer at 37 °C for 30 min, rinsed in the same buffer, and subsequently subjected to two consecutive rounds of incubation in the same conditions, first with rabbit anti-mouse serum conjugated to fluorescein isothiocyanate (FITC) at a dilution of 1:1000, and finally with FITC-conjugated goat anti-rabbit serum at the same dilution, to amplify the fluorescence signals. The slides were then washed once in blocking buffer, dehydrated through a graded ethanol series, and air dried. Metaphase preparations were counterstained with an antifade solution containing propidium iodide (50 μg/ml), examined under a microphot AFX Nikon fluorescence microscope, and photographed on Kodachrome color slide film (400 ASA). During characterization of a family of four Hin dIII satellite DNAs in the truncated wedgeshell, a DNA fragment of similar length to a satellite pentamer was released upon digestion of sperm DNA. This fragment resisted endonuclease fragmentation even under extensive digestion conditions. It hybridized positively in Southern blots of electrophoretically resolved restriction fragments from Hin dIII digests probed with the monomer clone DTHS1 (GenBankTM/EBI accession number X94534) of the characterized type-1 Hin dIII Donax satellite. The DNA in the corresponding band was recovered, cloned, and sequenced (GenBankTM/EBI accession number X94546) (21Plohl M. Cornudella L. J. Mol. Evol. 1997; 44: 189-198Crossref PubMed Scopus (22) Google Scholar). The cloned insert was 836 bp long and ended in a 66-bp tract that corresponded to the 3′-terminal half of the DTHS1 monomer unit. The latter sequence was preceded upstream by a 130-bp segment comprising 22 tandemly arrayed copies of the hexanucleotide C3TA2, which appeared to be the reversed complement of the vertebrate-type (TTAGGG) telomeric repeat, as well as that of the main subrepeat element found in the DTHS1 satellite DNA. The hexamer repeats were all perfect copies except for the 3′-penultimate repeat in which one C was lacking. A second internal deletion of a single T was observed 5′ contiguous to position 670 of the insert sequence. The clustered repeats were flanked on both sides by endonuclease recognition sequences for MseI and DdeI, with cleavage sites 3′ to positions 622 and 770, respectively (Fig. 1). These two restriction enzyme sites allowed to release from the original clone a DNA fragment encompassing the entire set of highly conserved tandem repeats. The excised 148-bp DNA fragment was blunt-ended and subcloned into the EcoRV site of the pBluescript +SK vector. In turn, the cloned insert was released with polylinker enzymes contiguous to the cloning site and used as a probe throughout the experimental work presented here. The genomic localization of (TTAGGG)n sequences was examined by following the time course of Bal31 degradation of sperm DNA from the wedgeshell clam. High molecular mass genomic DNA extracted from fresh sperm suspensions was subjected to Bal31 digestion with time. Phage λ-DNA Hin dIII fragments were added to the DNA samples to monitor the extent of exonucleolytic trimming. DNA aliquots were taken at intervals, and a fraction of each aliquot was subsequently digested to completion with AluI. DNA samples, prior and after AluI digestion, were then electrophoresed in parallel on a 0.8% agarose gel (Fig. 2 A) and Southern blotted to a nylon membrane. The half of the membrane containing the undigested Bal31-trimmed DNA was cut out and hybridized with fluorescein-labeled λ-DNA Hin dIII fragments (Fig. 2 B). It can be clearly observed the progressive shortening of the λ-Hin dIII fragment lengths. All fragment sizes gradually decreased with the course of Bal31 digestion as a consequence of the continuous exonuclease trimming of the λ-DNA fragment termini. Obviously, this pattern was more apparent in the smaller fragments, which tended to fade away at late time intervals. The second half of the nylon membrane, containing the Bal31-AluI-digested DNA fragments, was initially hybridized to the insert of the recombinant pUC19 clone carrying a 1.5-kb Hin cII fragment containing the entire sequence of the H. tubulosa histone H4 gene (GenBankTM/EBI accession number Z46226) (27Drabent B. Louroutziatis A. Prats E. Cornudella L. Doenecke D. DNA Sequence. 1995; 6: 41-45Crossref PubMed Scopus (4) Google Scholar), labeled with fluorescein (Fig. 2 C). As expected from the internal location of this histone gene in the genome, the DNA bands reacting with the histone H4 probe remained unaltered during the time course of Bal31 digestion except for a slight fall in signal intensity at longer times. The results of the hybridizations with the λ-Hin dIII DNA and histone H4 probes indicate the integrity of the extracted genomic DNA, ruling out any nicking or inner degradation of the DNA, while corroborating the exonucleolytic specificity of Bal31. Subsequently, the H4 probe was stripped off the membrane, which was rehybridized to the fluorescein-labeled (TTAGGG)22 cloned sequence (Fig. 2 D). In contrast to the electrophoretic patterns of the bulk DNA fragments shown in Fig. 2 A, where the effects of the digestions with Bal31 alone or in pairwise combination with AluI were hardly detected by the ethidium bromide staining, the double digestion of sperm DNA generated an uneven and dauby pattern of hybridization with the repeat probe. AluI digestion of genomic DNA at time 0 of the serial digestions with Bal31 produced a broad, intense hybridizing band of low mobility. A fast-moving band, comparable with the former but broader and less intense, was also seen near the migration front (lane 0′ in Fig. 2 D). Integration of the areas under the signal peaks in densitometer tracings of the autoradiogram yielded a DNA distribution in both peaks amounting, respectively, to approximately 45 and 35% of the total DNA hybridizing to the probe in the lane. Likewise, the estimated size of the DNA fragments under the peaks, as deduced from the scans, ranged from 10,000 to 20,000 bp and 500 to 1000 bp, respectively (data not shown). The signal intensity of the hybridizing DNA bands generated by AluI digestion gradually decreased during the Bal31 exonucleolytic trimming, shifting toward lower length distributions with a concomitant reduction of the hybridization signals at the longest times. However, the sensitivity of the upper DNA signal to Bal31 was slightly higher than that of the lower signal. The intensity of the former decreased drastically within 30 min of digestion, thereafter fading faster than the lower signal, traces of which still persisted at longer digestion intervals. The high level of hybridization of genomic DNA to the cloned (TTAGGG)22 sequence reveals the presence of arrays of this vertebrate-type hexameric repeat within Donax DNA. Furthermore, the preferential susceptibility of the DNA sequences positively reacting with the repeat probe to Bal31 exonucleolytic trimming favors a terminal positioning of the bulk of the repeat arrays and therefore allows to qualify them as true telomeric DNA located at the chromosome ends in this mollusc. To verify the occurrence of similar hexameric sequences in the genomes of taxonomically related molluscs and other marine invertebrates, sperm DNA samples from the blue mussel M. galloprovincialis and the echinoderm H. tubulosa were subjected to Bal31 digestion in the above conditions (Fig. 3). As expected, the histone H4 repeat taken as internal gene marker in the Donax genomic DNA showed comparable behavior in both the mussel and the sea cucumber sperm DNAs when subjected to serial digestions with Bal31 and to completion with AluI. The corresponding DNA restriction fragments reacting positively with the histone H4 probe remained insensitive to exonucleolytic trimming. However, the hybridization patterns of the histone H4 gene were not strictly identical in the three invertebrate species examined. The minor dissimilarities observed can be attributed to varying susceptibilities of the respective DNAs to AluI together with differing structural arrangements of histone H4 genes in these organisms (27Drabent B. Louroutziatis A. Prats E. Cornudella L. Doenecke D. DNA Sequence. 1995; 6: 41-45Crossref PubMed Scopus (4) Google Scholar). Both sperm DNAs reacted similarly to DonaxDNA (see Fig. 2 D), yielding broad patterns upon digestion with AluI and hybridization to the labeled (TTAGGG)22 probe. The size distribution of both blurred patterns gradually decreased with the digestion, displaying a clear tendency to fade with time. This indicates a marked sensitivity of the hybridizing DNA to exonuclease trimming. These data support tracing the Bal31-sensitive DNA to chromosomal termini in both the mussel and sea cucumber DNAs and unambiguously confirm the presence of a substantial proportion of (TTAGGG) repeats at the telomeres of these marine invertebrate DNAs. No positively hybridizing DNA bands resistant to Bal31 were seen in the DNAs analyzed except those of the mussel. The hybridization pattern of Mytilus DNA digested with AluI displayed a few discrete bands, albeit faint, in the range 500–1000 bp that were unaffected by the Bal31 digestion. The insensitivity to trimming of these DNA bands containing (TTAGGG) sequences suggests an internal positioning within genomic DNA, namely, at interstitial chromosome regions (2Biessman H. Mason J.M. Chromosoma (Berl.). 1994; 103: 154-161Crossref PubMed Scopus (63) Google Scholar). The presence of these internal hexameric sequences implies that they are represented within genomic DNA in the mussel, constituting discrete DNA elements that produce well defined bands upon digestion. The intensity of the hybridizing bands suggests the repetitiveness of these internal sequences. Concurrently, the failure to detect similar DNA bands in the hybridization patterns from D. trunculus and H. tubulosa does not preclude their existence provided that they are either organized in DNA tracts variable in length and/or so poorly represented in the genomes that they remain undetectable. To derive the content of (TTAGGG) repeated sequences at the ends of Donax chromosomes, the relative genomic abundance of the hexameric DNA sequence was determined from dot-blots of increasing amounts of the recombinant clone carrying the (TTAGGG)22 insert, together with graded amounts of total sperm DNA from the mollusc. Subsequently, the nylon membrane was hybridized to a 32P-labeled (TTAGGG)22 insert released from the same clone and the hybridization signals quantified using a laser densitometer (Fig. 4). The genomic abundance computed for the hexameric tandem repeat comprised 0.05% of the total sperm DNA. Since the size of the haploid DNA complement (C-value) of the wedgeshell clam has been estimated as 1.4 × 109 bp (28Hinegardner R. Comp. Biochem. Physiol. 1974; 47A: 447-460Crossref Scopus (104) Google Scholar), the former value roughly amounts to 4700 copies of the (TTAGGG)22 sequence per haploid genome, equivalent to approximately 100,000 hexamer copies. The genomic length of the putative telomeric arrays of (TTAGGG) repeats can be approximated assuming their preferential localization at the ends of the mollusc chromosomes and using the repeat copy number found, together with the chromosome number of the haploid cells from Donax (n = 19) (29Cornet M. Soulard C. Genetica. 1990; 82: 93-97Crossref Scopus (12) Google Scholar). Since the frequency found for the (TTAGGG) repeat amounts to 100,000 copies and taking into account the haploid chromosomal complement, the computation yields a value of 5200 repeats per chromosome or 2600 per chromosomal terminus, equivalent to a repeat tract length of 15.6 kb of DNA. This value is consistent with the size range of the upper DNA band generated by AluI at zero time of the Bal31 digestion (lane 0′ in Fig. 2 D), as well as with those from the four-cutter endonuclease digestions shown in Fig. 6, and falls within the range of lengths reported for telomeric repeats in mammalian cells (3Collins K. Curr. Opin. Cell Biol." @default.
• W2047423592 created "2016-06-24" @default.
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• W2047423592 date "2002-05-01" @default.
• W2047423592 modified "2023-10-18" @default.
• W2047423592 title "Telomeric Localization of the Vertebrate-type Hexamer Repeat, (TTAGGG) , in the Wedgeshell Clam Donax trunculus and Other Marine Invertebrate Genomes" @default.
• W2047423592 cites W1508644246 @default.
• W2047423592 cites W152055366 @default.
• W2047423592 cites W1533349658 @default.
• W2047423592 cites W1965666620 @default.
• W2047423592 cites W1967999707 @default.
• W2047423592 cites W1973552223 @default.
• W2047423592 cites W1986808310 @default.
• W2047423592 cites W1987958417 @default.
• W2047423592 cites W1989584159 @default.
• W2047423592 cites W2001499069 @default.
• W2047423592 cites W2004787540 @default.
• W2047423592 cites W2008314775 @default.
• W2047423592 cites W2010882280 @default.
• W2047423592 cites W2014669590 @default.
• W2047423592 cites W2020038227 @default.
• W2047423592 cites W2021910892 @default.
• W2047423592 cites W2027361898 @default.
• W2047423592 cites W2030541575 @default.
• W2047423592 cites W2033695054 @default.
• W2047423592 cites W2033721933 @default.
• W2047423592 cites W2035307518 @default.
• W2047423592 cites W2036289995 @default.
• W2047423592 cites W2041590824 @default.
• W2047423592 cites W2042377388 @default.
• W2047423592 cites W2063880804 @default.
• W2047423592 cites W2067668969 @default.
• W2047423592 cites W2070677673 @default.
• W2047423592 cites W2077889601 @default.
• W2047423592 cites W2078502421 @default.
• W2047423592 cites W2089362593 @default.
• W2047423592 cites W2091314517 @default.
• W2047423592 cites W2117448101 @default.
• W2047423592 cites W2400933137 @default.
• W2047423592 cites W39996336 @default.
• W2047423592 doi "https://doi.org/10.1074/jbc.m201032200" @default.
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