Molecular Cytogenetic Analysis and Isolation of a 5S Rrna-Related Marker in the Scleractinian Coral Platygyra Contorta Veron 1990 (Hexacorallia, Anthozoa, Cnidaria)

Molecular Cytogenetic Analysis and Isolation of a 5S rRNA-Related Marker in the Scleractinian Coral Platygyra contorta Veron 1990 (Hexacorallia, Anthozoa, Cnidaria)

Takahiro Taguchi1,2*, Satoshi Kubota1, Erika Tagami2, Takuma Mezaki3, Satoko Sekida4, Kazuo Okuda4 and Akira Tominaga1,2

1 Division of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi 783–8505, Japan 2 Department of Molecular & Cellular Biology, Kochi Medical School, Nankoku, Kochi 783–8505, Japan 3 Kuroshio Biological Research Foundation, Otsuki, Hata County, Kochi 788–0333, Japan 4 Division of Marine Bioresources, Graduate School of Kuroshio Science, Kochi University, 2–5–1 Akebono-cho, Kochi 780–8520, Japan

Received September 14, 2016; accepted January 30, 2017

Summary A molecular cytogenetic analysis was conducted on the scleractinian coral Platygyra contorta, which is commonly found along temperate coasts in Japan. P. contorta was karyotyped (2n=28) by conventional G- and C-bandings, and the karyogram revealed that about 50% of the metaphase spreads had a homogenously staining region (hsr) in the terminal portion of the long arm of chromosome 12. Fluorescence in situ hybridization (FISH) showed that this hsr consisted of rRNA genes (rDNA) stained by C-banding. The presence of an hsr, which is a highly ampliﬁed rDNA, may explain the molecular diversity of coral rDNA. FISH visualized and

demonstrated the presence of a telomere motif (TTA GGG)n in this coral using a human telomere probe. Further- more, we isolated a speciﬁc FISH marker (312 bp) designated PC-T1, which was located near the centromere of chromosome 11. A sequence analysis revealed that the terminal part of PC-T1 (51 bp from the 3′ end) was 90% homologous with a Montipora capitate microsatellite and the Actinia equine 5S rRNA gene. Isolating FISH markers will assist the classiﬁcation of scleractinian corals with the progression on describing their genome. The data obtained in this study will be valuable for discriminating species among scleractinian corals and understanding their genetics, including chromosomal evolution.

Key words Scleractinian coral, Karyotype, hsr, FISH, rRNA gene.

More than 800 species of scleractinian corals (stony led to several new findings, such as precise and con- corals) are found worldwide (Veron 2000). Many studies sistent numbers of chromosomes, rRNA gene loci, and have classified stony corals using morphological and mo- the presence of a homogenously staining region (hsr), lecular techniques. Nevertheless, the coral classification consisting of rDNA and some coral repeated sequences is somewhat controversial, as those studies occasionally shared with humans (Taguchi et al. 2013, 2014, 2016). did not achieve concordance between morphology and In this study, we report a detailed molecular cyto- DNA sequence homology (Fukami et al. 2004). genetic analysis of the stony coral Platygyra contorta A karyotype provides key data for classifying eu- (family Faviidae). P. contorta is commonly found only karyotes, in accordance with other morphological and around mainland Japan (Wallace 1999). The morpho- molecular studies. However, the cytogenetics of stony logical features of P. contorta include massive, encrust- corals, such as their karyotypes and gene mapping on ing, and columnar colonies; valleys that are usually long chromosomes, have not been investigated sufficiently. and relatively straight at the colony margins but become So far, we have reported molecular cytogenetic informa- short, sinuous, and contorted towards the center of the tion on three species of stony corals, such as Acropora colony; and thin walls with highly irregular septa (Veron solitaryensis (family Acroporidae), Coleostrea aspera 2000). This is our fourth study on a coral species, us- (family Merulinidae), and Echinophillia aspera (family ing both conventional banding (G- and C-bandings) and Lobophylliidae). Our studies on these three species have fluorescence in situ hybridization (FISH), to discover the detailed chromosomal characteristics of this coral. As a * Corresponding author, e-mail: [email protected] result of G- and C-banding analyses, we identified an hsr DOI: 10.1508/cytologia.82.205 on P. contorta chromosome 12 after karyotyping. An hsr 206 T. Taguchi et al. Cytologia 82(2) has been found previously in the corals C. aspera and Toyo Corp., Tokyo, Japan) to remove any external con- E. aspera (Taguchi et al. 2013, 2016). Conventional C- taminants. banding revealed that the hsr portion of P. contorta was C-band positive (darkly stained), indicating the presence Chromosome preparations and conventional G- and C- of specific proteins, such as centromere regions. bandings Furthermore, we successfully isolated a DNA marker, We reported the method for making coral chromo- designated PC-T1, which was utilized as a specific FISH some preparations previously (Taguchi et al. 2013). In probe (exclusively hybridized with the centromere region brief, 10–14 h after artificial fertilization, the embryos of chromosome 11). The location of this marker may were treated with 0.01% (v/v) colchicine (Sigma, St. be the locus of the 5S rRNA gene because PC-T1 was Louis, MO, U.S.A.) in filtered seawater for 1 h, followed cloned from the polymerase chain reaction (PCR) prod- by a hypotonic treatment (0.5× seawater diluted with uct using the 5S rRNA gene primer reported in another distilled water) to spread the chromosomes, which were cnidarian study (Stover and Steele 2001). The sequence fixed using a freshly mixed solution of absolute metha- analysis of the specific FISH marker (PC-T1) revealed nol and glacial acetic acid (3 : 1). The fixed embryos that this marker contained partial common 5S rRNA se- were soaked in diethyl ether overnight to remove intra- quences of Actinia equine (Actiniaria) and a Montipora cellular lipids and returned to the fixative. About 20–50 capitate (Scleractinia) microsatellite. embryos were cut into their constituent cells using a fine Here, we describe novel findings from a P. contorta needle. Suspensions containing embryonic cells were study obtained by conventional and molecular cytoge- transferred to a 1.5-mL tube filled with fixative. The netic techniques. We discuss how these findings will tube was centrifuged at 2000×g for 2 min, and the pel- assist the scleractinian coral classification, resulting in let was re-suspended in 0.5 mL of fresh fixative. One the contribution to understand their genetics and genome drop containing fragments of embryos and isolated cells (Shinzato et al. 2011). was placed on a clean slide and air- or flame-dried to spread the chromosomes. Slides with metaphase spreads Materials and methods were treated with 0.025% trypsin solution for 1 min for

G-banding (Seabright 1973) and with 5% Ba(OH)2 for Coral collection 5 min at 50°C, followed by 2× standard saline citrate P. contorta is a simultaneous hermaphrodite, and (SSC) for 1 h at 60°C for C-banding (Sumner 1978). spawning consists of the release of eggs and sperm Both sets of slides were stained with 5% Giemsa. packed together in discrete bundles from the mouths of fertile polyps. P. contorta gametes were collected at DNA extraction Nishidomari (32°46′N, 132°43′E), Kochi Prefecture, DNA from P. contorta sperm (about 0.1 mL) and em- Japan (Fig. 1). The release of gamete bundles was ob- bryos (about 0.5 mL) was extracted using a Wizard Ge- served between 9 : 00 p.m. and 9 : 45 p.m. on August 9, nomic DNA Purification kit (Promega Corp., Madison, 2015, during which the bundles were collected in plastic WI, U.S.A.), according to the manufacturer’s instruc- cups placed over some of the colonies during spawning. tions. After collection, the egg-sperm bundles were broken apart, and the fertilized eggs were rinsed in 0.2 µm fil- Generation of rRNA gene probes tered seawater (ADVANTEC cartridge filter; Advantec Probes for the putative 5S, 18S, and 28S rRNA gene-

Fig. 1. Collection map; Nishidomari, Otsuki-cho, Hata county, Kochi 788–0333, Japan (32°46′44″N, 132°43′57″E) (Left). Inset: enlarged collection area (arrow points to the Nishimidori collection site). Appearance of Platygyra contorta in the sea (right). Scale=5 cm. 2017 Molecular Cytogenetic Analysis of the Scleractinian Coral Platygyra contorta 207 derived DNAs were obtained from the PCR products (F-12-dUTP) or cyanine-3-dUTP (Cy-3-dUTP) in accor- generated using either a forward or reverse single primer dance with the kit protocol (Invitrogen, Tokyo, Japan). in a PCR analysis. The following were the primers The FISH analysis was carried out as reported previ- used for the rRNA genes: 5′-CTT CCG TGA TCG GAC ously (Taguchi et al. 1993), with slight modifications. In GAG AAC-3′ for the nuclear partial 5S ribosomal DNA brief, the metaphase preparations were denatured in 70% (rDNA) of cnidarians (Stover and Steele 2001) and both formamide/2× SSC at 73°C for 2 min. Then, 0.8 µL of 5′-GTC GTA ACA AGG TTT CCG TA-3′ for 18S and the probe was mixed with 10 µL hybridization solution 5′-GCT TTG GGC TGC AGT CCC AAG CAA CCC ACT (H7782; Sigma, Tokyo, Japan) and denatured at 80°C C-3′ for 28S of Acropora (Chen et al. 2000). P. contorta for 10 min. The probes were hybridized on chromosome sperm DNA was used as a template. The PCR was car- preparations at 37°C in a CO2 incubator for 12–15 h, fol- ried out using a thermal cycler (WK-0518; Wako, Osaka, lowed by two post-hybridization washes in 2× SSC and Japan). The PCR conditions for 5S rDNA were: pre- 1× PBD (0.05% Tween20/4× SSC) and counterstained heating for 2 min at 98°C, followed by 30 cycles at 98°C with 4′,6-diamidino-2-phenylindole (DAPI). The probes (10 s), 55°C (30 s), and 72°C (1 min). The PCR reaction were visualized under a fluorescence microscope (BX50; conditions for the 18S and 28S rDNAs were: pre-heating Olympus, Tokyo, Japan). for 2 min at 95°C, followed by four cycles at 94°C (30 s), 60°C (1 min) and 68°C (3.5 min), and 30 cycles at 94°C Image acquisition and processing (30 s), 56°C (30 s), and 72°C (1 min), with a final exten- The FISH slides were examined with a fluorescence sion at 72°C for 10 min. The amplified bands were cut microscope. Images of suitable metaphase spreads were out of the gel and purified using the QIAquick PCR Pu- acquired on an Olympus DP70 microscope workstation rification kit (Qiagen, Valencia, CA, U.S.A.), according equipped with a cooled charge-coupled device and FISH to the manufacturer’s instructions. analysis software. The Miller units used for each fluorescence light type (FITC, Cy-3, and DAPI) were U-NIBA, Cloning the FISH marker U-MWU, and U-MWIB (Olympus), respectively. After separating the bands, the extracted purified PCR products were ligated into a pGEM-T Easy- Results Vector (Promega, Madison, WI, U.S.A.) and 30 ng of the ligation products were used to transform com- P. contorta has a diploid karyotype petent cells (JM109, pGEM-T Easy-Vector Systems; Five metaphase spreads were karyotyped using con- Promega, Madison, WI, U.S.A.) that were plated on ventional trypsin G-banding, and an hsr was identified Lucia broth (LB) plates containing 100 µg mL-1 ampicil- in chromosome 12 in almost 50% of the metaphase -1 lin, 40 µg mL X-Gal (5-bromo-4-chloro-3-indolyl β-D- galactopyranoside), and 0.05 mM IPTG (isopropyl β-D-1- thiogalactopyranoside). The transformed clones were transferred to 15 mL test tubes containing 1.5 mL LB/ ampicillin medium and proliferated at 37°C overnight. The resulting clones were extracted as plasmid DNAs using a standard method.

DNA sequencing and homology search The fragment inserted in the clone was sequenced with M13 forward and reverse primers using an ABI Prism BigDye Terminator Cycle Sequencing Ready Re- action Kit v2.0 (PE Biosystems, Chiba, Japan) and the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). Computer-assisted DNA alignment and comparison was performed using Gapped BLAST and PSI-BLAST: a new generation of protein database search programs to search the GenBank database (http://www. ddbj.nig.ac.jp).

FISH analysis Fig. 2. (A) G-banded and (B) C-banded karyograms. Chromosomes A human digoxigenin-labeled telomere probe was were stained with Giemsa. Arrow (pale stained portion) purchased from Appligene Oncor (Lifescreen, Watford, indicates the homogenously staining region (hsr) in (A) and the slender arrows in (B) indicate rDNA regions (dark UK). Random prime labeling of the probe DNA from the stained; larger portion is the hsr; left) of chromosome 12, PCR products was performed with ﬂuorescein-12-dUTP which was C-band positive. 208 T. Taguchi et al. Cytologia 82(2)

Table 1. Relative lengths, centromere indices and types of the rRNA gene loci, and larger domains among these were five chromosome pairs; means and standard deviations derived from the hsr (Fig. 3A). The ratio of metaphase obtained from five metaphase spreads. Types were catego- rized according to references in Levan et al. (1964). spreads with the hsr was >50% (43/80). Several fragments with green signals (arrowheads) were occasionally Chromosome Relative length Centromeric index Type found and were probably derived from a broken hsr (Fig. 1 100.0±2.0 36.4±2.4 sm 3C). However, the 28S rDNA PCR product hybridized 2 80.3±1.4 29.5±3.1 sm not only with the same loci as 18S but also with all of 3 73.3±1.2 29.4±4.5 sm the centromere regions (Fig. 4A), whereas the 5S rDNA 4 67.1±1.0 32.4±3.7 sm 5 67.1±1.5 45.3±4.6 m PCR product hybridized with the centromere region of 6 61.2±1.1 44.4±4.1 m chromosome 11 (Figs. 3C, 4D, F). 7 61.2±0.9 38.6±5.3 m The FISH analysis revealed that the human telomeric 8 61.2±1.2 34.9±6.4 sm probe (TTA GGG) hybridized intensely to the telomeres 9 55.3±1.0 41.6±3.1 m n 10 54.6±0.9 37.6±3.6 m of all P. contorta chromosomes (red signals in Fig. 4C), 11 52.0±0.7 39.4±3.6 m suggesting that the telomere sequence of this coral is 12 49.3±0.7 38.9±5.0 m probably identical to that of humans. 13 44.1±1.2 42.9±5.7 m 14 37.5±0.7 45.3±5.0 m Sequence analysis of the isolated PC-T1 clone m: metacentric, sm: submetacentric. The part of an hsr on chro- The primary 5S rDNA PCR products were cloned mosome 12 was excluded. to define their specificity for chromosome 11 of P. contorta. All clones (19 colonies), which were ob- spreads (Fig. 2). The modal number of chromosomes per tained from a white and blue selection-plate, exhibited metaphase spread (100 cells examined) in P. contorta a hybridized signal at the centromere region of chromo- cells was 28 (2n=28), and a tentative karyogram is some 11 (Fig. 4D, F; red signals; arrowheads). FISH sig- shown in Fig. 2. The chromosomes were arranged in nals were observed at a single site, even in uncontracted decreasing order of chromosome length from 1 to 14 on prometaphase spreads with elongated chromosomes, so this karyogram. Chromosome 12 was easily identified specificity of the clones was predicted to be very high. due to the hsr (Fig. 2A), but the other chromosomes were We tested probe affinity on four other coral species, in- not as easy to distinguish because of the poor G-banding cluding A. solitaryensis, Acropora primosa, C. aspera, pattern and similarities in their length and centromere and E. aspera (data not shown) by FISH, but no hybrid- locations. Moreover, the variety of chromosome conden- ization signals were detected on these four corals. Then, sation patterns, which depends on the time of the cell the clone designated PC-T1 was sequenced. A PC-T1 se- cycle when the cells were fixed, made it difficult to pre- quence homology search analysis using the DDBJ (http:// cisely measure chromosome length. The lengths of the ddbj.nig.ac.jp/blast/) revealed that 51 bp from the 3′ end chromosomes and the arm ratios were finally measured of the total 322 bp sequence (Fig. 5; darkly highlighted) in five metaphase spreads. Based on the arm ratio (Le- was 90% homologous with A. equine 5S ribosomal RNA van et al. 1964), this karyogram consisted of five sub- (Actiniaria) and the M. capitate clone Mc0343 microsat- metacentric (1, 2, 3, 4, and 8) and nine metacentric (5, 6, ellite sequence (Scleractinia). 7, 9, 10, 11, 12, 13, and 14) chromosomes (Table 1). We also tried conventional C-banding using Ba(OH)2, 2× Discussion SSC, and Giemsa. Although the C-banding pattern was not as clear as that obtained in mammals, the hsr was More than 800 species of scleractinian corals have C-band positive (Fig. 2B). Some asymmetries in C-band- been identified worldwide (Veron 2000). To solve the positive regions were observed between homologous controversial taxonomic issue (Fukami et al. 2004) chromosomes. and develop an understanding of coral genetics, we have been accumulating molecular cytogenetic data of FISH mapping of rRNA genes and human telomere scleractinian corals. So far, we have published three mo- sequences lecular cytogenetic reports using FISH on scleractinian The locations of the 18S and 28S ribosomal RNA corals, such as A. solitaryensis, E. aspera, and C. aspera (rRNA) genes (rDNAs) by FISH are shown in Figs. 3A (Taguchi et al. 2013, 2014, 2016), which are commonly and 4A, respectively. The 18S rRNA gene loci were found on the western coast of Japan (Wallace 1999). mapped on the terminal end of homologous chromo- The present study is our fourth report and concerns some 12. Large reddish FISH signals (lower arrow and P. contorta; it is a different genus from our previous upper arrowhead in Fig. 3A) and small signals (upper studies but is classified in the same family Merulinidae arrow and lower arrowhead) were observed both in with C. aspera. interphase nuclei and on metaphase spreads. All of the First, we conducted conventional G- and C-bandings signal domains (arrows and arrowheads) indicated 18S to establish the P. contorta karyotype. In general, ob- 2017 Molecular Cytogenetic Analysis of the Scleractinian Coral Platygyra contorta 209

Fig. 3. Visualization of rRNA genes by fluorescence in situ hybridization (FISH). (A) 18S rDNA (red) was highlighted not only on the chromosomes (arrowheads) but also on interphase nuclei (arrows). The homogenously staining region (hsr) produced large signals (lower arrow and upper arrowhead). (B) Same image stained with DAPI. (C) Putative 5S rRNA gene (arrows: red) on normal chromosome 11 and 18S rRNA gene signals (green). Large arrowhead (green signal) shows the 18S rRNA gene locus on normal chromosome 12. The seven small arrowheads indicate fragmented 18S rRNA gene (green signals), possibly derived from the hsr. Arrows show the putative 5S rRNA gene. (D) Same image as C but stained with DAPI. Scale bars represent 5 µm. taining high quality G- and C-bandings in invertebrate It has been frequently found in human cancer cells as an chromosomes is somewhat difficult due to the relatively oncogene amplification product (Takaoka et al. 2012), small size of the chromosomes and the weaker effect of which is advantageous for cells that grow and proliferate trypsin on G-banding and that of Ba(OH)2 on C-band- rapidly. Based on our recent studies of three scleractin- ing. This is the first report on stony coral chromosomes ian coral species, including P. contorta, the hsr found in displaying conventional C-banding produced using the coral chromosome seems to be one of the character-

Ba(OH)2 treatment (Sumner 1978). Thus, we revealed istic features common to scleractinian corals (Taguchi the G- and C-banding patterns on P. contorta chromo- et al. 2013, 2016). Additionally, we also observed an somes, although they were not as clear as those seen on hsr-like structure in four other corals, such as Favia mammalian samples (Fig. 2A, B). We arranged the P. favus, Micromussa amakusensis, Pavona decussate, and contorta chromosomes in decreasing order of length us- Trachyphillia geoffroyi audouin (data not shown). We ing the G- and C-banding and identified chromosome 12 occasionally observed chromosome fragments that were with an hsr. In our previous studies (Taguchi et al. 2013, likely derived from an hsr because they displayed rDNA 2016), we revealed the presence of an hsr on chromo- FISH signals (Fig. 3C), suggesting that the hsr tends to somes 11 and 13 through a FISH analysis of two other be somewhat unstable. Interestingly, the hsr region of P. corals C. aspera and E. aspera, respectively, and now contorta chromosome 12 was C-band positive (Fig. 2B) on chromosome 12 in P. contorta. Because the lengths but not Ag- nucleolar organizing region-positive (Good- of chromosomes 11, 12, and 13 were so similar, chro- pasture and Bloom 1975) (data not shown). The mecha- mosome 12 may be arranged to be chromosome 11 or nism for the C-band pattern which appears by Ba(OH)2 13. An hsr was originally identified in mammalian cells treatment remains unclear, but particular non-histone after many gene amplifications through a drug-selection proteins closely bound to C-band heterochromatin may process using methotrexate (Biedler and Spengler 1976). stain darkly with Giemsa during C-band procedure 210 T. Taguchi et al. Cytologia 82(2)

Fig. 4. FISH images. Visualization of the 5S, 18S and 28S rRNA genes and telomere (TTA GGG)n by FISH. (A) 28S rDNA is highlighted not only on the centromere regions of most of the chromosomes but also the homogenously staining region (hsr) (arrowheads). (B) The same image stained with DAPI. (C) Telomere sequences (red ﬂuorescent signals) visualized by FISH. (D) Dual color ﬂuorescence in situ hybridization (FISH) image of Platygyra contorta using the cloned 5S (red; arrowheads) and 18S probes (green; arrows). Arrowheads indicate cloned 5S rDNA on the centromere portion of chromosome 11. Arrows indicate 18S rDNA on chromosome 12. A larger green signal was produced by the homogenously staining region (hsr). (E) The same image stained by DAPI. (F) Karyogram of Platygyra contorta (2n=28) obtained from a dual color FISH image using rRNA gene primers for cloned 5S (red; arrowheads) and 18S rDNA (green; arrows). Scale bars represent 5 µm.

Fig. 5. Full sequence of the PC-T1 clone from the polymerase chain reaction (PCR) product. PC-T1 was 322 bp (66 A, 86 T, 78 G, and 92 C; GC content: 53.4%). The bases are aligned from the 5′ to the 3′ end. The 21 bases from the 5′-end (1–21; dark highlight) indicate the primer sequence used. The 51 bases from the 3′-end (272–322; dark highlight) were 90% homologous with the Montipora capitate (rice coral) clone Mc0343 microsatellite sequence and Actinia equine 5S rRNA by a DDBJ blast search analysis (http://ddbj.nig.ac.jp/blast/).

(most of the stained regions are centromeres) (Hsieh and component, which are sensitive to C-band producing

Brutlag 1979). Thus, the hsr of this coral may contain chemicals, such as Ba(OH)2. heterochromatin with non-histone proteins as a major FISH signals of 18S rRNA gene were located on the 2017 Molecular Cytogenetic Analysis of the Scleractinian Coral Platygyra contorta 211 terminal end of the long arm of chromosome 12 (12qter) considered to contain at least a representative sequence (Fig. 3A), whereas the 28S rDNA signals were located of the partial 5S rRNA gene. As the representative se- not only at the same loci of 18S RNA gene but also at quence may have been duplicated many times in tandem the centromeres of all chromosomes (Fig. 4A). These within this region, the copy number of the sequence ap- FISH signals on the centromeres are also observed in A. peared to be large. So, the size of the FISH signals was solitaryensis using the 28S rDNA probe, as reported pre- almost the same as that of the C-band, which reminded viously (Taguchi et al. 2014). We chose a single primer us of repetitious sequences, such as alphoid repeat DNA to amplify the repeated sequences, because a primer set in primates (Jørgensen et al. 1992). Therefore, the cen- (forward and reverse) is not necessarily needed for the tromere portion of chromosome 11 (FISH signal loca- reaction (Watson et al. 1992). The single primer used for tion) seemed to be the P. contorta 5S rRNA gene locus. 28S rDNA may have annealed to centromeric-specific Overall, these new findings have led us to survey repeated sequences, such as α-satellite sequences, which other scleractinian coral species and look for clues to may be homologous with 28S rDNA because FISH sig- solve the difficulties related to stony coral taxonomy and nals were seen on most of the centromeres and the rDNA genomics. (Fig. 4A). FISH mapping revealed that the telomere repeated Acknowledgements sequences (mini-satellite) were quite similar to those of humans (TTA GGG)n because the telomere probe This study was supported by grants from the used here was a commercial product for human use. It Japan Society for Promotion of Science (26292107 and is interesting that stony corals and humans share some 15K14789: to T.T.). repeated sequences, such as human satellites, telomeres, and Alu repeats, as reported in our previous studies References (Taguchi et al. 2013, 2016). Information on chromosome mapping of the 5S rRNA Biedler, J. L. and Spengler, B. A. 1976. A novel chromosome abnor- gene in stony corals has so far been limited. Therefore, mality in human neuroblastoma and antifolate-resistant Chinese hamster cell lives in culture. J. Natl. Cancer Inst. 57: 683–695. we tried several rDNA primers, which have been report- Chen, C. A., Wallace, C. C., Yu, J. K. and Wei, N. V. 2000. Strategies ed for scleractinian corals (Stover and Steele 2001) but for amplification by polymerase chain reaction of the complete also for hydrozoans, jellyfish, and eleutherozoans, such sequence of the gene encoding nuclear large subunit ribosomal as starfish (Hori et al. 1982, Walker and Doolittle 1983, RNA in corals. Mar. Biotechnol. 2: 558–570. Hendriks et al. 1987). Consequently, the primers utilized Fukami, H., Budd, A. F., Paulay, G., Solé-Cava, A., Chen, C. A., Iwao, K. and Knowlton, N. 2004. Conventional taxonomy obscures for cnidarian 5S rRNA repeats (Stover and Steele 2001) deep divergence between Pacific and Atlantic corals. Nature 427: were suitable to make the 5S rDNA FISH probe for P. 832–835. contorta. The PCR product, which was obtained using Goodpasture, C. and Bloom, S. E. 1975. Visualization of nucleolar or- the cnidarian 5S rRNA primer, exclusively hybridized ganizer regions in mammalian chromosomes using silver stain- near the centromere region of chromosome 11 (Figs. 3C, ing. Chromosoma 53: 37–50. 4D, F). However, we detected no hybridization signals Hendriks, L., De Baere, R., Vandenberghe, A. and De Wachter, R. 1987. The nucleotide sequences of the 5S ribosomal RNA of on four other coral species, such as A. solitaryensis, A. Actinia equine and Sepia officinalis. Nucleic Acids Res. 15: 2773. primosa, C. aspera, and E. aspera, when we tested the Hori, H., Ohama, T., Kumazaki, T. and Osawa, S. 1982. Nucleotide FISH probe (data not shown), suggesting that this marker sequences of 5S rRNAs from four jellyfishes. Nucleic Acids Res. is species-specific. Cloning was conducted to specify 10: 7405–7408. the base sequence of the FISH probe. The probe was Hsieh, T. and Brutlag, D. L. 1979. A protein that preferentially binds Drosophila satellite DNA. Proc. Natl. Acad. Sci. U.S.A. 76: cloned, sequenced, and designated PC-T1. The total size 726–730. of PC-T1 was 322 bp (66 A, 86 T, 78 G, 92 C; GC con- Jørgensen, A. L., Laursen, H. B., Jones, C. and Bak, A. L. 1992. Evo- tent: 53.4%). The 21 bases from the 5′-end (1–21; dark lutionarily different alphoid repeat DNA on homologous chromo- highlight; Fig. 5) were used for the primer sequence. somes in human and chimpanzee. Proc. Natl. Acad. Sci. U.S.A. The 51 bases from the 3′-end (272–322; dark highlight; 89: 3310–3314. Levan, A., Fredga, K. and Sandberg, A. A. 1964. Nomenclature for Fig. 5) were unique but showed 90% sequence similarity centromeric position on chromosomes. Hereditas 52: 201–220. with the A. equine (Actiniaria) 5S rRNA gene and the Seabright, M. 1973. Improvement of trypsin method for banding chro- M. capitate (Scleractinia) clone Mc0343 microsatellite mosomes. Lancet 1: 1249–1250. sequence after a DDBJ blast search (http://ddbj.nig.ac.jp/ Shinzato, C., Shoguchi, E., Kawashima, T., Hamada, M., Hisata, K., blast/). We have not confirmed that the entire sequence Tanaka, M., Fujie, M., Fujiwara, M., Koyanagi, R., Ikuta, T., Fu- jiyama, A., Miller, D. J. and Satoh, N. 2011. Using the Acropora of this probe matches P. contorta 5S rRNA because digitifera genome to understand coral responses to environmen- we did not have information on the full P. contorta 5S tal change. Nature 476: 320–323. rRNA sequence. However, it is clear that this probe Stover, N. A. and Steele, R. E. 2001. Trans-spliced leader addition exclusively hybridized to chromosome 11 near the cen- to mRNAs in a cnidarian. Proc. Natl. Acad. Sci. U.S.A. 98: tromere region of the long arm. The PC-T1 clone was 5693–5698. 212 T. Taguchi et al. Cytologia 82(2)

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