Chromosome Science 14: 23-28, 2011 Cho et al. 23

Regular Article

Effects of the cytidine analogue zebularine on wheat mitotic chromosomes

Seong-Woo Cho, Takayoshi Ishii, Naoya Matsumoto, Hiroyuki Tanaka, Amin Elsadig Eltayeb and Hisashi Tsujimoto

Received: August 31, 2011 / Accepted: October 5, 2011 © 2011 by the Society of Chromosome Research

Abstract et al. 2005). Zebularine is stable in aqueous solution, with a longer half-life (Marquez et al. 2005) and lower toxicity Zebularine, cytidine analog is known as DNA than 5-azacytidine (Rao et al. 2007). Zebularine can sustain methylation inhibitor such as 5-azacytidine and 5-aza- demethylation for a long time and prevent remethylation 2’-deoxycytidine, and it is more stable in aqueous (Cheng et al. 2004). solution than 5-azacytidine and 5-aza-2’-deoxycytidine. The first discover of the methylated base We investigated effects of zebularine on plant mitotic 5-methylcytosine is in calf thymus (Hotchkiss 1948). DNA chromosomes. A wheat disomic addition line carrying a methylation occurs by the addition of a methyl group pair of alien chromosomes (Leymus racemosus chromosome to the 5 position of cytosine in higher eukaryotic cells, ℓ) was treated with zebularine at various concentrations. vertebrates, plants, and many lower eukaryotes (Attwood The alien chromosomes were discriminated from the et al. 2002; Castiglione et al. 2002; Clark and Melki 2002). wheat chromosomes by genomic in situ hybridization, Between 60 and 90% of CpGs are methylated at the 5 facilitating observation of any rearrangements between the position on the cytosine ring in vertebrate genome (Bird wheat and alien chromosomes. Root growth was obviously 1986). In plants, the methylated cytosine can be at a variety inhibited by zebularine because of reduction of the mitotic of cytosine-containing nucleotides such as C-G, C-A, C-T, division cells. Rearrangements such as ring chromosomes, C-C, C-A-G, C-T-G, and C-A-T sites (Gruenbaum et al. insertions, deletions, and translocations were observed 1981). in the treated mitotic chromosomes. The aberrations In this study, we investigated effect of zebularine on were increased in a concentration-dependent manner of wheat mitotic chromosomes. The genome size of wheat is zebularine. large, approximately 16,000 Mb and it is about 40 times larger than that of rice (Arumuganathan and Earle 1991). Keywords: zebularine, wheat, chromosomal rearrange- Wheat can survive even under the chromosome deletion ments, chromosome breakage, alien chromosome addi- in homozygous state (Endo and Gill 1996) because it tion line is an allohexaploid. Homoeologous genes on the other genomes with similar function complement the genes on Introduction the lost chromosomes (Kikuchi et al. 2009). Hence, the wheat is suitable material for studying on chromosomes. Zebularine is a cytidine analog like 5-azacytidine and We used a wheat disomic addition line carrying one 5-aza-2’-deoxycytidine (Jones and Taylor 1980; Harris 1982; pair of chromosomes from Leymus racemosus. The alien Gowher and Jeltsch 2004). Zebularine lacks the amino chromosomes were used as marker chromosomes (Kikuchi group at position 4 of the pyrimidine ring (Champion et al. et al. 2009) because they can be distinguished from the 2010) and was originally known as the cytidine deaminase wheat chromosomes by Genomic in situ hybridization inhibitor 1-β-D-ribofuranosyl-2(1H)-pyrimidinone (GISH). In addition, we investigated various concentrations (McCormack et al. 1980; Kim et al. 1986). In addition to of zebularine and treatment times for wheat, and inhibiting cytidine deaminase, zebularine is known as documented the types of chromosome rearrangements DNA methylation inhibitor (Zhou et al. 2002; Ben-Kasus induced by this substance.

Seong-Woo Cho, Takayoshi Ishii, Naoya Matsumoto, Materials and methods Hiroyuki Tanaka, Amin Elsadig Eltayeb and Hisashi Tsujimoto (*) Plant materials Laboratory of Molecular Breeding, Arid Land Research Center, Tot- tori University, Hamasaka, Tottori 680-0001, Japan For all of the experiments in this study, we used a e-mail: [email protected] wheat (Triticum aestivum L. cv. Chinese Spring) disomic Hiroyuki Tanaka addition line carrying one pair of chromosomes [ℓ] from Laboratory of Plant Genetics, Faculty of Agriculture, Tottori Univer- Leymus racemosus (2n = 42+2, AABBDD+ℓ ℓ, Kishii et al. sity, Tottori 680-8553, Japan 2004). This line combined with GISH as described later 24 Wheat chromosome aberrations induced by zebularine is adequate to observe chromosome aberration in detail For measurement of chromosome aberrations in (Kikuchi et al. 2009). metaphase, a total number of 500 cells per slide were scored, and 10 slides were scored for each concentration Treatment of seeds with zebularine after in situ hybridization. The ratio of cells in metaphase was calculated as the number of cells in metaphase divided Seeds of the wheat-Leymus racemosus disomic addition by the total number of cells. The ratio of metaphase cells line were incubated for 3 days at 4°C on wet filter paper and with chromosome aberrations was calculated as the then transferred to dark condition at 20°C to germinate. number of cells with abnormal structure chromosome The 15 seeds selected by the same length of coleoptile by chromosome aberrations between wheat and alien (about 2 mm) were treated on the wet Petri dishes with chromosomes in metaphase divided by the total number of various concentrations of zebularine (lower concentrations: metaphase cells. 2.5, 5.0, 7.5, and 10.0 μM and higher concentrations: 25, 50, 75, and 100 μM) in dark condition at 20°C. The Petri dishes Labeling of DNA probes were wrapped with parafilm to prevent evaporation. The length of roots was measured after treatment of zebularine Genomic DNA of L. racemosus and L. mollis was for 24, 36, 48, and 60 h. Zebularine (Wako) was first labeled with tetramethyl-rhodamine-5-dUTP (Roche) and dissolved in dimethyl sulfoxide (DMSO) and then further fluorescein-12-dUTP (Roche) by random-primer labeling diluted in water. The final concentration of DMSO was to distinguish between euchromatic and heterochromatic 0.5% for all concentrations of zebularine. regions of chromosomes of L. racemosus. This is possible because the chromosomes of L. racemosus have Preparation of slides with mitotic chromosomes heterochromatic regions at the chromosome ends, but L. mollis chromosomes do not (Kishii et al. 1999). To prepare slides for in situ hybridization, at least 10 In addition to the probes of genomic DNAs, we used roots were selected from each treatment concentration after the wheat centromere probe to detect the centromeric treatment for 36 h. Primary roots were incubated for 24 h regions. The sequences of wheat centromeric region were in ice water to accumulate mitotic metaphase cells and then amplified by oligonucleotide primers (Fukui et al. 2001), fixed in ethanol: acetic acid (3:1 v/v) fixative solution at forward (5’-ATGATACTGATTTCAAAGAT-3’) and room temperature for 5 days. Root tips were stained using reverse (5’-ACCATACACAATTTCAAAAGG-3’). The PCR 2% acetocarmine solution then squashed in 45% acetic acid product was cloned using pGEM®-T and pGEM®-T Easy on microscope slides and then covered with cover slips. Vector Systems (Promega) in ligation reaction and then The slides were frozen at -80°C to remove the cover transformed using E. coli DH5 α Competent cell (TaKaRa). slips and air-dried for GISH treatments. In case of mitotic The clone DNA was labeled with tetramethyl-rhodamine- index, primary roots were prepared without ice-water 5-dUTP by the random-primer method and used as probe pretreatment and fixed in fixative solution. Slides were of fluorescence in situ hybridization (FISH). prepared by the same process described above. Sequential in situ hybridization Cytological analysis

Figure 1. Root growth of the wheat–Leymus racemosus disomic addition line under zebularine treatment at various concentrations. Each bar indicates the standard error (n=15).

In mitotic index, cells undergoing mitotic division were Sequential GISH and FISH were performed to counted to assess the effects of zebularine. A total number distinguish the pair of alien chromosomes in the wheat– of 1,000 cells were scored per slide, and measurement of Leymus racemosus disomic addition line and to identify mitotic index was performed 3 times. Mitotic index was centromeric region on wheat chromosomes following measured for the 5 and 10 μM zebularine treatments at 24 Dou et al. (2009). Briefly chromosome slides prepared and 36 h of treatment. by the squash method were denatured by using 0.2 M Cho et al. 25

NaOH in 70% ethanol at room temperature for 10 min. temperature and air-dried. The second hybridization was After denaturation, the slides were dehydrated in cold performed by the same process of FISH described above ethanol series (70, 90, and 99.5% ethanol, for 5 min at for GISH. each concentration). In the first hybridization for FISH, hybridization solution (50% formamide, 10% dextran sulfate, 50–500 ng labeled probe, 2 х SSC) was applied to Results the slides, which were then incubated for at least 20 h at Root growth under various concentrations and 37°C in a humid box. After hybridization, the slides were treatment times washed for 5 min in 2 х saline sodium citrate (SSC) with 0.1% Triton X-100, followed by 5 min in 2 х SSC. The We first treated 25-100 μM of zebularine and observed slides were briefly dried and then covered with a drop of the root growth at 24-60 h (Fig. 1a). After treatment for 24 Vectashield (Vector) containing 1 ng/μl 4’, 6-diamidino- h, root growth was similar to untreated control. However, 2-phenylidole (DAPI). Images were captured using at 36 h and beyond, root growth was seriously inhibited a fluorescence microscope (Olympus BX61) with a for all concentrations. Although the roots of the untreated cooled CCD camera (Photometrics CoolSNAP fx; Roper control elongated over the time, even in the concentration Scientific) and processed using Meta imaging series 5.0 of zebularine at 25 μM, the roots were significantly software (Universal Imaging Corporation). After the first inhibited in t-test (P<0.01). in situ hybridization, the cover glasses were removed and To know the specific effect of zebularine, we lowered the slides were washed in 2 х SSC for 30 min at room the concentration to 2.5 to 10.0 μM and observed root growth (Fig. 1b): The root in concentration of 2.5 μM was slightly inhibited over the time of treatment. According to increase of zebularine, the roots were significantly inhibited (P<0.01). At 7.5 μM, the length of roots were about half of control.

Mitotic index of treated root tips cells The mitotic index was affected by both treatment time and concentration. The mitotic index of control cells was 27.4 ± 0.35% (± standard error) after 24 h. At 5.0 and 10.0 μM, the indices were reduced to 24.2 ± 2.29% and 23.8 ± 2.35%, respectively, but these were not significantly different from the control in t-test (P>0.01). After 36 h, the indices at 5.0 and 10.0 μM were 20.9 ± 0.66% and 10.9 ± 1.25%, respectively, while the control index was 24.3 ± 1.63%. The index at 10.0 μM was significantly less than that in the control (P<0.01).

Chromosome rearrangements caused by zebularine After 36 h of zebularine treatment, chromosomes exhibited abnormal structures such as gaps (Fig. 2a, arrowhead) and abnormally short chromosomes (Fig. 2a, hollow arrowhead). GISH/FISH analyses revealed more precise rearrangements. For example, as for the cell in Fig. 2a and b, the structure of one alien chromosome was normal but another chromosome was highly rearranged: the euchromatic region was inserted into a wheat chromosome losing subtelomeric heterochromatin regions. Figure 2. Chromosome rearrangements between wheat and Leymus racemosus chro- The subtelomeric regions are remained as mosomes in metaphase after zebularine treatment for 36 h. a small chromosome (Fig. 2b). In the cell a, c, and e: Different interference images of the chromosomes. b, d, and f: The same cells as in a, c, and e, respectively, after GISH/FISH. Centromeric regions (green) of shown in the images of Figs. 2c and d, two wheat chromosomes (blue), heterochromatic regions of the alien chromosomes (red) alien chromosomes were separated into four and the euchromatic regions (white) were observed. Arrows in each pair images indi- parts, a deleted chromosome lacking one cate the same positions on the same chromosomes. Arrowheads and hollow arrow- of the telomeric heterochromatin and three heads are remarkable chromosomes with rearrangement(s) (See text). Scale bar, 10 µm wheat-alien translocated chromosomes of 26 Wheat chromosome aberrations induced by zebularine

Figure 3. Examples of rearranged chromosomes after treatment of zebularine for 36 h. a and b: Normal chromosomes of Leymus racemosus and wheat chromosomes (blue), respectively, as controls. c, d and f: Rearranged L. rac- emosus chromosomes. e and g-n: Rearranged chromosomes consisting of both wheat and L. racemosus chromosomes. Heterochromatic re- gions (red), euchromatic regions (white) and centromeric region (green) were detected by sequential GISH/FISH method. which two carries only the portion of heterochromatin. In result, those caused insertions or long and multicentric images of Figs. 2e and f, one very long chromosome was chromosomes. shown (Fig. 2e, hollow arrowhead). Sequential GISH/FISH revealed that this chromosome was a resultant of fusion of Frequency of cells with abnormal chromosomes in an alien chromosome and wheat chromosomes, including several centromeric regions (Fig. 2f). Here another alien metaphase chromosome seemed to be normal. In addition to these We observed cells with abnormal structure chromosomes alien chromosomes, a chromatid-type aberration was by chromosome aberrations between wheat and alien observed (Fig. 2e, arrowhead), Fig. 3 shows examples of chromosomes in metaphase. The frequency of cells with chromosome aberrations observed in the treated plants. abnormal structure chromosomes as a percentage of total Chromosomes in Figs. 3c-e are truncated of which those metaphase cells was approximately 0.5% in control (Fig. in c are interstitial deletion of the alien chromosome, 4). At concentrations of zebularine ranging from 2.5 to those in d may be of one arm, and that in e is produced 10.0 μM, the frequency of cells with abnormal structure wheat-alien translocation carrying a heterochromatic chromosomes increased from 3.9 to 16.9% of total cells. The region of the alien chromosome and euchromatic wheat frequency of cells with abnormal structure chromosomes chromatin. Chromosomes in Fig. 3f are ring form: Upper was roughly proportional to the dose of zebularine used. chromosome loses the heterochromatic region, whereas Reduction of metaphase cells frequency was also observed lower one carried a heterochromatic region. Figs. 3g-n are with increasing concentration (Fig. 4). translocations between wheat and alien chromosomes. The lengths of the translocated regions were various in chromosome: Some chromosome might be produced by a Discussion single translocation event (g-i), whereas others produced We found in wheat that elongation of roots was inhibited by complex events of chromosome aberrations, and as a after 36 h of treatment at more than 5.0 μM of zebularine. Recently, it is reported that growth of Arabidopsis thaliana is inhibited on medium with 20, 40, and 80 μM of zebularine (Baubec et al. 2009). Most of the roots did not elongate at all concentrations above 10.0 μM of treatment even after 36 h in wheat. In addition, the proportion of dividing cells (mitotic index) decreased with increasing of concentration and treatment time. In human breast cancer cells, the cell viability is inhibited by zebularine treatment in dose and time dependently (Billam et al. 2010). In addition, zebularine induces DNA damage in human leukemic T cells (Ruiz-Magaña et al. 2011) and causes cell death in Escherichia coli (Betham et al. 2010). In wheat disomic addition line, the concentration over 10.0 μM seemed to be too strong to observe real effect of zebularine on mitotic cell division. Here, in this study, we found chromosome breakage Figure 4. Frequencies of cells with abnormal structure chromo- somes by chromosome aberrations between wheat and alien chro- on wheat mitotic chromosomes, treated zebularine using mosomes in metaphase as a % of total metaphase cells (A) and well-established system to observe chromosome breakage. metaphase cell as a % of total cells (B) after treatment with various Previously Kikuchi et al. (2009) used this system to observe concentrations of zebularine for 36 h. Each bar indicates standard effect of heavy ion beam on chromosome aberration. The deviation (n=10). chromosome breakage is reported in DNA-protein kinase- Cho et al. 27 deficient human glioblastoma cells under zebularine and mutagenicity of deoxycytidine analogs in Escherichia coli. treatment (Meador et al. 2010). We investigated cells DNA Repair (Amst) 9: 153-160 in metaphase after zebularine treatment and identified Billam M, Sobolewski MD, Davidson NE (2010) Effects of a novel various types of chromosomal aberrations (Fig. 3). 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