Proc. Nati Acad. Sci. USA Vol. 78, No. 1, pp. 338-342, January 1981 Cell Biology Sequence arrangement of a highly methylated satellite DNA of a plant, Scilla: A tandemly repeated inverted repeat (plant chromosomes/DNA sequences/gene arrangement/methylcytosine/mutation) BARBARA DEUMLING Department of Membrane Biology and Biochemistry, Institute of Cell and Tumor Biology, German Cancer Research Center, D-6900 Heidelberg, Federal Republic of Germany Communicated by Joseph G. Gall, September 25, 1980 ABSTRACT G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the MOnocotyledonousant, ScitasibeDica, byusingA+-Cs SO4gra- diemnottedonou is satellite DNAhad an unusi hi melting point and a high methylcytosine (m5C) content (-25% of total bases; m5C/cytosine ratio 1L.5) and was localized, by in situ hy- bridization, in the heterochromatin regions of the chromosomes. Digestion with restriction endonuclease Hae m yielded a series of fragments rangingfrom 35 to several hundred nucleotide pairs. The major fragments, I-IV (35, 50, 59, and 69, nucleotide pairs, respectively), were isolated, and their nucleotide sequences were determined. The dominant fragment I was a highly symmetrical molecule, with a basically palindromic arrangement. This se- quence represented the basic unit of Scilla satellite DNA and was tandemly repeated many times, with some base substitutions and multiple successive insertions of the tetranucleotide G-T-C-C. The dinucleotide CpG was the commonest nearest-neighbor sequence. Thin layer chromatography, DNA sequence analysis, and gas chromatography combined with mass spectrometry showed the high m5C content (m5C/Cyt = 2.2 and 2.8, respectively, for frag- ments II and IEl). Identical cleavage fragments were found in sat- ellite DNAs from two other species of this genus (S. amoena and S. ingridae), which suggests that this constitutively methylated se- quence is evolutionarily stable. The sequence arrangement of this plant satellite DNA is compared with those reported for several animal satellite DNAs. Temperature, OC The nuclear DNA of most, probably all, eukaryotic cells con- tains fractions of highly reiterated, relatively simple sequences FIG. 1. (A) Separation of satellite DNA (arrow) of S. siberica by that, in many species, appear as DNA satellites separable from preparative Ag'-CsSO4 gradient centrifugation. (B) Melting profiles the bulk of the DNA (1). In a variety of animals, the nucleotide of total satellite DNA (. ) and the most thermostable component ob- tained after partial denaturation and treatment with nuclease S1 sequence arrangement of such satellite DNAs has been eluci- (-). Dots denote tm points. Temperatures were calculated for 0.15 dated; as a general principle, they contain tandem repeats of a M NaCl/0.015 M Na3 citrate, pH 7.4. specific sequence unit, which is different in composition and size in different organisms (1-7). Satellite DNAs have also been described (8-11) in several plants that are enriched in hetero- sand. Tissues ofS. amoena and S. ingridae were kindly provided chromatin. Among the plants that show conspicuous hetero- by J. Greilhuber (Department of Botany, University ofVienna, chromatin arrays, the monocotyledonous bluebell species, Austria). Scilla siberica, has been studied in detail (9, 10, 12-14). The sat- Isolation and Fractionation ofDNA. DNA was extracted and ellite DNA in the heterochromatin ofthis plant (9, 10) has been purified by chromatography on hydroxyapatite as described analyzed, and the nucleotide sequence of a part of it has been (12). Total DNA was separated in Ag'-Cs2SO4 density gradients determined. The results show some unusual features ofsatellite (molar ratio of Ag+/DNA = 0.27) into satellite and main-band DNA organization-multiple tandem arrays of an inverted re- fractions (Fig. la). This satellite fraction was still heteroge- peat, multiple insertions of a tetranucleotide, and remarkably neous, as indicated by its complex meltingprofile (Fig. lb). En- high contents of 5-methylcytosine (m5C) and the dinucleotide richment ofthe most thermostable component was achieved by CpG. partial denaturation (by 30 mM NaCl/10 mM NaOAc, pH 4.6) at 650C and subsequent digestion, also at 650C, with nuclease SI MATERIALS AND METHODS (Fig. 1B). Plants. Bulbs, leaves, and root tips were used. Bulbs of S. Restriction and Sequence Analysis. Satellite DNAs from all siberica were obtained commercially and grown in soil or wet three species, including the nuclease Sl-treated fractions, were digested with restriction endonuclease Hae III in 6 mM each The publication costs ofthis article were defrayed in part by page charge Tris HCl, pH 7.4/NaCI/MgCI2/mercaptoethanol at 370C for payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: m5C, 5-methylcytosine; bp, base pair(s). 338 Downloaded by guest on October 2, 2021 Cell Biology: Deumfing Proc. Natl. Acad. Sci. USA 78 (1981) 339 1 2 3 4 5 k. 6 -1. " 4 4 _. Aw vj ~ ~ ~ ~ . 14~~~~~~~~~~li.s .1 _.'..d IF t 4: .; 4 -'Er - I 4 q i A Wt I FIG. 3. DNA fragments obtained by digestion of satellite DNAs from S. siberica (lanes 1-3), S. ingridae (lane 4), and S. amoena (lane * 5) with Hae III were separated by electrophoresis on 8% polycrylamide gels and stained with ethidium bromide. Concentration ofHae III used fordigestion of DNA shown in lane 2 was 10-fold higher than that used for DNA in lane 1. Major bands: I, 35 base pairs (bp); II, 50 bp; III, 59 bp; IV, 69 bp. Identical Hae III-digestion patterns were obtained with ", satellite DNA treated with S1 nuclease as in Fig. 1B. ! .*i i, tot~~~~~~~~~~~f RESULTS All three species ofScilla had large proportions ofsatellite DNA that could be separated in Ag'-Cs2SO4 density gradients (see B Fig. LA; ref. 9). In S. siberica, the satellite DNA represented 19% ofthe total DNA, whereas less satellite DNA was found in the two other species (10.5% in S. ingridae and -6.5% in S. FIG. 2. In situ hybridization of [3H]-labeled Scilla satellite cRNA amoena). In all species, the satellite DNA included a component to chromosomes of S. siberica (A) and S. ingridae (B). Arrows denote an = and the contour of an interphase nucleus. Note strong labeling in distinct that had unusually high melting point (ti 102.5°C) heterochromatic bands on chromosomes, or chromocenters in nuclei. could be further purified by partial denaturation and treatment The pattern of satellite DNA-containing bands is different in the two with nuclease S1 (see Fig. 1). The high G+C content (78%) species. suggested by this high thermostability was confirmed by anal- ysis of the base composition by thin layer chromatography, which also showed that the m5C content was unusually high various times. The fragments obtained were separated by elec- (25% of total bases). In all three species, the satellite DNA trophoresis on 8% polyacrylamide (15), using 1-2 ,ug of DNA wAas located by in situ hybridization (see Fig. 2) in certain te- per lane for analysis and 200-250 ,ug for preparative gels. Re- l meric and intercalary heterochromatin regions stained by the striction fragment bands were visualized by UV absorption (16) n-banding technique (14). The relative positions of the satellite and eluted for 8 hr at 50°C. After end labeling with [y- DNA-containing heterochromatin bands, however, were dif- 32P]phosphate, the strands were separated in 30% dimethylsulf- ferent in the metaphase chromosomes of the three species (Fig. oxide (2 min at 95°C) and analyzed by electrophoresis on 8% and 2 A and B; unpublished data). 12% polyacrylamide gels (16). Fast and slow strands were se- Because of its high degree of methylation, Scilla satellite quenced (15, 17), including by purine-specific cleavage (16). DNA was not cleaved by many of the restriction endonucleases The cleavage products were separated by electrophoresis (15, that are specific for G+C-rich sequences. Cleavage of satellite 17) on 10% and 20% polyacrylamide gels and autoradiographed. DNA with Hae III, however, yielded a number of distinct frag- Determination of Methylcytosine. The m5C contents in the ments that were identical in sizes in all three species (Fig. 3). DNA fractions and the isolated restriction fragments were de- Sixteen Hae III cleavage fragments could be distinguished (for termined after hydrolysis with 98% formic acid by thin-layer numbering of major fragments, see Fig. 3). Four major frag- chromatography on cellulose, by gas chromatography, and by ments (I-IV) were isolated, and their separated slow and fast combined gas chromatography and mass spectrometry (18, 19). strands were subjected to sequence analysis. Some of the typ- Sample injection was as described by Ros (20). An OV-101 glass ical features in the sequence analysis of this DNA are shown capillary was used as the column (J. Stadler, unpublished data). in Fig. 4, and the sequences of fragments I-IV are shown in In Situ Hybridization. Synthesis ofc[3H]RNA from total sat- Figs. 5 and 6. Fragment I (35 nucleotide pairs), the quantita- ellite DNA or isolated Hae III restriction fragments and hy- tively predominant fragment, had a dyad symmetry and rep- bridization in situ (21) was as described (12). resented an inverted repeat that could be written into a hairpin Downloaded by guest on October 2, 2021 (1981) %.f-xw Cell Biology: Deumling Proc. Natd Acad. Sci. USA 78 A C A C M * *M M M *M + + 5'- CCCATGCACC GAACCGCCCG CGGCTCGTCC GTGGG G G A C T G G A C T I .,. GG GGGTACGTGG CTTGGCGGGC GCCGAGCAGG CACCC -5' AT K. GG C T 3'- G G G T G C T G CT GGCGC m 5'- C C C A T G A C G A C C GC G C C A low~~~~~~a,_ _0 mc 0* FIG.