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G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the monocotyledonous plant, Scilla siberica, by using Ag(+)-Cs(2)SO(4) gradient techniques. This satellite DNA had an unusually high melting point and a high methylcytosine (m(5)C) content ( approximately 25% of total bases; m(5)C/cytosine ratio approximately 1.5) and was localized, by in situ hybridization, in the heterochromatin regions of the chromosomes. Digestion with restriction endonuclease Hae III yielded a series of fragments ranging from 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 sequence 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 m(5)C content (m(5)C/Cyt = 2.2 and 2.8, respectively, for fragments II and III). Identical cleavage fragments were found in satellite DNAs from two other species of this genus (S. amoena and S. ingridae), which suggests that this constitutively methylated sequence is evolutionarily stable. The sequence arrangement of this plant satellite DNA is compared with those reported for several animal satellite DNAs.

Sequence arrangement of a highly methylated satellite DNA of a plant, Scilla : A tandemly repeated inverted repeat