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

Abstract

G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the MOnocotyledonousant, ScitasibeDica, byusing A+-Cs SO4gra-diemnot tedonou is satellite DNA had 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 hybridization, 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 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 m5C content (m5C/Cyt = 2.2 and 2.8, respectively, for fragments II and IEl). 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. Temperature, OCThe nuclear DNA of most, probably all, eukaryotic cells contains fractions of highly reiterated, relatively simple sequences that, in many species, appear as DNA satellites separable from the bulk of the DNA (1). In a variety of animals, the nucleotide sequence arrangement of such satellite DNAs has been elucidated; as a general principle, they contain tandem repeats of a 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 heterochromatin. Among the plants that show conspicuous heterochromatin arrays, the monocotyledonous bluebell species, Scilla siberica, has been studied in detail (9,10,(12)(13)(14). The satellite DNA in the heterochromatin of this plant (9, 10) has been analyzed, and the nucleotide sequence of a part of it has been determined. The results show some unusual features ofsatellite DNA organization-multiple tandem arrays of an inverted repeat, multiple insertions of a tetranucleotide, and remarkably high contents of 5-methylcytosine (m5C)

A Chromosome Phylogeny of the Droseraceae by Using CMA-DAPI Fluorescent Banding.

“…CMA binds specifically to guanine regions in helical DNA (Ward et al 1965) or heterochromatin (Deumling 1981, Deumling andGreilhuber 1982) , while DAPI binds specifically to AT base pairs in the minor groove of DNA (Portugal and Waring 1988) . Therefore, a CMA-positive or DAPI-negative (CMA+DAPI-) segment is high GC-content .…”

DNA methylation in wheat

“…In higher plants the distribution of methylcytosines in the genome has been analyzed in cotton [9], tobacco [lo, 111, melon and cucumber [12, 131, radish [14], flax [15], rice [16], wheat [ll] and the bluebell (Scilla sp.) [17]. Preferential methylation of rRNA genes and of satellite DNA fractions, and developmental changes of the methylation patterns have been observed in several species.…”

Methylation Pattern of Radish (Raphanus sativus) Nuclear Ribosomal RNA Genes

“…It is therefore relevant to investigate the methylation pattern of specific plant genes and compare it with the animal situation. Preliminary observations have suggested that plant rDNA is almost completely methylated at Hpa II sites (15,27,30) and more detailed studies have revealed a complex methylation pattern for flax 5S rRNA genes (17) and for a satellite DNA sequences from Scilla (12). A recent study dealing with flax rDNA has established that some specific Hpa II sites are hypomethylated (14).…”