Tight DNA-protein Complexes in Barley Seedlings are Formed by GC-rich Sequences Enriched in Guanine Quadruplexes

Bulk genomic DNA was extracted according to the protocol of a chloroform-isoamylic alcohol extraction with some modifications. Plant tissues were frozen in liquid nitrogen and grinded in a mortar up to a fine powder. The powder was suspended in 25 ml of the pre-warmed (65ºC) extraction buffer (100 mM Tris HCl pH7.5; 500 mM NaCl, 50 mM Na2EDTA, 1.25% SDS, 3.8 g/l Na bisulfite). The mixture was incubated for 30 minutes at 65ºC in the same buffer with occasional shaking. DNA was extracted with the same volume of chloroform/isoamilic alcohol mixture (24:1), the suspension was centrifuged, the water phase was separated. RNA was precipitated with concentrated LiCl solution (up to 4M with subsequent incubation for 1 h on ice). The sample was centrifuged to pellet the RNA. DNA was precipitated with 1 volume of butoxyethanol. DNA was (1) digested with Hind III and Pst 1 restrictases or alternatively with Alu I restrictase. The solution was pressed through nitrocellulose filter pre-soaked with filtration buffer supported in Swinnex filter holde. The filter retained DNA fraction enriched in the tightly bound proteins. The DNA content in filtered fraction (F), low-ionic strength eluted fraction (R1) and alkali-eluted fraction (R2) was measured spectrophotometrically. Cloning for sequencing was performed with pooled F fraction DNA (FF), coleoptile R1 and R2 fraction DNA extracted from the coleoptiles, leaves and roots (CR1, CR2, LR1, LR2, RR1, RR2 fractions respectively). (2) DNA was digested with Dnase I. To obtain the residual DNA fragments the digest was incubated with Proteinase K and SDS and deproteinized with chloroform. Remnants of DNA were precipitated with ethanol. DNA fragments were obtained from coleoptiles, first leaves and roots, fractions were called Cd, FLd and Rd respectively. The sequencing reaction was performed with primer M13 21uni and 5 ml of the purified product. Sequencing was performed on the MegaBace 1000 Sequencing System (Amersham Biosciences, Piscataway, USA). All sequences obtained were compared with those in the database using BLAST. Sequences without complementation and longer than 30 bp are submitted.