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.

批量基因组DNA（bulk genomic DNA）的提取参照经改良的氯仿-异戊醇（chloroform-isoamyl alcohol）提取方案进行。将植物组织置于液氮中速冻，随后在研钵中研磨至细腻粉末状。将粉末悬浮于25 mL预热至65℃的提取缓冲液中，该缓冲液成分为：100 mM Tris-HCl（pH 7.5）、500 mM NaCl、50 mM 乙二胺四乙酸二钠（Na₂EDTA）、1.25% 十二烷基硫酸钠（SDS）以及3.8 g/L 亚硫酸氢钠（Na bisulfite）。将混合液置于65℃水浴孵育30分钟，期间间断振荡混匀。采用等体积的氯仿/异戊醇混合液（体积比24:1）进行DNA抽提，随后离心悬浮液并分离水相。使用终浓度达4M的浓氯化锂（LiCl）溶液沉淀RNA，将混合物置于冰上孵育1小时后，通过离心收集RNA沉淀。随后用1倍体积的丁氧基乙醇沉淀DNA。（1）使用限制性内切酶（restriction enzyme）Hind III与Pst I对DNA进行酶切，或单独使用限制性内切酶Alu I进行酶切。将酶切后的溶液通过经过滤缓冲液预浸的硝酸纤维素滤膜（nitrocellulose filter），滤膜搭载于Swinnex滤器支架（Swinnex filter holder）中，滤膜将富集紧密结合蛋白的DNA组分截留。采用分光光度法测定过滤组分（F）、低离子强度洗脱组分（R1）以及碱洗脱组分（R2）中的DNA含量。测序克隆所用的DNA样本包括：混合过滤组分DNA（FF）、从胚芽鞘（coleoptile）中提取的胚芽鞘来源R1、R2组分DNA，以及从叶片、根系中提取的对应组分DNA，分别记为CR1、CR2、LR1、LR2、RR1、RR2。（2）使用脱氧核糖核酸酶I（DNase I）对DNA进行酶切。为获取残留的DNA片段，将酶切产物与蛋白酶K（Proteinase K）及SDS共同孵育，随后通过氯仿抽提去除蛋白质。用乙醇沉淀回收残留的DNA。从胚芽鞘、第一片真叶以及根系中分别获取DNA片段，对应的组分依次记为Cd、FLd与Rd。以M13 21uni为引物，取5 mL纯化产物进行测序反应。测序工作在MegaBace 1000测序系统（MegaBace 1000 Sequencing System，安玛西亚生物科学公司，美国皮斯卡塔韦）上完成。使用BLAST工具将所有获得的序列与数据库中的序列进行比对，最终仅提交无互补序列且长度大于30 bp的序列。