Sjakste Tatjana, Leonova Elina, Petrovs Rudolfs, Trapina Ilva, Röder Marion S, Sjakste Nikolajs
Genomics and Bioinformatics Group, Institute of Biology, University of Latvia, Riga, Latvia.
Faculty of Medicine, University of Latvia, Riga, Latvia.
PeerJ. 2020 Feb 18;8:e8569. doi: 10.7717/peerj.8569. eCollection 2020.
The concept of chromatin domains attached to the nuclear matrix is being revisited, with nucleus described as a set of topologically associating domains. The significance of the tightly bound to DNA proteins (TBP), a protein group that remains attached to DNA after its deproteinization should be also revisited, as the existence of these interactions is in good agreement with the concept of the topologically associating domain. The work aimed to characterize the DNA component of TBP isolated from barley seedlings.
The tight DNA-protein complexes from the first leaves, coleoptiles, and roots of barley seedlings were isolated by purification with chromatography on nitrocellulose or exhaustive digestion of DNA with DNase I. Cloning and transformation were performed using pMOSB Blunt Ended Cloning Kit. Inserts were amplified by PCR, and sequencing was performed on the MegaBace 1000 Sequencing System. The BLAST search was performed using sequence databases at NCBI, CR-EST, and TREP and Ensembl Plants databases. Comparison to MAR/SAR sequences was performed using http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi database. The prediction of G quadruplexes (GQ) was performed with the aid of R-studio library pqsfinder. CD spectra were recorded on a Chirascan CS/3D spectrometer.
Although the barley genome is AT-rich (43% of GC pairs), most DNA fragments associated with TBP were GC-rich (up to 70% in some fractions). Both fractionation procedures yielded a high proportion of CT-motif sequences presented predominantly by the 16-bp CC(TCTCCC) TC fragment present in clones derived from the TBP-bound DNA and absent in free DNA. BLAST analysis revealed alignment with different barley repeats. Some clones, however, aligned with both nuclear and chloroplast structural genes. Alignments with MAR/SAR motifs were very few. The analysis produced by the pqsfinder program revealed numerous potential quadruplex-forming sites in the TBP-bound sequences. A set of oligonucleotides containing sites of possible GQs were designed and ordered. Three of them represented the minus strand of the CT-repeat. Two were derived from sequences of two clones of nitrocellulose retained fraction from leaves and contained GC-rich motifs different from the CT motif. Circular dichroism spectroscopy revealed profound changes in spectra when oligonucleotides were incubated with 100 mM KCl. There was either an increase of positive band in the area of 260 nm or the formation of a positive band at 290 nm. In the former case, changes are typical for parallel G-quadruplexes and, in the latter, 3 + 1 structures.
The G-quadruplexes anchor proteins are probably involved in the maintenance of the topologically associated domain structure.
附着于核基质的染色质结构域的概念正在被重新审视，细胞核被描述为一组拓扑相关结构域。与DNA紧密结合的蛋白质（TBP）的重要性也应重新审视，TBP是一组在DNA脱蛋白后仍与DNA结合的蛋白质，因为这些相互作用的存在与拓扑相关结构域的概念高度契合。这项工作旨在表征从大麦幼苗中分离出的TBP的DNA成分。
通过在硝酸纤维素上进行色谱纯化或用DNase I彻底消化DNA，从大麦幼苗的第一片叶子、胚芽鞘和根中分离紧密的DNA-蛋白质复合物。使用pMOSB平端克隆试剂盒进行克隆和转化。通过PCR扩增插入片段，并在MegaBace 1000测序系统上进行测序。使用NCBI、CR-EST和TREP以及Ensembl Plants数据库中的序列数据库进行BLAST搜索。使用http://smartdb.bioinf.med.uni-goettingen.de/cgi-bin/SMARtDB/smar.cgi数据库与MAR/SAR序列进行比较。借助R-studio库pqsfinder预测G-四链体（GQ）。在Chirascan CS/3D光谱仪上记录圆二色光谱。
尽管大麦基因组富含AT（GC对占43%），但与TBP相关的大多数DNA片段富含GC（某些组分中高达70%）。两种分级分离程序都产生了高比例的CT基序序列，主要由存在于源自TBP结合DNA的克隆中且不存在于游离DNA中的16 bp CC(TCTCCC)TC片段呈现。BLAST分析显示与不同的大麦重复序列比对。然而，一些克隆与核结构基因和叶绿体结构基因都比对上了。与MAR/SAR基序的比对非常少。pqsfinder程序的分析揭示了TBP结合序列中众多潜在的四链体形成位点。设计并订购了一组包含可能的GQ位点的寡核苷酸。其中三个代表CT重复序列的负链。另外两个源自叶片硝酸纤维素保留组分的两个克隆的序列，包含与CT基序不同的富含GC的基序。圆二色光谱显示，当寡核苷酸与100 mM KCl孵育时，光谱发生了深刻变化。要么在260 nm区域正带增加，要么在290 nm形成正带。在前一种情况下，变化是平行G-四链体的典型特征，而在后一种情况下，是3 + 1结构的典型特征。
G-四链体锚定蛋白可能参与维持拓扑相关结构域的结构。
