Screening And Cloning Of The Random MARs Of Human Genome And Study Of It's Functions

Background and objectives:With the sketch of human genome having been finished and the genomics developed extensively, the studies of gene expression and regulation already have beening changed from spot and line regulation of the individual gene to the regulation network of the three-dimensional polygene, gene cluster and whole genome. In the process of genetic regulation, the interaction between trans-acting factors and cis-acting elements constructs the network foundation of gene expression and regulation.Matrix attachment regions (MARs) are DNA fragments which can bind with the nuclear matrix in eukaryotic genome. MARs can make chromatin form loop domains and regulate DNA replication, RNA synthesis and hnRNA machining with other cis-acting elements and trans-acting factors together and have important roles in regulation of gene expression. In addition, the inserted MARs can eliminate the sensitivity of the transgene to the integrated sites and the effect of cis-acting elements around of inserted sites, and then improve the efficiency and stability of the transgene expression in transfected cells in the study of the genetic engineering, which may make the gene therapy to be applied successfully in clinic. Nowadays, in the S/MARDB library, there are only 117 of MARs from the human, but in human genome there are more than hundred thousands of MARs needed to be furtherly investigated. Therefore, this study tries to isolate and screen random MARs from human genome and establish a random MARs library, then these fragments which have the characteristics of MARs identified by binding assay in vitro are screened to sequence and analyze by bioinformatics methods in order to prognosticate the functional structure of these MARs and study the actions and functions of MARs in human geneome furtherly and provide new theoretic evidences to understand the rules and molecular mechanisms of MARs in the expression regulation of eukaryotes gene deeply.Methods:1. Screening and cloning of the random MARs and identification of these MARS:(1) Screening and isolation of the random MARs: The random MARs of human genome were screened and isolated from CD3+-Jurkat cells of T lymphocyte clone by these characteristics that MARs binded with nuclear matrix can resist to DNase L high concentration of salt.(2) Construction of MARs library: The extracted MARs were filled up and inserted into the PUC19 vector. The recombination plasmids were transformed into the bacteria and then the positive clones were screened.(3) Preparation of nuclear matrixes (NM): The cultured cells were collected and nuclei were isolated. Nuclear matrixes were prepared from the isolated nuclei which were processed by DNase U high concentration of salt.(4) Identification of MARs: The positive recombination plasmids were identified by PCR and digesting test. The bioactivity of MARs which bind with nuclear matrixes were identified by the binding assay and agarose gel electrophoresis.(5) Sequencing of MARs: These MARs which can bind with nuclear matrixes in vitro were sequenced.2. Relative bioinformatics analysis of MARs(1) Analysis of the specific motifs of MARs: The specific motifs of MARs were analyzed by MAR-Wiz (http://www.futuresoft.org/modules/Mar Finder/index.html, MAR-Wiz 1.0) software.(2) Forecast of the biding sites of transcription factor in MARs: The biding site of transcription factor in MARs were analyzed and forecasted by the TFSEARCH software of the transcription factor binding site profile database ( http://www.cbrc.jp/ research/db/TFSEARCH.html) (version 1.3) of the Japanese Bioinformatics Research Institute.(3) Homologous comparing of the isolated MARs in the human genome library and locating in chromosome of genome: These relative analysis were completed by the BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/) of American National Bioinformatics Center.Results and Conclusions:LA large number of random MARs have been isolated and extracted from human T lymphocyte by these characteristics that MARs binded with nuclear matrix proteins canresist to DNase L high concentration of salt and the first random MARs library consisted of 238 MARs clones with the sequence length of 100 bp to 3kb also has been established constructed successfully.2.The 12 human MARs which yet were not reported in the S/MARtDB library have been first isolated and sequenced successfully. The results of binding assay have indicated clearly that these isolated MARs have the bioactivity which can bind with nuclear matrix proteins in vitro. Then , The sequenced results of random 12 MARs which have higher affinity with nuclear matrix have shown that the length of the fragments of the isolated MARs were between 100bp~3kb with the content of A+T between 52-66%, all which are consistent with the rules of the base sequence constitutes of MARs. Moreover, the affinity between MARs and nuclear matrix is not accordant with the A+T content of MARs.3.The results of bio-informatics blasting have shown that most of 12 MARs obtained from the human geneome are homologous with the sequence in human genome. 9 MARs fragments whose homology were more than 99% were located in lq22^ 7pl2>20ql3> Ip22> 19ql2. Ip22^ 20qlK 4pl4> 18p 11 respectively.4.The functional structure motifs of MARs in expression and regulation of the eukaryote gene have been prognosticated by the bioinformatics methods successfully. These results of bioinformatics analysis have indicated that the same one MAR not only contains A C and A T A T motifs, but also includes many origins of transcription/replication, enhancer, curved DNA, kinked DNA regions and numerous reverse repeated sequences. The potential ability binding with nuclear matrixes proteins are different with the potential value more than 0.95 or very lower in different MARs. The binding sites of transcription factors in every MAR are richer so that the binding sites of transcription factors are more than 10 in a sequence of several hundreds base-pairs.5.Any one of the obtained MARs includes many different cis-acting elements and can react to different transcription factors at one time, which indicates that the functions and roles of MARs hi gene expression regulation are complicated and multiform and may be worth to be investigated furtherly.