Construction Of Recombinant Plasmids And The Bioinformative Analysis Of Binding Sites Of Transcription Factors Of POLH, I, K Gene In Mammalian Cells

Alkylating agents especially the N-Nitroso alkylating agents are widely present in the environment and DNA alkylation seems to be a common event. The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a model chemical of N-Nitroso alkylating agent is a mutagen/carcinogen that targets cellular DNA and proteins to generate adducts. Among the adducts, O~6-alkyl guanine is the predominantly mutagenic lesion because of its mispairing properties, and these adducts can eventually lead to chromosomal aberrations, point mutations, and cell death. This lesion also appears to be involved in tumor initiation, particularly in gastric carcinogenesis.In our laboratory, a unique mutation detection system using a shuttle vector plasmid has been established to demonstrate that a low concentration of MNNG (0.2 μM) can induce nontargeted mutation in mammalian cells: the mammalian cells were exposed to 0.2μM MNNG for 2.5 h, then a shuttle plasmid pZ189 carrying supF tRNA gene was transfected into cells after 24h culture. We found a 5-fold higher mutation frequency of the plasmid replicated in pretreated cells than the spontaneous mutation frequency of the plasmid replicated in control cells. This kind of mutation did not occur immediately after MNNG exposure. Time-course analysis showed that the frequency of MNNG induced nontargeted mutation increased gradually, reached the peak at 12 h after MNNG treatment, and then declined. Thespecific nontargeted mutation spectrum is different from that of targeted mutation, whereas the mutation occurs at damaged DNA site.We have found that low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) led to the decrease of DNA replication fidelity and the upregulation of POLB of X family DNA polymerase and POLZ(REV3) of B family DNA polymerase in cultured human amnion FL cells. And our publishing data showed that the expression of POLE and POLI were increased to MNNG exposure, as well POLK was decreased. When POLK was blocked by anti-sense mRNA, the level of spontaneous mutation frequency increased significantly. The blockage of POLZ(REV3) and POLE can inhibit MNNG induced untargeted mutagenesis.To study the mechanism of transcription regulation and the activity of transcription factors response in cell, we used bioinformatic analysis to predict the promoters and putative binding sites for different transcription factors in the trancription regulation region of these three polymerases. Promoters were predicted by online software such as Promoterlnspector> PromoterScanN Promoter 2.0 and so forth. Furthermore, we obtained putative transcription factor binding sites on the three predicted promoters by phylogenetic footprinting and online software Match 1.0. Promoter fragments of POLE, POLI and POLK were amplified by PCR from genome DNA, and inserted into the pSeap-Basic reporter vector. It was planned to measure the response of reporter gene expression driven by indicated promoter sequence to low concentration MNNG exposure, after transfecting the recombinant plasmids into the culture FL cells.1. Pridiction of promoter and transcription factor binding sites of POLH, POLI, and POLK with the methods of bioinformatics.With the completion of genome sequence of human and several other animals, and with the development of computer aid analysis metheds, bioinformatics has been widely used in biological research. Exact expression of gene depends on a complex regulation system, and the relationship of transcript factors and their binding sites is very important in this system. With the stimulation of different factors or signals,they will activate or inhibit the transcription and bring defferent expression. So, to understand the regulation of transcription of the gene, we have to confirm the TF binding sites. Mutation is largely affected by environmental factors, and some of them are lethal, and those mutations will be cleared in the evolution. Thus, more and more non-functional bases accumulated, as they are harmless. We can conclude that highly conserved squences always have function. People analyse such case with DNAse I footprinting, which named phylogenetic footprinting by Tagle. Here we analyze the obtained conserved sequence, and we obtained promoter regions in it. Furthermore, we pridict TF binding sites in these regions with bioinformatics methods. Establishment of orthologous relationships between human and mouse genesThe mRNA sequences of the genes, POLE, POLI and POLK were extracted from the GenBank database. Each of the human mRNA sequences was BLASTed against mouse mRNA sequences and the top hit was BLASTed back to the original human mRNA database. If the top human mRNA hit is the same as the original human mRNA, we assumed an orthologous relationship between the human and mouse genes. To minimize the redundancy, only human mRNA in GenBank RefSeq section was used to establish any human-mouse ortholog relationships. Transcript mapping and construction of ortholog promoter relationshipTo ensure that the 5' end of a mRNA is close to the transcription initial site, the mRNAs of any gene were aligned by Clustal W and the 5' end position was identified by the alignment of the 5' end of the cDNA with the genome sequence by MegaBLAST. For each human or mouse gene, sequences assumed to contain promoters are fetched by taking the 1000 bp upstream and 2000bp downstream sequence of the transcriptional start site, as identified by the transcript mapping procedure. The promoters of the human genes were testified by promoter prediction software, such as Promoterlnspector, PromoterScan, and so on, and the human-mouse ortholog promoter relationship was established thereafter. Comparative promoter TF she analysisFor each human—mouse orthologous promoter pair, DBA software was used toextract the conserved blocks of nucleotide sequences using the default parameter settings with the assumption that conserved regulatory regions might be important for regulation of the gene expression (refer to p56, appendix 1). The promoter sequences were then checked for TF binding sites by running MATCH against TRANSFAC TF binding site matrices. The output file from MATCH was parsed and stored in Excel tables. Matrix similarity scores range from 0.0 to 1.0. Matrix similarity scores of 0.8 were used as cutoff scores in the analyses and a matrix similarity score of 0.8 was considered to be significantly high. Only TF site matches that exist in the conserved blocks of both human and mouse promoters within 1000 bp upstream and 2000bp downstream of the 5' end of a specific gene were extracted as ultimate output consequence. Then, the common TF binding sites in the promoter regions of these genes were sought out.The putative TF binding sites and their sequences in the promoter regions of human POL H, I and K gene were listed as table 3 in p34.2.Constructing the recombinant reporter plasmids driven by POLH, POLI, POLK promoterWith the designed primers, we amplified the predicted promoter regions of human POL Ht /and K gene by PCR from extracted genomic DNA. After collecting the purified PCR products, they were inserted into the pGEM-T vector, then E. coli DH5 ct were transformed with these recombinant plasmid DNAs. Sequencing report from Huada Gene Company confirmed our predicted result. The promoter sequences harvested from the recombinants by endonuclease cleavage were then inserted into the corresponding polycloning site of pSeap-basic reportor vector.Three pSeap-basic recombinant reportor plasmid driven by POLH, POLI, POLK promoter respectively were successfully constructed in this study..