Cloning And Sequence Analysis Of DNA Polymerase β Gene Of Three Cell Lines

BACKGROUND: DNA polymerase was identified in Escherichia coli by Komberg firstly in 1956. In prokaryote and microorganism it was found sequently later. DNA polymerase is a large family. So far at least 14 kinds of DNA polymerases were found in eukaryote, named polymerase α,β ,γ ,δ, ε, ζ, η, θ,ι ,κ ,λ , μ, REV1 and MUS308. They play an important role in many courses of biologic metabolizability about replication, restoration, recombination of DNA and the control of cell cycle. Weissbach reported that DNA polymerase β is a kind of low molecule protein with a single peptide chain in the moggy thymus gland cells, and it consist in mammiferous nucleolus widely. The coding gene of human cell polymerase β consist in 8P12-P11. It is 35kb long and composed of 14 exons and 13 introns. DNA polymerase β is considered as housekeeping gene and it is highly conservative from yeast to mammalian cell. The primary structure of DNA polymerase β in human and rodent is a multipeptide consisted of 335 amino acide. At the present time it is the minimum polymerase admittedly. To the pathogenesis of malignant tumor , the research previously concentrated on the mutation about the tumor susceptibility gene and the tumor suppressing gene, but the original molecule vice which lead these gene mutation accumulated to tumor is unknown clear so much. With the development of molecular biology, people found that the mutation of polβ gene in the colon cancer, rectum cancer, prostate cancer, stomach cancer, mammary cancer, lung cancer, kidney cancer, bladder cancer and oesophagus cancer one after the other.Then the relationship between polβ gene and tumor catch people's attention gradually. The research indicated that DNA polymerase β in mammalianparticipate in some courses of DNA's synthesization and restoration. They can supervise the error of DNA, excise the scathing DNA segment, restore the gap of DNA, ensure the DNA's stability and prevent the cell mutation which arose from the gene mutation and the tumor's resultant. The purpose of this study is to clone and analyse the sequence of DNA polymerase {3 gene of three cell lines EcalO9, Eca9706 and NIH3T3, supply a credible evidence and investigation background for further research of the relationship between pol 3 and cancer.METHODS: The total RNA from the three cell lines of human esophageal cancer about EcalO9, Eca9706 and NIH3T3 were extracted, and amplified the whole sequence of polp by reverse transcriptase-polymerase chain reaction (RT-PCR). The gene was linked into pGEM —T Easy plasmid and the recombinant pGEM —T Easy- poip was transformed into JM109.By using of Amp fastness and blue/white color screening the positive colonies of transformed bacteria were selected. The primer designed according to T7/SP6 sequence of pGEM—T Easy plasmid was applied to identify the linking correctness. Identified positive clone was sent to do the sequencing and compared with the corresponding sequence in the GeneBank . RESULTS:1 .Amplification of target gene and enzyme cutting identification: the full length segments of pol 3 cDNA segments from these three cell lines was amplified by RT-PCR and were identified by the enzyme cutting with EcoR I , the results was correct. 2.Result of identification of clone:Target gene linked with plasmid pGEM — T Easy. Then recombinant was transformed into JM109. After blue-white selection, 6 positive colonies were selected at random to undergo the PCR amplification with primer T7/SP6. The amplified fragments at 1008bp long were obtained from all samples.3.As compared with the corresponding sequence in the GeneBank, there were two mutations in the EcalO9 cell line at no.71 from Glu to Gly and no.203 from Glu to Gly,and there were four mutations in the Eca9706 cell line at no.203 from Glu to Gly,no.219 from Gin to nonsense mutation,no.230 from Lys to Glu and no.247 fromGlu to Gly,but there was no any mutations in the NIH3T3 cell line. CONCLUSIONS: This test is successful in cloning the cDNA of DNA polymerase pGene of three cell lines about EcalO9, Eca9706 and NIH3T3 .Found that there were some point mutations in the EcalO9 and Eca9706 cell lines,but there was not any mutation in the NIH3T3 cell line by analyzing the cDNA sequence of DNA polymerase P gene about these three cell lines.