| Objective: Tumor development involves a multistage cascade of events progressing from benign adenoma to malignant carcinoma, and it seems to be the consequence of accumulation of mutation of protooncogenes and tumor suppressor genes such as p53, DCC, or APC. Spontaneous mutation rate appears to be insufficient to account for the observed multiple mutation. The highly protective cellular DNA repair system could remove most of the damage or mutation in the genome and thus would be able to prevent tumorigenic alterations. A genetic error of defective DNA repair has been implicated in the causation of xeroderma pigmentosum, a rare genetic disease.To examine the potential defect in DNA repair system related to esophageal carcinoma, DNA polymerase 13 (polp) has been chosen to investigate the relationship between the alterations in this enzyme and human esophageal carcinoma, polpis a member of DNA polymerase family which has ten members. The Mr 39000 polp (355amino acids) is well-characterized nuclear protein, known to be involved in chemically induced DNA damage, such as base exercion " repair (BER). The mDNA sequence and genomic sequence of human pol have been reported. The enzyme has two domains: the NH2-terminal segent (about 75amino acids ) with a strong affinity for DNA terminal, and the COOH-terminal segment (about 250amino acids) amino acids) with polymerase activity. The pol P gene is localized on chromosome 8p. Another objection was to build some eukaryotic expression vector which involves classic mutation type observed above for further research of the enzymatic function.Materials and Methods: Twenty fresh esophageal carcinoma tissues were obtained from the People's hospital of Linzhou city, and were frozen by liquid nitrogen immediately and then stored at -80癈. Corresponding normal epithelial tissues were obtained at the same time. All the tumors were diagnosed as infiltrative carcinoma. Two pieces of six-months-old embryonic esophagus used as a normal control were obtained by induction of labor with water bag method from the First Appendicular hospital of Zhengzhou university and the Women and Children's hospital of Zhengzhou.Total RNA was isolated by Total RNA Extraction kit, and 2 u g RNA was used for reverse transcription with a oligo d(T) primerdesigned according to the sequence of polpmRNA. The first strand of complementary DNA(cDNA) was directly used 35 cycles of polymerase chain reaction(PCR) amplification, using Taq polymerase with seven pairs of primers designed according to seven different tandem fragments in the sequence of polpmRNA. The products of PCR were checked for mutation by single strand conformation polymorphism (SSCP). Then the mutated samples were chosen, and their cDNAs were directly used 35 cycles of PCR amplification with a pair of primers designed according to the original and terminal fragment sequence of polpmRNA. The wild pol(3RNA was also amplificated.The products were directly ligated to PUC18 plasmid vector and transfected into JM109 Escherichia coli cells. Plasmid DNAs with polnsertion were islated by QIAGEN tip 20 mini plasmid purify kit and sequenced by DNA sequencer.Following the same method, plasmids vr1012 with polinsertion were built as eukaryotic expression vector. However, recombinated pcDNA3 plasmid vectors were not built that method. The inserted polp fragments were not PCR products but were obtained by digestion and purification from the PUC 18 plasmid DNAs with pol(3insertion.Result: there was no mutation in EE1,EE2. But obvious mutation was detected by SSCP in 9 of 20 esophageal infiltrative cancer tissues (with mutation rate 45%) and 1 of 20 cancer corresponding normal tissue. Within 4 esophageal cancer specimens, 58bp (177nt to 234nt) deleted mutation of polpwas founded. There were 8 point mutation forms: (1) A-G at 375nt (Ile-Val) , (2) T-C at 454nt (Phe-Ser) , (3) G-T at 462nt (Glu-terminal code) , (4) G-A at 466nt (Gly-Glu) , (5) A-T at 613nt (Lys-Ile) , (6) G-C at 648nt (Gly-Arg) , (7) A-G at 660nt (Arg-Gly)(8) A-Gat... |
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