FUNCTIONAL MUTATION OF P53 GENE DETECTED BY NOVEL YEAST ASSAY IN THE STOMACH OF MONGOLIAN GERBIL INFECTED WITH H.P

Abstract

Aim:Helicobacter pylori (Hp) is a definite carcinogen in human gastric cancer and is proven to induce gastric cancer in Mongolian gerbil (MG) infected with H. pylori. Major disadvantage in MG model is to lack specific reagents and genetic informations for investigation. In this study, we cloned p53 gene of MG and established a functional mutation assay by using yeast system and detected it in the stomach of MG infected with H. pylori.Methods:1) RNA was extracted from MG liver and p53 cDNA was cloned by RT‐PCR and RACE methods. 2) cDNA of p53 in the stomach, intestine and other tissues were amplified by RT‐PCR and was transfected into yeast IG397 with pLSGP53 vector. After transfection, cDNA of p53 was ligated in the vector by homologous recombination and produced p53 protein in yeast. 3) The stomach of MG infected with H. pylori up to 142 weeks was studied.Results:1) p53 protein of MG had 76.2% homology to human p53, 61.4% in N‐terminal, 87.8% in DNA binding domain and 72.2% in C‐terminal. 2) Functional mutation rate (red yeast colony) of p53 in stomach, intestine, liver, brain without H. pylori infection were less than 10%. 3) Two of 16 stomachs infected with H. pylori showed significantly high percentage of red colony, 117 week‐infected MG. 4) p53 cDNA with red yeast colonies derived from 106 week‐infected MG stomach was sequenced and they showed clonal mutation.Conclusion:One of 16 stomachs infected with H. pylori demonstrated clonal mutation of p53 and lost the transcriptional activity of wild type p53. This novel assay is useful for detecting a functional mutation of p53 of MG infected with H. pylori