Study Of P53 And DNA Damage Repair

Study of P53 and DNA damage repairObjective:P53 in response to DNA damage is a hot area under research. This study try to (1) Establish a set of p53 truncated mutants and analyse these mutants' effects on expression of p53-related genes in reponse to DNA damage;(2)Analyse DNA damage and apoptosis in Hela cell under ultraviolet light radiation, especially analyse the expression of p53 and downstream related genes in UV treated cells; and(3)Clone those p53 most related genes.Methods:(1) Using PCR-base mutagensis method, a set of truncated mutants were amplified from wild type p53 gene cDNA by use of 7 primers' cross-pairing. All mutated gene were sequenced and subcloned into mammalian cell expression vector pcDNA3.1 or Adenovirus transfer vector. These recombinant expression plasmids were used to tranfect Hela cell and stable expression cell lines were established by using O418 selection. The expression of truncated p53 mutants and their effects on other p53-related genes were analysed by RT-PCR in Hela cells. (2) Hela cells treated with ultraviolet light for different time were analysed DNA damage and apoptosis, and p53 and related genes expression were also analysed in those treated cells. (3) Using RT-PCR method to clone some of most important p5 3-related gene such as mdm2, p21, pi6, gadd45 and bax.Results:(1) Twelve clones of p53 and its mutated gene were cloned by PCR-based mutagensis method. All clones were sequence confirmed. Then, these genes were subcloned into mammalian cell expression vector pcDNAS. 1. The mutated genes were inserted in pcDNA3. 1 with two orientation. Those positive clones were used to transfect Hela cell and stable cell lines expressing transfected gene were established by G418 selection.(2) All transfected cell lines were analysed for gene expression, especially the effects of these mutated genes on the expression of p53-related downstream genes were analysed.(3) A set of recombinant adenovirus plasmids harboring these p53 mutated truncated genes were also established. They should be convenient with further analysis in a variety of cell lines.(4) Hela cells treated with ultraviolet light were analysed for DNA damage and apoptosis. When treated with UV radiation for 15 min or longer most Hela cell behave serious DNA damage and apoptosis were induced. In those cells p53 and its mostrelated gene such as p21, gadd45, bax were highly expressed but with no detectable mdm2 gene expression, indicated that cell were very likely apoptosis-induced under the situation.(5) Those p53-related gene, such as mdm2, p21, pi6, gadd45 and bax gene were cloned using RT-PCR method from UV-treated Hela cells. Hela cell lines stably expressing mdm2 and p21 were also established by using gene transfer method.Conclusion:A set of P53 mutated gene were cloned by PCR-based mutagenesis, and stably transfected Hela cell lines carrying these mutated p53 genes were established. Their exprssion and their effects on other p53 related genes expression were analysed in Hela cell. The effects of ultraviolet light on Hela cell DNA damage and cell apoptosis were also performed, especially the expression of p53 and p53 related genes were analysed in this condition. Some of the most p53 related genes were cloned. These work setup a solid base for further research on p53 in response to cell DNA damage.