The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain glioma cell line U251 while exogenous p53 gene is overexpressed. The constructed cDNA library contained 1.3×106 directional recombinants, and its insert size ranged from 0.5 to 2.0 kb. Screening the cDNA library, we obtained two novel p53 downstream genes, PAP1 and PAP2. Polymerase chain reaction (PCR) analyses of the library for specific genes revealed the presence of cDNAs for p53 downstream genes such as p21, gadd45, and PCNA. These results demonstrate the sequence complexity and relatively low redundancy of our cDNA library. It is a valuable and unique resource for studying p53 gene expression, regulatory mechanisms and screening p53 downstream genes.
Key words: p53 Gene, p53 downstream gene, cDNA library, normalization.
LDPCR, Long distance polymerase chain reaction; MCK, muscle creatine kinase; MDM2, murine double minute 2; PCNA, proliferating cell nuclear antigen; PGM, phosphoglycerate mutase; TIGAR, TP53-induced glycolysis and apoptosis regulator; SCO2, synthesis of cytochrome c oxidase 2; TRE,tetracycline-response element; Dox, doxycycline; FBS, fetal bovine serum; PBS,phosphate buffered saline; DSN, duplex-specific nuclease; ds, double stranded;PCR, polymerase chain reaction.
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