Changing of the promater selectivity by p53 point nutations

将含有p53突变蛋白表达质粒的酵母文库（配种类型a）与含有含有WAF1、MDM2、BAX、14-3-3σ、p53AlPl、GADD45、NOXA或p53R2启动子的GFP或DsRed报告质粒的酵母文库（配种类型α）进行配种，并通过定量荧光强度测定每个p53突变蛋白对每个启动子的转录激活能力。结果，2314个p53突变体被分类如下：(1)对相当于野生型p53的所有8个启动子都具有完全转录活性的突变体（1,278）；(2)对相当于普通p53突变体的所有启动子都没有转录活性的突变体（400）；(3)对启动子具有变异活性的突变体（636）。将所得结果与国际癌症研究机构（IARC） p53突变数据库进行比较，在分类(1)的突变体中，临床肿瘤样本中报道了373个p53突变体。然而，这些突变的报道频率很低（1-17次），这表明还有其他靶基因对肿瘤形成很重要，或者这些突变是偶然报道的。另一方面，在分类的突变体(2)中，有39个p53突变体未在临床资料中报道。这些数据表明DNA损伤应激对p53基因的影响是不同的。最后，将分类为(3)的一些突变体用含有WAF1、MDM2、BAK、14-3-3σ、GADD45或p53R2启动子的荧光素酶质粒导入SaOs2细胞。p53突变体的启动子选择性并不总是与酵母系统中观察到的数据一致，但在哺乳动物细胞中确实观察到启动子选择性。提示启动子的选择性来自于p53蛋白的点突变引起的高层结构的改变。

The yeast library (mating type a), containing p53 mutant protein expression plasmid accompanied by all the 1 amino-acid substitution resulting from 1 nucleotide substitution, and the yeast (mating type α), containing GFP or the DsRed reporter plasmid which has WAF1, MDM2, BAX, 14-3-3σ, p53AlPl, GADD45, NOXA, or p53R2 promoter, were mated and the transcriptional activation ability of each p53 mutant protein towards each promoter was measured by quantified fluorescence intensity. As a result, 2,314 of p53 mutants were classified that as follows;(1) mutants which have fufl transcriptional activities towards all examined 8 promoters equivalent to wild type p53 (1,278),(2) mutants which have no transcriptional activities towards all promoters equivalent to common p53 mutants (400),(3) mutants which have variation activities depends on the promoters (636).Comparing the obtained results with p53 mutation data base of International Agency for Research on Cancer (IARC), among the mutants classified (1), 373 of p53 mutants were reported in clinical neoplasm samples. However, reported frequency of these mutants was low (1-17 times), it suggests that there are other target genes important for neoplasm formation or these mutations are accidentally reported. On the other hand, among the mutants classified (2), 39 of p53 mutants were not reported in clinical materials. These data suggest that the DNA damaging stress influences differently on p53 genes. Finally some mutants, which classified (3), were introduced into SaOs2 cell with luciferase plasmids containing WAF1, MDM2, BAK, 14-3-3σ , GADD45, or p53R2 promoter. The promoter selectivity of p53 mutants did not always coincident with the data observed in yeast system, but indeed the promoter selectivity was observed in mammalian cell. It is suggested that the promoter selectivity comes from changing higher structure of p53 proteins due to point mutation.