Changing of the promater selectivity by p53 point nutations

将含有p5 3突变蛋白表达载体的酵母文库(交配型a)和含有绿色荧光蛋白的酵母文库(交配型α)和含有waf1、mdm2、bax、14-3-3σ、p53AlP1、GADD45、noxA或p53R2启动子的酵母文库(交配型酵母)进行杂交，用定量荧光强度法检测各突变型p5 3蛋白对各启动子的转录激活能力。结果，2,314个突变体被归类为：(1)对所有被检测的8个启动子都有转录活性的突变体(1,278)，(2)对所有启动子都没有转录活性的突变体(400)，(3)与启动子有关的突变体(636)。与国际癌症研究机构(IARC)的p53突变数据库比较，在分类的突变体(1)中，有373个突变体是在临床肿瘤标本中报道的。然而，这些突变的报道频率很低(1-17倍)，这表明还有其他对肿瘤形成重要的靶基因或这些突变是偶然报道的。另一方面，在被归类的突变型(2)中，有39个突变型p53突变型在临床资料中未见报道。这些数据表明，DNA损伤应激对p53基因的影响是不同的。用含有WAF1、MDM2、BAK、14-3-3、σ、GADD45或p53R2启动子的荧光素酶载体，将突变株(3)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，但确实在哺乳动物细胞中观察到了启动子的选择性。推测启动子的选择性来自于点突变改变了P53蛋白的高级结构。

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.