Molecular analysis of novel PDZ domain-containing gene encoding autoantigen of autoimmune enteropathy

LEC大鼠是一种突变品系，其中肝炎和肝癌以极高的发病率自发发展。使用酵母为基础的分析，得分的比例突变p53基因转录为红色菌落，我们检测到频繁的突变，在LEC大鼠的肝脏。大多数（50-65%）是由额外的腺嘌呤（A）插入引起的移码突变，所有的A插入都发现在含有6个连续腺嘌呤的区域。额外腺嘌呤的插入几乎只在mRNA（cDNA）中发现，特别是在三个（A）6片段（外显子4、7和8）中位于最5 '侧（外显子4）的（A）6片段中，而在相应基因组DNA部分的克隆中很少发现。用SP 6 RNA聚合酶人工合成p53 mRNA，在12个分析的克隆中，有2个克隆在外显子7和1个在外显子8插入了额外的腺嘌呤，但在外显子4没有插入。占转录mRNA的1.6%，这表明腺嘌呤插入外显子4（A）6区是一种体内现象，而不是逆转录中的人工产物。A插入的发生率在急性肝炎期急剧增加，高达约20%，在慢性肝炎期恢复到对照水平，并在肿瘤期再次略有增加。突变率与血清AST水平相关（r = 0.96，p<0.001），而与肝铜和8-羟基脱氧鸟苷含量无关。对培养的肝源性细胞进行乙醇处理也增加了（A）6道处的转录单克隆滑移速率，这共同表明非特异性细胞损伤是转录突变比率增加的原因。这些结果表明，细胞损伤降低了转录保真度，从而进一步增强了细胞功能的损害。

The LEC rat is a mutant strain in which hepatitis and hepatoma spontaneously develop at an extremely high incidence. Using a yeast-based assay which scores the ratio of mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority of them (50-65%) were frameshift mutations caused by insertion of an extra adenine (A) and all the A insertions were found in the regions containing 6 consecutive adenines. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especlally in the (A)6 tract located in the most 5'-side (exon 4) among the three (A)6 tracts (exon 4, 7, and 8), and rarely in the clones of the corresponding genomic DNA part. Artifrcially synthesized p53 mRNA with the use of SP6 RNA polymerase gave insertions of an extra adenine in two clones in exon 7 and one in exon 8, but none in exon 4 out of 1 2 analyzed clones recovered from red colonies; overall accounting for 1.6% of the transcribed mRNA, indicating that the adenine insertion in exon4 (A)6 tract was an in vivo phenomenon rather than an artifact in reverse-transcription. The incidence of A insertion sharply increased up to about 20% in acute hepatitis stage, returned to control level in chronic hepatitis stage, and increased again slightly in neoplastic stage. The mutation rate correlated with serum AST level (r = 0.96, p<0.00 1) but not with contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of cultured liver-derived cells also increased the rate of transcriptlonal slippage at the (A)6 tract, together suggesting that non-specific cellular damage is responsible to the increased ratios of the transcriptional mutations. These results suggest that cellular damage reduces transcriptional fidelity, thereby further enhancing the impairment of cellular functions.