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

LEC大鼠是一种突变株，在这种突变株中，肝炎和肝癌的自发性发病率极高。使用基于酵母的检测方法，将p53基因转录本中的突变比例作为红色菌落进行评分，我们在LEC大鼠的肝脏中检测到频繁的突变。大多数(50%~65%)是由于插入额外的腺嘌呤(A)而引起的移码突变，所有的A插入都发生在连续6个腺嘌呤的区域。在3个(A)6区(外显子4、7和8)中的(A)6区(A)6区(外显子4、7和8)中，几乎不存在额外的腺嘌呤插入。用SP6RNA聚合酶人工合成p53基因，在外显子7的两个克隆和外显子8的一个克隆中插入了额外的腺嘌呤，但在12个红色克隆中的外显子4中没有插入额外的腺嘌呤，总共占转录mRNA的1.6%，表明在外显子4(A)6区域插入腺嘌呤是一种体内现象，而不是逆转录的伪迹。A插入率在急性肝炎时急剧增加，达20%左右，在慢性肝炎时恢复到对照水平，在肿瘤期略有增加。LEC大鼠的突变率与血清AST水平呈正相关(r=0.96，p&lt；0.00 1)，而与肝组织中铜和8-羟基脱氧鸟苷的含量无关。乙醇处理培养的肝源性细胞也增加了(A)6区转录滑移的速率，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的保真度，从而进一步加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.