ALTERNATIVE SPLICING OF THE P53 TUMOR-SUPPRESSOR GENE IN THE MOLT-4 T-LYMPHOBLASTIC LEUKEMIA-CELL LINE
ALTERNATIVE SPLICING OF THE P53 TUMOR-SUPPRESSOR GENE IN THE MOLT-4 T-LYMPHOBLASTIC LEUKEMIA-CELL LINE
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DOI：
10.1016/0304-3835(93)90256-9
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发表时间：
1993-09-30
期刊：
影响因子：
9.7
通讯作者：
TOCK, EPC
中科院分区：
文献类型：
--
作者：
CHOW, VTK;QUEK, HH;TOCK, EPC
采用逆转录、聚合酶链反应（PCR）和直接测序技术，研究了p53肿瘤抑制基因在10个人类细胞系（9个肿瘤细胞系和1个正常细胞系）中的表达。使用P53U和P53D引物扩增371碱基对（bp）的目标片段，该片段横跨p53 cDNA外显子7-10，从9个细胞系中扩增出正常大小的PCR产物，但未从Hep3B肝细胞细胞系中扩增出。在Molt-4 t淋巴母细胞白血病细胞系中观察到一个更大的条带（504 bp）。利用P531和P53D引物对76 bp的p53 cDNA片段进行PCR扩增，得到76 bp和209 bp的产物。504 bp和209 bp条带的直接测序证实，在异常转录本的外显子9和10之间存在133 bp的插入。除了2个点突变和3个碱基插入外，该插入与野生型p53内含子9内的130 bp序列同源。Molt-4基因组DNA的P53U/P53D PCR产物测序显示，在133 bp插入的下游有8 bp的缺失，在内含子9内创建了一个新的供体剪接位点。该位点与133 bp插入上游的固有受体剪接位点相结合，表明133 bp的拉伸代表了另一个外显子。在这个替代转录本中终止信号的发生预计最终会导致截断的p53翻译产物。Molt-4、HT-1080、SiHa、CaSki、HeLa和MRC-5细胞系的371 bp PCR产物序列与野生型p53 cDNA相符。在Mahlavu和PLC/PRF/5 HCC细胞系中检测到p53密码子249第三碱基的G -> T突变，而在Raji Burkitt淋巴瘤细胞系的一个等位基因中观察到密码子234位点的TAC到CAC突变。
The expression of the p53 tumor suppressor gene in ten human cell lines (nine cancers and one normal) was studied using reverse transcription, polymerase chain reaction (PCR) and direct sequencing. Using P53U and P53D primers for amplifying a 371-base pair (bp) target fragment spanning exons 7-10 of p53 cDNA, normal-sized PCR products were amplified from 9 cell lines but not from the Hep3B hepatocellular carcinoma (HCC) cell line. An additional larger band (504 bp) was observed for the Molt-4 T-lymphoblastic leukemia cell line. Employing P531 and P53D primers which flank a 76-bp p53 cDNA fragment, 76 bp as well as 209 bp products were generated by PCR of Molt-4 cDNA. Direct sequencing of the 504 bp and 209 bp bands confirmed the presence of a 133 bp insertion between exons 9 and 10 in the aberrant transcript. This insertion was homologous to a 130-bp sequence within the wild-type p53 intron 9, except for 2 point mutations and 3 base insertions. Sequencing of P53U/P53D PCR products of Molt-4 genomic DNA revealed an 8 bp deletion just downstream to the 133 bp insertion, creating a novel donor splicing site within intron 9. This site, coupled with an inherent acceptor splicing site just upstream to the 133 bp insertion, suggests that the 133 bp stretch represents an alternative exon. The occurrence of a termination signal within this alternative transcript is predicted to culminate in a truncated p53 translational product. The sequences of the 371 bp PCR products of Molt-4, HT-1080, SiHa, CaSki, HeLa and MRC-5 cell lines corresponded with the wild-type p53 cDNA. G --> T transversions at the third base of codon 249 of p53 were detected in Mahlavu and PLC/PRF/5 HCC lines, while a TAC to CAC mutation at codon 234 was observed in an allele of the Raji Burkitt lymphoma line.