Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line Y79.
Molecular mechanism of retinoblastoma gene inactivation in retinoblastoma cell line Y79.
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视网膜母细胞瘤细胞系Y79中视网膜母细胞瘤基因失活的分子机制。
DOI：
10.1073/pnas.85.16.6017
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发表时间：
1988
影响因子：
11.1
通讯作者：
Lee,WH
中科院分区：
文献类型：
--
作者：
Lee,EY;Bookstein,R;Young,LJ;Lin,CJ;Rosenfeld,MG;Lee,WH
视网膜母细胞瘤是一种发生在儿童视网膜的癌症，它的形成是由分子克隆的常染色体基因(Rb)的突变失活决定的。虽然所有正常组织和许多肿瘤细胞都表达4.7kb的Rb基因，但6个视网膜母细胞瘤中有6个要么缺乏Rb基因表达，要么Rb转录本长度异常(缩短)。为了进一步研究后一种类型的突变，我们选择了视网膜母细胞瘤细胞系Y79，它表达了大约4.0kb的缩短的Rb mRNA。从Y79基因文库中分离到的Rb基因克隆在5‘端的内部缺失了470个核苷酸，这与外显子2-6的缺失相对应。含有缺失连接的基因组克隆是从Y79 DNA文库中分离出来的，这使得对内含子1和6的缺失终点进行了精确的定位和测序。这些区域之间没有明显的同源性，也没有与Alu重复序列家族的明显同源性，这意味着缺失可能是通过同源序列重组以外的机制发生的。外显子2-6的缺失会打断Rb mRNA的开放阅读框架，导致翻译提前终止。由于特异性抗体免疫沉淀法未检测到正常的Rb蛋白，Y79细胞中另一个看似正常的Rb等位基因必然被不同的突变灭活。
Formation of retinoblastoma, a cancer arising in the retinas of young children, is determined by mutational inactivation of an autosomal gene (RB), which has been molecularly cloned. Whereas all normal tissues and many tumor cells express an RB mRNA of 4.7 kilobases, six of six retinoblastomas were previously found either to lack RB gene expression or to have RB transcripts of abnormal (reduced) length. To further characterize the latter type of mutation, we chose to examine retinoblastoma cell line Y79, which expressed a shortened RB mRNA of about 4.0 kilobases. RB cDNA clones isolated from a library constructed with Y79 mRNA demonstrated an internal loss of 470 nucleotides near the 5' end, which corresponded to a deletion of exons 2-6. Genomic clones containing the deletion junction were isolated from a library made with Y79 DNA, which allowed precise localization and sequencing of deletion endpoints in introns 1 and 6. These regions had no apparent homology to each other or to the Alu family of repetitive sequences, implying that the deletion must have occurred by a mechanism other than recombination of homologous sequences. Deletion of exons 2-6 would interrupt the open reading frame in RB mRNA and would result in premature termination of translation. Since no normal RB protein was detected by immunoprecipitation with specific antibody, the other, apparently normal RB allele in Y79 cells was necessarily inactivated by a different mutation.