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全基因组捕获被致癌信号抑制的编码分泌蛋白和跨膜蛋白的基因。

Genomewide trapping of genes that encode secreted and transmembrane proteins repressed by oncogenic signaling.

作者信息

Gebauer M, von Melchner H, Beckers T

机构信息

ASTA Medica AG, Department of Cancer Research, University of Frankfurt Medical School, Frankfurt am Main, Germany.

出版信息

Genome Res. 2001 Nov;11(11):1871-7. doi: 10.1101/gr.202601.

DOI:10.1101/gr.202601

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC311167/

Abstract

A retroviral gene trap containing a human CD2 cell surface antigen/neomycin-phosphotransferase fusion gene in the U3 region of its LTR (U3Ceo) was used to screen the mammalian genome for genes encoding secreted and/or transmembrane proteins that are repressed by oncogenic transformation. From an integration library consisting of cells transformable by the insulin-like growth factor 1 (IGF-1), a collection of neomycin resistant (Neo(R)) clones was obtained; 86% also expressed the CD2 cell surface antigen. Molecular analysis of a random sample of Neo(R) clones revealed that the U3Ceo gene trap preferentially disrupted genes coding for secreted and transmembrane proteins. In each case, the signal sequence of the endogenous gene was fused in-frame to the CD2/neomycin-phosphotransferase reporter gene due to a cryptic splice acceptor site embedded in the coding region of the CD2 cDNA. When the library was transformed by IGF-1 and selected against CD2 expression, integrations were obtained in genes that are repressed by transformation. Molecular analysis of six randomly chosen integrations revealed that, in each case, U3Ceo captured a signal sequence from proteins involved in oncogenic transformation and metastatic spread.

摘要

一种逆转录病毒基因捕获载体，其长末端重复序列（LTR）的U3区域含有一个人CD2细胞表面抗原/新霉素磷酸转移酶融合基因（U3Ceo），用于筛选哺乳动物基因组中编码被致癌转化抑制的分泌蛋白和/或跨膜蛋白的基因。从由胰岛素样生长因子1（IGF-1）可转化的细胞组成的整合文库中，获得了一组新霉素抗性（Neo(R)）克隆；86%的克隆也表达CD2细胞表面抗原。对Neo(R)克隆的随机样本进行分子分析表明，U3Ceo基因捕获载体优先破坏编码分泌蛋白和跨膜蛋白的基因。在每种情况下，由于CD2 cDNA编码区中嵌入了一个隐蔽的剪接受体位点，内源基因的信号序列与CD2/新霉素磷酸转移酶报告基因读框融合。当文库用IGF-1转化并针对CD2表达进行筛选时，在被转化抑制的基因中获得了整合。对六个随机选择的整合进行分子分析表明，在每种情况下，U3Ceo都从参与致癌转化和转移扩散的蛋白质中捕获了一个信号序列。