Gonsky R, Knauf J A, Elisei R, Wang J W, Su S, Fagin J A
Division of Endocrinology and Metabolism, University of Cincinnati School of Medicine, Cincinnati, OH 45267-0547, USA.
Nucleic Acids Res. 1997 Oct 1;25(19):3823-31. doi: 10.1093/nar/25.19.3823.
The mRNAs of transiently expressed proteins such as cytokines and proto-oncogenes are commonly subject to rapid transcriptional activation and degradation. Transcript turnover is determined in part by association of certain proteins with consensus AU-rich motifs (AUUUA) in the 3'-untranslated region of the transcripts. Here we report a modification of differential RNA display (DRD) to detect differentially expressed rapid turnover mRNAs containing AU-rich motifs from thyroid cancer tissues and cell lines. RNA of normal and thyroid cancer tissues was differentially displayed using a 3'anchor primer to the poly(A) tail and an arbitrary 5'primer incorporating an AUUUA sequence. The appropriateness of the strategy was established by its ability to display known early response genes, such as c- fos, using partially degenerate primers. To test whether the novel cDNAs isolated coded for transcripts subject to rapid turnover, they were used as probes for Northern blots of RNA from clonal human thyroid carcinoma cell lines treated for varying periods with either cycloheximide or actinomycin D. A number of novel differentially expressed cDNA fragments were isolated from human papillary thyroid carcinoma tissues, among them a cDNA with zinc finger motifs and homology to other zinc finger proteins. Using this fragment to probe a cDNA library, a full-length cDNA (ZnF20) was isolated that was 4333 bp in length and contained an open reading frame of 1029 amino acids. The ZnF20 cDNA hybridized to multiple transcripts in a thyroid cancer cell line (8.0, 4.5 and 2 kb) that increased after cycloheximide treatment and decayed <2 h after addition of actinomycin D. The ZnF20 mRNA was overexpressed in three of six thyroid papillary carcinomas as compared with paired normal thyroid tissue controls. The data presented here support the use of a targeted DRD approach for the isolation of rapid turnover mRNAs, many of which may be interesting candidate oncogenes.
诸如细胞因子和原癌基因等瞬时表达蛋白的信使核糖核酸（mRNA）通常会经历快速的转录激活和降解。转录本的周转部分取决于某些蛋白质与转录本3'非翻译区中富含AU的共有基序（AUUUA）的结合。在此，我们报告了一种差异RNA显示（DRD）方法的改进，用于检测来自甲状腺癌组织和细胞系的含有富含AU基序的差异表达快速周转mRNA。使用与聚（A）尾结合的3'锚定引物和包含AUUUA序列的任意5'引物对正常和甲状腺癌组织的RNA进行差异显示。通过使用部分简并引物显示已知的早期反应基因（如c-fos）的能力，确定了该策略的适用性。为了测试分离出的新cDNA是否编码快速周转的转录本，将它们用作探针，对来自克隆人甲状腺癌细胞系的RNA进行Northern印迹分析，这些细胞系用放线菌酮或放线菌素D处理不同时间。从人甲状腺乳头状癌组织中分离出许多新的差异表达cDNA片段，其中一个具有锌指基序且与其他锌指蛋白具有同源性的cDNA。用该片段探测cDNA文库，分离出一个全长cDNA（ZnF20），其长度为4333 bp，包含一个1029个氨基酸的开放阅读框。ZnF20 cDNA与甲状腺癌细胞系中的多个转录本杂交（8.0、4.5和2 kb），这些转录本在放线菌酮处理后增加，在添加放线菌素D后<2小时衰减。与配对的正常甲状腺组织对照相比，ZnF20 mRNA在六个甲状腺乳头状癌中的三个中过表达。本文提供的数据支持使用靶向DRD方法分离快速周转的mRNA，其中许多可能是有趣的候选癌基因。
