The Structure and Function of Adenovirus ElA Enhancer-Binding Protein.

腺病毒立即早期基因ELA是一种病毒转录单位，首先在生产性感染中被激活，ELA产品刺激自身基因和病毒基因组上其他早期基因区域的转录。通过DNase I足迹分析，我们将21个HeLa细胞核蛋白结合位点定位在Ad5EIA基因的上游区域。我们发现，先前由其他组定义的ELA增强子的两个不同元件与A/CGGAA/TGT的结合特异性结合了一个公共因子(称为ELA因子：EIA-F)。利用西南克隆技术，从HeLa细胞mRNA的lambdagtll基因文库中分离到一个可能编码ELA-F的lambda3-16克隆。竞争凝胶迁移率漂移实验和甲基化干扰实验表明，lambda3-16噬菌体的溶原能产生与ELA增强子特异结合的蛋白。因此，Oflambda3-16的CDNA似乎编码ELA增强子结合因子。核苷酸序列分析表明，该基因与人Ets-1和Ets-2癌蛋白的Ets结构域有62%的同源性。Ets结构域长约90个氨基酸，负责序列特异性结合，并与Ets癌蛋白家族共享。我的结论是，EIA-F是ETS癌蛋白家族的新成员。Northern印迹杂交显示，在不同的人类细胞系(KB、HeLa、HT1080和MG63)中都有一种长度为2.5kb的mRNA。在Ad5感染早期，该基因的表达增加了3~5倍。一个新分离的ETS癌基因的转录意义目前正在研究中。将进行比较分析，以确定ETS转录因子的细胞水平是否与腺病毒的宿主范围相关。

The adenovirus immediate early gene, ElA, is a viral transcription unit first activated in productive infection and ElA products stimulate transcriptions from its own gene and other early gene regions on the viral genome. We mapped 21 binding sites of HeLa cell nuclear proteins on the upstream region of the Ad5 EIA gene by using DNase I footprint assay. We found that two different elements of the ElA enhancer, previously defined by other groups, bound a common factor with the binding specificity of A/CGGAA/TGT(called ElA factor : EIA-F). By the south-western cloning technique, I have isolated a lambda3-16 clone possibly coding for ElA-F from the lambdagtll cDNA library of HeLa cell mRNA. The lysogen of lambda3-16 phage produced the protein with the binding specificity to the ElA enhancer, as revealed by the competition gel mobility-shift assay and methylation interference assay. Thus the CDNA oflambda3-16 seems to be encoding ElA enhancer-binding factor. Nucleotide sequence analysis of the cDNA showed a homology(62% identity)in amino acid sequences with the ETS domain of human ets-1 and ets-2 oncoprbteins. The ETS domain is about 90 amino acid long, responsible for the sequence-specific binding and shared with the ets oncoprotein family. I concluded that EIA-F is a new member of the ets oncoprotein family. Northern blot hybridization indicated a single species of mRNA with 2.5kb length in various human cell lines(KB, HeLa, HT1080, and MG63 cells). This mRNA increased 3 to 5 folds during early times of Ad5 infection. Transcriptional significance of a newly isolated ets oncogene is currently under-investigated. Comparative analysis will be performed to determine whether a cellular level of the ets transcription factor correlates with the host-range of adenovirus.