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

腺病毒即时早期基因ElA是一个病毒转录单元，首先在生产感染中激活，ElA产品刺激其自身基因和病毒基因组上其他早期基因区域的转录。我们利用dna酶I足迹法在Ad5 EIA基因上游定位了21个HeLa细胞核蛋白的结合位点。我们发现ElA增强子的两个不同元件（先前由其他组定义）结合具有a /CGGAA/TGT结合特异性的共同因子（称为ElA因子：EIA-F）。利用西南克隆技术，从HeLa细胞mRNA的lambdagtll cDNA文库中分离出一个可能编码ElA-F的lambda3-16克隆。竞争凝胶迁移实验和甲基化干扰实验表明，lamda3 -16噬菌体的溶原产生了对ElA增强子具有结合特异性的蛋白。因此，flambda3-16的CDNA可能编码ElA增强结合因子。核苷酸序列分析表明，该cDNA序列与人类ETS -1和ETS -2癌蛋白的ETS结构域具有同源性（62%）。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 blot杂交结果显示，在多种人类细胞系（KB、HeLa、HT1080和MG63细胞）中均存在一种长度为2.5kb的mRNA。该mRNA在Ad5感染早期增加了3 ~ 5倍。一个新分离的ets致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.