| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
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.
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