|Budget Amount *help
¥6,800,000 (Direct Cost : ¥6,800,000)
Fiscal Year 1994 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1993 : ¥5,100,000 (Direct Cost : ¥5,100,000)
The adenovirus early region 1A (E1A) gene plays a central role in productive infection. The E1A gene is expressed immediately after infection, and E1A gene products activate transcription of other early genes through trans-activation function. Focusing on host cell factors which modulate expression of the E1A gene itself, we have identified a cellular E1AF protein bound to E1A enhancer sequences located upstream of the E1A gene. Here, we have isolated genomic DNA and cDNA clones encoding E1AF protein to analyze structure and function of the E1AF gene. Also, roles of the E1AF gene in productive infection of adenovirus have been studied.
E1AF was found to be a new human ets gene family member because of the ETS domain present in the C-terminal region.Analysis of N-terminal deletion mutants revealed transcriptional activation domain in the acidic amino acid- and glutamine-rich regions. Four genomic DNA clones, which carry the C-terminus, ETS domain and glutamine-rich region, were isolated
from a human genome library, and exon/intron structure of the E1AF gene was partially determind.
During early times of infection, amounts of 1.5kb E1AF mRNA were increased. Cotransfection with the E1AF gene resulted in 5 fold increase of E1A promoter activity. The E1AF gene was expressed in a variety of cells while not in human breast cancer MCF7 cells. To examine whether E1AF affects production of virus, we isolated MCF7 cells stably expressing the exogenously introduced E1AF gene. These cells were infected with virus, and viral mRNA and DNA were measured at the early and late times of infection, respectively. Unexpectedly, E1A mRNA and viral DNA were synthesized at the similar level in MCF7 cells and E1AF-expressing MCF7 cells. Possibly, small amounts of endogenous E1AF in MCF7 may give rise to an initiation of E1A gene transcription and subsequent replication of viral DNA.Using transdominant negative mutants of the E1AF gene lacking transcriptional activation domain, we are currently investigating whether these constructs suppress virus production. Less