Preliminary Characterisation of the Adenovirus Type 40 E1A Region

To investigate the involvement of the E1A region in the restricted growth of Ad40, a preliminary characterisation of sequences which were important for basal and trans-activated transcription was undertaken. Comparison of the intact Ad40 E1A promoter with the intact Ad5 E1A promoter in transient transfection assays, revealed that basal transcription from the Ad40 E1A promoter was lowered by approximately 6 fold when compared to basal transcription from the Ad5 E1A promoter. To identify sequences important for basal transcription within the Ad40 E1A promoter, a series of promoter deletions were constructed using Bal 31 nuclease digestion, revealing a region between -349 to -140, relative to the cap site at +1, to be important for basal transcription from the Ad40 E1A promoter. Comparison of the Ad40 E1A promoter with the transcription factor database, held at EMBL (Ghosh, 1990), revealed that this region contained a number of possible transcription factor binding sites similarly arranged to the Ad5 E1A promoter. To map sequences within the region -349 to -140, a series of deletions were constructed which deleted transcription factor binding sites known to be important for basal transcription from the Ad5 E1A promoter. The deletions were characterised in parallel with the intact and Bal 31 deletion mutants of the Ad40 E1A promoter by transient transfection assays, which revealed that the Ad40 E1A transcriptional control region contains an enhancer element located between -328 to -235 relative to the Ad40 E1A cap site at +1 similar to that in the Ad5 E1A promoter (Hearing and Shenk, 1983a, 1986; Bruder and Hearing, 1989, 1991). To investigate whether the Ad40 E1A 249R (equivalent to the Ad5 E1A 13S mRNA which encodes a 289R protein) and 22IR (equivalent to the Ad5 E1A 12S mRNA which encodes a 243R protein) proteins were involved in the aberrant expression of the Ad40 E1A region, a cDNA equivalent to the Ad5 E1A 13S mRNA was generated by RT-PCR of Ad40 infected cell extracts, then cloned into a CMV expression plasmid. An Ad40 equivalent to the Ad5 E1A 12S mRNA was not generated by RT-PCR of Ad40 infected cell extracts However, a library of Ad40 E1A specific cDNAs were generated, and four novel Ad40 E1A specific cDNAs were characterised. Comparison of trans-activated transcription from the Ad40 E1A promoter by the Ad40 E1A 249R protein and the Ad5 E1A 289R protein, revealed that trans-activated transcription from the Ad40 E1A promoter was approximately 6 fold lower in the presence of the Ad40 E1A 249R protein. To map sequences which were important for trans-activated transcription from the Ad40 E1A promoter, cells were cotransfected with either the intact or deleted Ad40 E1A promoter and the Ad5 E1A 289R protein or the Ad40 E1A 249R E1A protein, revealing a region between -328 to -235, relative to the Ad40 E1A cap site at +1, to be important for Ad5 E1A 289R activated transcription from the Ad40 E1A promoter. Ad40 E1A 249R activated expression for both the Ad5 and Ad40 E5A promoters was not discussed, due to an equipment (incubator) problem which rendered the experimental data unreliable. Comparison of the ratios of Ad5 E1A 289R activated expression to basal expression within the Ad5 and Ad40 promoter constructs indicated that no single sequence element could be identified which was uniquely important for trans-activated expression. Rather those elements which affected the level of basal expression seemed to have an effect on the level of trans-activated expression observed in the presence of the Ad5 E1A 289R protein. Thus the Ad5 E1A 2S9R protein probably activates gene expression through the basal transcription apparatus, as demonstrated by the Ad5 E1A promoter. (Abstract shortened by ProQuest.)