coccidiosis vaccine iminent, page-10

re: earlier success holds key The region of the fowl adenovirus serotype 10 (FAV-10) genome containing the major late promoter (MLP) and leader sequences was determined and appropriate genomic fragments were cloned and sequenced. A TATA box was identified and the location of the putative transcription start site was determined. By using synthetic primers from the transcription start site in conjunction with oligonucleotides from the coding regions of the penton base and hexon genes, cDNA was produced from late mRNA isolated from cell cultures infected with FAV-10 at 24 h post-infection. The resulting cDNA was cloned and sequenced and the leader sequences thus identified. It was found that the FAV-10 MLP utilized only two leader sequences (a bipartite leader). By comparison with human adenoviruses (HAVs) it appeared that the second leader in HAVs was absent from the FAV-10. The second leader sequences of FAV-10 was larger than either the second or third leaders of HAVs, but was 29 basepairs shorter than the combined size of the leader sequences 2 and 3 from HAV-2. To confirm the transcription start site and leader sequences, single stranded cDNA was produced from mRNA using the primers from within the coding sequence for the penton base or hexon. A tail of dGTP's was added and cDNA synthesis was completed using an oligonucleotide from within the hexon or penton base coding sequence and a second poly-dCTP oligonucleotide. Sequencing of the resultant G-tailed DNA confirmed the location of the transcription start site as an adenosine residue 24 basepairs upstream from the 3-prime (3') end of the TATA box. Sequencing 5' of the TATA box failed to reveal any sequence similarity with the human adenovirus upstream stimulatory factor (USF). Various plasmids were constructed which placed the determined sequences of the MLP, leader, and the region upstream of the TATA box linked to the co-acetyl acid transferase (CAT) gene. These expression plasmids in transient expression assays of CAT activity in primary chicken kidney cell culture with or without FAV-10 co-infection were determined. These experiments showed that the cassette containing sequences 5' of the TATA box expressed CAT to a much greater level than cassettes not containing this upstream region and that the presence of virus significantly increased the activity of the promoter following the onset of viral DNA replication. Without the 5' region, cassettes failed to express above background levels. These results suggest that the basic structure of the fowl adenovirus MLP is similar to that of the human adenovirus although it utilizes a bipartite rather than a tripartite leader sequence.