Large numbers of random point and cluster mutations within the adenovirus VA I gene allow characterization of sequences required for efficient transcription.

We have isolated clones with well over 100 randomly dispersed point mutations distributed throughout the 5' half of chemically synthesized adenovirus type 2 VA I genes. In addition, we have isolated clusters of mutations targeted to the regions corresponding to the A and B block consensus sequences of eukaryotic tRNA and adenovirus VA genes. In vitro analyses of these constructs have allowed us to survey in detail the importance of DNA sequence to transcriptional efficiency. Our analyses demonstrate that certain constructs with radically substituted A block regions can be transcribed efficiently. In contrast, there is little tolerance for variation in the sequence within the B block region. We propose that the B block sequence should be R-G-A/T-T-C-R-A-N-N-C for optimal transcriptional efficiency of the VA I gene in mammalian cells.