Nucleic Acids Research, 1987, Vol. 15, No. 20 8293-8303
© 1987
Articles |
Large numbers of random point and cluster mutations within the adenovirus VA I gene allow characterization of sequences required for efficient transcription
1Department of Pathology, University of North Carolina Chapel Hill, NC 27514, USA 2Department of Microbiology and Immunology, University of North Carolina Chapel Hill, NC 27514, USA 3Program in Molecular Biology and Biotechnology, University of North Carolina Chapel Hill, NC 27514, USA 4The Curriculum in Neurobiology, University of North Carolina, Chapel Hill, NC 27514, USA
*To whom correspondence should be addressed at: Department of Pathology, CB 7525, University of North Carolina, Chapel Hill, NC 27514, USA
Received July 17, 1987. Revised September 25, 1987. Accepted September 25, 1987.
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.