Ultraviolet mapping of the adenovirus 2 early promoters

Abstract

HeLa cells were infected with adenovirus 2 (Ad2) virions irradiated with different doses of ultraviolet light. The concentration of early viral mRNA accumulating over 8 hr in the cytoplasm of infected cells was found to fall as a function of ultraviolet dose, and the inhibition displayed first-order kinetics. Transcripts from each of the four early regions of the viral genome exhibit differing sensitivities to ultraviolet light. For three of the four early regions, the rate constant for the ultraviolet-induced inhibition of mRNA production is proportional to the physical size of the genome represented as mRNA in the cytoplasm. DNA extracted from irradiated virions was digested with restriction enzymes, and the fraction of each restriction fragment containing one or more ultraviolet-induced pyrimidine dimers was determined by treating the DNA with T4 ultraviolet endonuclease and assaying the products on alkaline agarose gels. In this manner, the rate of pyrimidine dimer formation per nucleotide was found to be approximately constant throughout the genome. From these data and the previously mapped positions of the early regions, we conclude that the mRNAs coded in each of the four early regions are transcribed from separate promoters. For three of the four early regions, the promoter is within 1000 nucleotides of the 5′-most sequences found in the cytoplasm. In the case of the fourth early region, the initial transcript may be approximately 3 times the size of the transcripts found in the cytoplasm, or transcription of this region of the genome may be influenced by one or more of the early gene products. The concentration of early mRNAs observed in cells mixedly infected with unirradiated and irradiated virions suggests that initiation of transcription is the rate-limiting step in the production of the early mRNAs, and that this rate is independent of the number of viral templates within the nucleus.