No abstract

This study defines a DNA sequence upstream from the mRNA cap site required for in vivo expression of the adenovirus 2 pre-early region. The adenovirus 2 pre-early region and flanking sequences were cloned in Escherichia coli plasmid pBR322. Derivatives of the plasmid lacking portions of the upstream viral DNA sequence were constructed. An assay was devised to test the ability of these plasmid DNAs to complement an adenovirus 5 mutant with a deletion in the pre-early region. Plasmids that retained at least 38 base pairs upstream from the mRNA cap site had complementing activity similar to that of the original plasmid, which contains 229 base pairs of upstream viral sequence. However, plasmids retaining 23 or fewer base pairs of viral sequence upstream from the cap site had significantly reduced complementing activity. These results indicate that a portion of the adenovirus 2 sequence between 23 and 38 base pairs upstream from the mRNA cap site is required for expression of the pre-early region. This interval includes the GoldbergHogness box-like sequence T-A-T-T-T-A-T-A.In eukaryotes and prokaryotes, transcription begins at specific regions on template duplex DNAs (1-7). In prokaryotes, DNA sequences in the immediate vicinity of start sites regulate transcription initiation (6, 7). However, in eukarvotes, the locations of DNA sequences that regulate mRNA transcription initiation have not been determined. One feature common to most eukaryotic genes whose sequences have been determined is an A+T-rich sequence closely resembling T-A-T-A-A-A-T-A (the Goldberg-Hogness box) centered approximately 27 base pairs upstream from the site coding for the first nucleotide of the mature mRNA (cap site; reviewed in ref. 8). The ubiquity and location of this sequence suggest that it may play a significant role in transcription initiation, but its precise role has not been defined. In this work we determine the upstream DNA sequence required for the expression of one eukaryotic transcriptional unit, adenovirus 2 (Ad 2) early-region IA (9, 10). Our approach was to clone Ad2 early-region IA, devise an assay for the expression of this cloned viral DNA in human cells, construct derivatives of the clone that have deletions in the upstream viral sequence, and test the expression of these altered templates in vivo. Our results indicate that the viral DNA sequence between 23 and 38 base pairs upstream from the mRNA cap site, including the Goldberg-Hogness box, is required for efficient expression of this eukaryotic transcriptional unit. However, even deletions that extend to 62 nucleotides into the transcribed region allow a low level of gene expression.Ad functions encoded in early-region IA are required for production of other early mRNAs (in a low-multiplicity infection; refs. 11 and 12) and are, therefore, called pre-early functions. In Ad2, significant amounts of two spliced mRNAs encoded in this region are produced during the early phase of infection (13-15) (Fig. 1). These mRNAs have identical 5' and 3' sequences encoded i...