Transcription function of each base pair in the control region of the adenovirus VARNA1 gene.

ABSTRACT

Eighty-seven mutants with single-base substitution in the control region (from -44 to +70) of the adenovirus VARNA1 gene were generated, including nearly every base pair, to examine the role of DNA sequences within this control region for regulating transcription. The effect of these mutations on transcription of the gene was determined in vitro using cytoplasmic S100 extracts from human KB cells. Mutations at -37T, -35A, -29T, -28A, -25C, -18A, -17A, -16A, -13A, -9C, -8C, and -1C in the 5'-flanking region reduced transcription of the gene. Thus two positive regulatory elements, from -44 to -25 and from -18 to +2, interspersed with a putative negative regulatory element were defined. Furthermore, a distinct A-rich purine stretch in the -18 to +2 element was identified. Point mutations in the pyrimidine-rich sequence immediately upstream of the A block promoter element reduced transcription of the gene. Mutations in the GTGG direct repeats of the A block promoter element drastically decreased transcription. Furthermore, mutations that altered the AT-rich sequence immediately downstream of the A block element to become less AT-rich decreased transcription. Mutations of the base pairs at +43C, +45T, and +51A in the inter-block element moderately reduced transcription efficiency of the gene. Mutations at the central four base pairs, GTTC, of the B block palindrome severely affected transcription. These unique sequence motifs and their exact base pairs were proven to be important for regulating transcription of the VARNA1 gene.