Regulation Of Transcription By The Polyomavirus Enhancer And The Adenovirus E1a Gene Products.

Enhancers are cis-acting regulatory sequence elements that increase expression of RNA polymerase II-transcribed genes. To help to define enhancer function, the effect of the polyomavirus enhancer on expression of RNA polymerase III-transcribed genes (the adenovirus VA genes) was tested, using DNA transfection and transient expression assays. The polyomavirus enhancer had little effect upon transcription of VA genes by RNA polymerase III in murine, hamster or human cells. Thus, cis-enhancement of transcription occurs for only specific classes of genes. The polyomavirus enhancer is required in cis for efficient replication of the viral genome, in addition to promoting high level expression of early gene transcripts. The ability of mutants containing enhancers with multiple nucleotide substitutions, deletions or an insertion to promote RNA synthesis or DNA replication was measured. Specific nucleotides were identified that are essential for transcription or replication, including nucleotides 5134, 5135 and 5140 in the BclI-PvuII fragment of the enhancer, and nucleotides 5215 and 5218 in the PvuII-PvuII fragment. Most enhancer mutations altered both transcription and replication in parallel. Significantly, both processes were affected by a change at nucleotide 5140, indicating dependence of transcription and replication upon a common sequence element. One exception was a mutant containing multiple nucleotide substitutions in the enhancer, which replicated at an efficiency similar to that of wild-type, but was deficient in induction of transcription. The difference between RNA and DNA synthesis dependent on the enhancer in this mutant is likely to be quantitative. Taken together these results support the hypothesis that transcriptional and replicative functions are linked in the enhancer of polyomavirus. The trans-acting adenovirus E1A gene products stimulate expression of viral and heterologous RNA polymerase II-transcribed genes. The effect of E1A gene products on expression of RNA polymerase III-transcribed genes (the adenovirus VAI and a bacterial tRNA gene) was assayed. Transcription was stimulated in adenovirus-infected cells or in cells which constitutively produce E1A. Thus, unlike the polyomavirus enhancer, E1A gene products induce expression of both RNA polymerase II- and III-transcribed genes.Ph.D.Biological SciencesMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127994/2/8712071.pd