Functional anatomy of the simian virus 40 late promoter.

Abstract

We have examined the control sequences for the late promoter function of simian virus 40 (SV40) in COS-1 cells which produce SV40 T antigen constitutively. Plasmids were constructed by cloning mutant late promoter segments upstream from sequences coding for the bacterial chloramphenicol acetyltransferase (CAT) gene, and were converted to "double-origin" type by inserting functional replication origin segments downstream from the CAT gene for replicative competence when necessary. The late promoter activity was determined by transient expression assay of the CAT mRNA and enzyme activity levels following DNA-mediated gene transfer into COS-1 cells. We find that the minimal replication origin and the 21-bp repeat containing T antigen and transcription factor Sp1 binding sites, respectively, are dispensable for late promoter function provided that one copy of the 72-bp repeat enhancer is present. We have mapped within the 72-bp repeat the major late promoter component in a 68-bp fragment (located between nucleotides 205 and 272), and found an overlapping 55-bp fragment (located between nucleotides 179 and 234) to have about one-fifth of the late promoter activity. Both the 68- and 55-bp fragments lack some of the core sequence elements required of the 72-bp repeat for transcriptional enhancer activity, and lack the ability to enhance the activity of the SV40 early promoter. The results suggest that the organization of functional units of the 72-bp repeat required for transcriptional enhancement of the early promoter is different from that required for late promoter function. The 21-bp repeat was found to have some late promoter activity located within the origin-distal copy in the absence of the 72-bp repeat. In association with the 21-bp repeat, the otherwise dispensable origin-proximal 22-bp of the 72-bp repeat containing activator protein AP-1 binding site augmented late promoter activity by three- to fourfold.