Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

Abstract

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.