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 TnS 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.

Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.

“…The importance of these sequences in the control of polyoma early gene activity and viral DNA replication is clearly documented. However, it is not known whether late gene expression also depends on enhancers, since a significant level of viral late expression during lytic infection or in virus-transformed cells requires extrachromosomal virus DNA replication (16,35,53). Replication is dependent on the expression of the early gene for the viral large T antigen (3,21,53) and, as noted above, requires a functional enhancer element (14).…”

“…Also shown are regions duplicated, deleted, and mutated in naturally occurring variants of polyomavirus (49) or in variants capable of growth in F9 (20,50) (51). scripts have heterogeneous initiation sites (11,35,54) (15) boxes that do form part of the polyoma early transcription unit (29,30). We considered it therefore of interest to determine whether the same sequences within the regulatory region regulate the levels of both early and late transcription.…”

A distinct basic fibroblast growth factor (FGF-2)/FGF receptor interaction distinguishes urokinase-type plasminogen activator induction from mitogenicity in endothelial cells.

“…GM Transfections were carried out with the calcium phosphate precipitation protocol (Sambrook et al, 1989). CHO cells were transfected with 91023b-flg, 91023b-bek, MomFR3SV, or LTR2HX in association (10:1, wt:wt) with pCB7 (Kern and Basilico, 1985) and selected with 250 ,tg/ml of G418 (Sigma). Both CHO and GM 7373 cells were transfected with pCEP4-flg 1.2 and selected with 500 or 200 ,tg/ml of hygromycin B (Boehringer Mannheim GmbH, Mannheim, Germany), respectively.…”

Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae.

“…(ii) An enhancer region, originally defined as the 244 bp between the BcII (bp 5021) and PvuII (bp 5264) restriction enzyme sites (17,81), has been subdivided into two unrelated but functionally redundant elements, enhancer A and enhancer B, both of which are capable of stimulating transcription of heterologous genes in an orientation-and position-independent manner (42). Studies with gel retention and nuclease mapping assays have identified several cellular protein factors that bind specifically to the B enhancer (7,26,70 (9,38) and in vivo (2,32,40) and that at least some of the elements of the polyomavirus early promoter, including portions of the enhancer, may regulate the levels of transcription from the late promoter of that virus (8,13,(52)(53)(54).…”