Lien vers Pubmed [PMID] – 6329714
EMBO J. 1982;1(8):895-900
Eucaryotic expression vectors containing two selective markers, the herpes simplex 1 thymidine kinase gene (tk) and the Escherichia coli gpt gene (Eco gpt) coding for a xanthine-guanine phosphoribosyl-transferase ( XGPRT ) were constructed. These plasmids were used to transfect mouse Ltk- cells followed by selection of either tk+ or XGPRT + colonies. The transcription and maturation of the Eco gpt mRNA is dependent on the presence of a eucaryotic promoter sequence at its 5′ end and on the presence of a viral intron and a poly(A) addition site at its 3′ end ( Mulligan and Berg, 1980). Here, we report that both simian virus 40 (SV40) and polyoma early and late promoters permit the transcription of this gene integrated into the cellular genome. Polyoma DNA fragments lacking the TATA box, or both the TATA and CAAT boxes directing early transcription, efficiently promote Eco gpt expression. Furthermore, a fragment terminating approximately 300 nucleotides upstream of the initiation site of early RNA permits Eco gpt synthesis when the early strand is joined to the Eco gpt-coding strand. The SV40 early promoter is 2- to 3-fold more efficient than the controlling sequence of late transcription. These results strongly suggest that the switch from a predominance of early RNA to that of late RNA occurring after the onset of DNA replication is caused by the increase in the abundance of template and by the concomitant repression of early transcription by the T antigen. The presence of the tk gene in all the plasmids constructed permits the analysis of Eco gpt expression as a non-selected marker in tk+ clones selected for growth in HAT medium.(ABSTRACT TRUNCATED AT 250 WORDS)