An Amplification-Promoting Sequence from Mouse Genomic DNA: Interaction with a Trans -Acting Factor That Also Affects Gene Expression

Abstract

We previously identified murine DNA sequences that stimulate the amplification of cis-linked plasmid DNA in mouse cells under selective conditions (Hoist et al., 1988). Here we focus on the structural features of one of these elements, the 229-bp element 5. The amplification-promoting activity was fully recovered from the middle part of element 5. The active region interacted in a sequence-specific way with a protein from nuclear extracts. Using footprinting analyses the binding region was characterized and subsequently shown to be functionally active as an amplification-promoting sequence, whereas a mutated binding site was inactive. Therefore, cis-acting element 5 functioned via interaction with a trans-acting factor. The same binding site was also active as a promoter element for RNA polymerase II transcription, because it efficiently reconstituted the activity of a truncated herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene promoter lacking the distal Spl binding site. Thus, the same protein seems to function in both RNA polymerase II transcription and DNA amplification. Possible relationships between both functions are discussed.