A negative regulatory sequence near the mouse beta-maj globin gene associated with a region of potential Z-DNA

The possible regulatory role of DNA sequences situated 5' to the beta-maj globin gene was investigated by two types of assay. First, a long term transformation assay was used to measure the efficiency of transformation of TK- mouse (LATK-) and hamster (BHKTK-) fibroblast cells with DNA molecules made by covalently linking mouse and human DNA fragments to the herpes simplex virus (HSV-1) thymidine kinase (tk) gene in the plasmid pTK1. When the promoter regions from the mouse beta-maj globin or the human epsilon-globin genes are substituted for the viral promoter in the tk gene transformation occurs with 10-20% of the efficiency of the original plasmid. A fragment (H1), containing sequences between 344 and 1413 bp upstream from the mouse beta-maj globin cap site, almost completely abolishes transformation when inserted next to hybrid tk genes containing the mouse beta-globin or human epsilon-globin promoter but has no effect on the intact tk gene. The effect can be demonstrated with the H1 fragment in either orientation relative to the tk gene. Secondly, in transient expression assays the H1 fragment strongly inhibits transcription when covalently linked to the tk gene under control of either globin promoter, but not when linked to the tk gene with its own promoter. The H1 fragment contains 53 bp of purine-pyrimidine alternation (ACAT)n as part of a larger region potentially capable of adopting a Z-DNA conformation.