In order to analyze the sequence requirements for induction of the mouse beta(maj)-globin gene, we have developed a transient assay system in mouse erythroleukemia (MEL) cells. These cells, which have been transformed by the Friend virus complex, can be chemically induced to undergo terminal differentiation during which transcription of endogenous alpha and beta globin genes is greatly increased. If we can mimic this effect in a transient assay, which only requires 4 to 5 days, then it should be possible to quickly and accurately analyze plasmid constructions with varying amounts of DNA 5' or 3' to the beta-globin promoter to determine what regions are required for induction. Transient assay conditions have been optimized for both uninduced and induced MEL cells and we currently wish to determine if induction affects genes located on transfected plasmids which in these experiments remain episomal. We have previously shown that DNA sequences known as enhancers increase the activity of the mouse beta-globin promoter in transient assays. Enhancers are cis-acting DNA sequences which act at the level of transcription to increase gene expression. They can function in either orientation both 3' and 5' to the target gene and their level of activation is relatively independent of position. While there is no high degree of sequence homology among the presently identified enhancers, two categories of short """"""""core"""""""" regions have been observed. We are interested in determining if any other common features of enhancer DNA sequences exist and, if so, whether they might suggest possible mechanisms of enhancer activation of the mouse beta-globin promoter as well as other enhancer activated promoters. Analysis of five enhancers has shown that each exhibits dyad symmetry; we are extending this analysis to include additional enhancers. We have also shown that known enhancer mutants exhibit less dyad symmetry than the wild type enhancer, suggesting there may be a correlation between enhancer function and degree of dyad symmetry.
