A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced Murine Erythroleukemia cells.
We report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and polyadenylation signals were included to produce a mature transcript coding for t-PA, whose activity can be detected in single cells by a fibrin-agarose plaque assay. Stable murine L-cell transfectants of the gamma.t-PA and beta.t-PA hybrid genes were fused to various cell lines to show that t-PA expression is increased specifically by erythroid MEL, HEL, and K562 cell fusion. The analogous H-2Kb.t-PA construct was not inducible under the same conditions. Interestingly, uninduced MEL cells increased beta.t-PA expression to the same extent as induced MEL cells. Chemiosmotic permeabilization of the beta-globin tester cell line and exposure to nuclear extracts were used to assay for trans-acting factors capable of stimulating beta.t-PA expression. Such factors were shown to be present in the nuclei of uninduced MEL cells.
|Keywords||0 (DNA, Recombinant), 0 (Recombinant Fusion Proteins), 0 (Transcription Factors), 9004-22-2 (Globins), Animals, Biological Assay, Cell Line, Cell Nucleus/physiology, DNA, Recombinant, EC 188.8.131.52 (Tissue Plasminogen Activator), Gene Expression Regulation, Globins/*genetics, Leukemia, Erythroblastic, Acute/*genetics, Mice, Recombinant Fusion Proteins/analysis, Support, Non-U.S. Gov't, Tissue Plasminogen Activator/genetics, Transcription Factors/*genetics, Transcription, Genetic, Tumor Cells, Cultured|
Wrighton, N., & Grosveld, F.G.. (1988). A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced Murine Erythroleukemia cells.. Molecular and Cellular Biology, 8, 130–137. Retrieved from http://hdl.handle.net/1765/2419