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.
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000032:analysis; D010959:Tissue Plasminogen Activator / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005914:Globins; D010959:Tissue Plasminogen Activator