title: Analysis of human interleukin-5 gene transcription by a novel nuclear run on method based on the polymerase chain reaction.
abstract: The total abundance of any mRNA is determined by several factors, principally the rate of new gene transcription and the stability of the mRNA. Interleukin-5 (IL-5) is a cytokine with an important role in supporting the proliferation, survival and activation of eosinophils. Gene transcription of IL-5 mRNA in human T cells was assessed by the conventional nuclear run on assay, but the signal strength was too low for satisfactory analysis. A novel run on assay was developed in which nuclei were incubated with and without nucleotides, and transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR). The difference between the samples with and without nucleotides was a measure of the amount of new transcription. IL-5 gene transcription was not detected in unstimulated T cell line HSB-2 cells or in unstimulated human T cells prepared from peripheral blood. Transcription was rapidly induced by a variety of stimuli, and ceased by 4-6 h after activation. This method is applicable to other genes expressed at low abundance, such as cytokine genes. mRNA stability was measured by quantitative RT-PCR. After activation with phorbol myristate acetate and ionomycin, the half-life of IL-5 mRNA was 2.6 h in HSB-2 cells and 4.0 h in T cells prepared from human blood. These data, taken together, indicate that human IL-5 mRNA is predominantly regulated at the level of gene transcription.
authors.affiliation: Centre for Immunology, University of New South Wales, Sydney, Australia.
authors.forename: F G
authors.initials: FG
authors.lastname: Rolfe
authors.forename: W A
authors.initials: WA
authors.lastname: Sewell
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D015848:Interleukin-5 / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D015848:Interleukin-5; D012333:RNA, Messenger