Analysis of human interleukin-5 gene transcription by a novel nuclear run on method based on the polymerase chain reaction.
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.
Rolfe
F G
FG
Centre for Immunology, University of New South Wales, Sydney, Australia.
Sewell
W A
WA
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
D015848:Interleukin-5; D012333:RNA, Messenger