ANALYSIS OF HUMAN INTERLEUKIN-5 GENE-TRANSCRIPTION BY A NOVEL NUCLEARRUN ON METHOD BASED ON THE POLYMERASE CHAIN-REACTION

The total abundance of any mRNA is determined by several factors, prin cipally the rate of new gene transcription and the stability of the mR NA. Interleukin-5 (IL-5) is a cytokine with an important role in suppo rting the proliferation, survival and activation of eosinophils. Gene transcription of IL-5 mRNA in human T cells was assessed by the conven tional nuclear run on assay, but the signal strength was too low for s atisfactory analysis. A novel run on assay was developed in which nucl ei were incubated with and without nucleotides, and transcripts were d etected by reverse transcription-polymerase chain reaction (RT-PCR). T he difference between the samples with and without nucleotides was a m easure of the amount of new transcription. IL-5 gene transcription was not detected in unstimulated T cell line HSB-2 cells or in unstimulat ed human T cells prepared from peripheral blood. Transcription was rap idly induced by a variety of stimuli, and ceased by 4-6 h after activa tion. This method is applicable to other genes expressed at low abunda nce, such as cytokine genes. mRNA stability was measured by quantitati ve RT-PCR. After activation with phorbol myristate acetate and ionomyc in, 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.