Isolation of interleukin 2-induced immediate-early genes.
Clonal expansion of antigen-reactive T lymphocytes is driven by the lymphokine interleukin 2 (IL-2). To further elucidate the mechanisms of IL-2 action, we have utilized a differential hybridization procedure to clone IL-2-induced immediate-early genes from an IL-2-stimulated human T-cell cDNA library. To increase the frequency of IL-2-induced transcripts represented in the library, the protein synthesis inhibitor cycloheximide was included during the 2-hr IL-2 stimulation to superinduce gene expression, and the uridine analogue 4-thiouridine was utilized to enable selective purification of newly synthesized transcripts. From the enriched library, we have isolated eight IL-2-induced genes, six of which represent previously unrecognized human sequences. Northern blot analysis revealed that the induction of seven of the genes is specific to the IL-2-mediated G1 "progression" phase of the cell cycle, in that only one gene is also induced during the T-cell receptor-triggered G0-G1 "competence" phase. These results indicate that the effects of IL-2 are mediated by the specific induction of a number of immediate-early genes and provide a means with which to further delineate the mechanisms whereby IL-2 stimulates T-lymphocyte proliferation and differentiation. The methods described in this report should also be of general utility in the dissection of the signaling pathways activated by diverse cytokine receptors.
Beadling
C
C
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755-3833.
Johnson
K W
KW
Smith
K A
KA
D001483:Base Sequence; D015152:Blotting, Northern; D017252:CD3 Complex / Q000502:physiology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004261:DNA Replication; D015870:Gene Expression / Q000187:drug effects*; D015723:Gene Library; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D007700:Kinetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000502:physiology
D017252:CD3 Complex; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D003513:Cycloheximide