Identification of genes induced by interleukin-3 and erythropoietin via the Jak-Stat5 pathway using enhanced differential display-reverse southern.
Cytokines mediate their effects on growth and maturation of hematopoietic cells by binding to their cognate receptors and activating target genes. Interleukin-3 (IL-3) and erythropoietin (Epo) induce signal transduction via the Jak-Stat pathway. We report here on the identification of several known and novel genes induced by IL-3 and Epo, using a modified version of the PCR-based technique, enhanced differential display (EDD). We modified the technique to facilitate the screening and verification of the differential expression of the genes by using reverse Southern blotting (RS) and PCR-Southern blotting, and we called it EDD-RS. From the initial 110 genetags that were identified as differential expressed genes, 14 contained more than one gene. Among the differentially expressed genes, 24 are known genes and 39 are novel genes. Several of the known genes, such as IRF-1 and P21waf, were previously observed by others to be induced by IL-3 and Epo, but their dependence on Stat5 activation in cytokine-dependent cells was unknown. Other known genes, such as crp and Mssp2/1, were not described previously as target genes for cytokine induction. The results demonstrate that EDD-RS is an efficient method to identify cytokine-induced genes and can be productive in delineating the signal required for their induction.
Ofir
R
R
Department of Microbiology & Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Qing
W
W
Krup
M
M
Weinstein
Y
Y
D015139:Blotting, Southern; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004921:Erythropoietin / Q000494:pharmacology*; D016762:Genes, fos; D016758:Genes, jun; D050856:Interferon Regulatory Factor-1; D007377:Interleukin-3 / Q000494:pharmacology*; D008894:Milk Proteins; D010750:Phosphoproteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000502:physiology*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; D007377:Interleukin-3; D008894:Milk Proteins; D010750:Phosphoproteins; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D004921:Erythropoietin