Functional analysis of a single nucleotide polymorphism in a potential binding site for GATA transcription factors in the ovine interleukin 2 gene.
The transcription factor GATA-3 is one regulator of Th1/Th2 differentiation. In sheep, we recently discovered a putative GATA-binding site (WGATAR) in the second intron of the Th1-cytokine gene interleukin 2 (IL2), showing a single nucleotide polymorphism (G/C). As genetic variations in cytokine genes are thought to regulate cytokine production, we studied the significance of this polymorphism for IL2 transcription. Sheep with different IL2 genotypes were identified by single-strand conformation polymorphism (SSCP)-analysis and IL2 transcription levels in peripheral blood mononuclear cells (PBMC) isolated from these animals were compared. For this purpose, transcription of IL2 mRNA was quantified by real-time polymerase chain reaction in unstimulated PBMC and in PBMC incubated for 4h in the presence of concanavalin A (ConA) or phorbol 12-myristate 13-acetate plus ionomycin (PMA/I). Compared to unstimulated cells, stimulation with ConA and PMA/I increased the IL2 mRNA transcription in average by 300- and 20-fold, respectively. Nevertheless, no significant differences in IL2 transcription between the genotypes could be detected. These findings were confirmed by band shift studies using different oligonucleotides containing variations of the potential binding motif, which showed no differences in the gel mobility after incubation with nuclear extract containing GATA-3. The obtained results argue against an impact of this polymorphism on the IL2 transcription and the genetic disease resistance in sheep.
Lühken
Gesine
G
Department of Animal Breeding and Genetics, Justus-Liebig-University, Ludwigstrasse 21B, 35390 Giessen, Germany. Gesine.Luehken@agrar.uni-giessen.de
Stamm
Ivonne
I
Menge
Christian
C
Erhardt
Georg
G
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050990:GATA3 Transcription Factor; D007376:Interleukin-2 / Q000235:genetics*; D007438:Introns; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012756:Sheep / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D018417:Th1 Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018418:Th2 Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D050990:GATA3 Transcription Factor; D007376:Interleukin-2; D012333:RNA, Messenger; D015534:Trans-Activators; D004247:DNA