Expression of Irga6 in Liver Figure 3. Irga6 expression in different mouse tissues is influenced differentially by IFNs. Western blots for Irga6 protein on detergent lysates of organs from WT 129Sv mice or type I IFN receptor knock-out (AR), type II IFN receptor knock-out (GR), or double knock-out (AGR) mice, on the same genetic background. Blots were probed with mAb 10D7. The organs indicated were taken from 4 individuals for the AGR mice and 2 for the WT, AR and GR mice. The loading for all tissue lysates was 150 mg/lane except for liver, which was 80 mg/lane. L929 mouse fibroblasts (5,000 cells/ lane) unstimulated (2) or stimulated (+) with 200 U/ml IFN-c for 24 h were used as controls. Calnexin protein was probed as loading control. doi:10.1371/journal.pone.0006787.g003 To identify the promoter usage responsible for the constitutive and IFN-inducible Irga6 in liver, two primer pairs were designed with 59 primers specific for exon 1A and exon 1B respectively, and a common 39 primer in exon 2 (see Materials and Methods) for use in quantitative real-time PCR on cDNA from uninfected and L. monocytogenes-infected mouse livers. The result (Fig. 4C), expressed in relative units, showed that essentially all the Irga6 transcripts in uninfected liver were initiated from the 1B promoter, while essentially all of the Irga6 transcripts in L. monocytogenes-infected liver were initiated from the 1A promoter. The relative expression of the 1A-derived transcript was increased approximately 800-fold by L. monocytogenes infection. Perhaps unexpectedly, the expression of the 1B-derived transcript was apparently not noticeably affected by the enormous increase in transcription from the 1A promoter. Indeed, a marginal increase was measured. A differential PCR analysis of the use of the 1A and 1B promoters for constitutive Irga6 expression in other organs of uninfected animals showed extremely low transcript levels compared with liver, and no consistent organ-specific preference for the 1A or 1B promoters (Fig S2). Unexpectedly, constitutive expression of Irga6 in the heart was not restricted to use of the 1B promoter, thus the explanation for this constitutive expression is clearly different, and would justify an intensive analysis. Exon1B, associated with the hepatocyte-specific Irga6 promot- er, has homologues in the 59 regions of two other genes of the IRGA cluster on mouse chromosome 18. Irga1 and Irga2, whose coding sequences cluster phylogenetically with Irga6 [10], both possess potential 59 exons with strong sequence homology to exon 1B of Irga6 (Fig. 5A and B). In the case of Irga1 the homology to the exon 1B region of Irga6 continues upstream across the whole region populated by HNF binding sites, while in Irga2 only the distal cluster of HNF binding sites is conserved (Table S1). Both Irga1 and Irga2 are probably functionally pseudogenes [10]. For unknown reasons, Irga1 fails to splice normally from the 59-UT to the 39 coding exon, and also has two short frame-shifting deletions in exon 2 which would lead to an abnormal truncated protein. Irga2 has a surprising splicing arrangement, using exclusively the 59-UT exon of Irga1 rather than its own 59 exon (which is identified in Fig. 5A by homology), and then leapfrogging the Irga1 coding exon and the entire intergenic region before splicing PLoS ONE | www.plosone.org 5 August 2009 | Volume 4 | Issue 8 | e6787