Expression of Irga6 in Liver Figure 4. Irga6 constitutive expression in liver uses a different promoter from that used for IFN induction. A. Scheme of Irga6 gene and promoter structure. The lengths of exons 1A, 1B and the long coding exon 2 are 104 bp, 107 bp and 2233 bp respectively (see also Figure S1). The distance between 1A and 1B is ,6.5 kb; between 1B and coding exon is ,7.0 kb (see also Table S2). Putative transcription factor binding sites (GAS, ISRE, C-Fos and HNF) within 500 bp and 600 bp upstream respectively of the Irga6 1A and 1B exons are indicated. Binding sites on the negative strand are marked by (2). B. Putative HNF sites in the Irga6 1B promoter. HNF-1 and HNF-3b sites identified as described in Materials and Methods were aligned to the consensus sequences and the position of each sequence upstream of the putative transcription start is given. Bases identical to consensus sequences are shaded in black. Binding sites on the negative strand are marked by (2). D = A/G/T, H = A/C/T, N = A/C/G/T, W = A/T, K = G/T, Y = C/T. C. Real-time PCR analysis of transcript expression of Irga6 carrying the 1A or 1B exons. mRNA from liver of C57BL/6J mice either untreated (Liver) or infected with Listeria monocytogenes at LD50 for 24 h (Liver+Listeria) were subjected to real-time RT-PCR. Relative units here were defined by the expression level of Irga6 transcript normalized to that of the mouse HPRT. The left hand graph with an expanded scale shows the predominant usage of the 1B promoter in the control liver (Liver). In the right hand graph, the date from the control liver (Liver) is redrawn on the reduced scale necessary to display the enormous increase in transcription of the 1A promoter in the infected liver (Liver+Listeria). It is important to take account of the scale difference between the two graphs. doi:10.1371/journal.pone.0006787.g004 correctly into the long coding exon 2 of Irga2. The products of this unusual splicing arrangement are apparently complete, but we have till now been unable to detect an Irga2 protein product in IFNc- induced cells derived from Irga62/2 fibroblasts using a rabbit anti- Irga6 antiserum that cross-reacts strongly on bacterially-expressed Irga2 (unpublished results). Irga2 transcripts of this type are strongly induced by IFNc, presumably from the ISRE and GAS sites located in the Irga1 exon 1 promoter [10]. The reason for the absence of an Irga2 protein product in vivo is not known. The bottom line, however, is that Irga6 is the only functional IRGA protein expressed from its constitutive liver-specific 1B promoter. The remaining genes in the Irga cluster have 59 untranslated exons homologous to the 1A exon of Irga6, preceded by clusters of canonical interferon- inducible elements ([10] and Fig. 5A alignment). The complete exon organization of the Irga cluster on chromosome 18 is given in Fig. 5C, along with a detailed description PLoS ONE | www.plosone.org 6 August 2009 | Volume 4 | Issue 8 | e6787