Expression of Irga6 in Liver Figure 5. Phylogenetic relationships among the 59-UT exons of the IRGA genes. A. Alignment of the putative 59 exons of the IRGA genes. Irga5, which is not listed, is a degraded pseudogene without transcript [10] (see also below). The 59 exons fall into two sequence classes homologous to the exon 1A and exon 1B of Irga6 respectively. Exon 1A and 1B of Irga6 have been presented in detail in Figure S1. Exon 1 of Irga3, Irga4, and Irga8 are all inferred from EST sequences and confirmed against the chromosome 18 genomic contig NT_039674.7. The predicted exon 1 of Irga7 is derived by homology from the other 1A exons. It is positioned and oriented correctly relative to the Irga7 gene but has not yet been recovered from an EST pool. B. Average distance phylogram based on percentage identity, relating the exon 1 sequences of the IRGA family of genes shown in Fig. 5A above. The numbers recorded above each branch give the percentage difference in sequence along the branch. The distinction between 1A and 1B exons is clear. The predicted exon 1 of Irga7 is an outlier in the 1A group, consistent with the possibility that Irga7 is a pseudogene (see Fig. 5C ). C. Scale map of the IRGA gene cluster on chromosome 18 of the C57BL/6 mouse. The complete sequence domain of the IRGA genes was extracted from the chromosome 18 genomic contig, NT_039674.7. The length of the whole cluster from the 59 nucleotide of exon 1 of Irga1 to the 39 nucleotide of the ORF of Irga8 is 230,910 nucleotides. In each case, the position of the 59-UT exon 1 is shown in black and the long exon 2 containing the ORF in red. Arrows above each 59 exon indicate the direction of transcription. The 39-UT region of exon 2 is not indicated. The homology group, 1A or 1B (see Fig. 5A and 5B above), is indicated for each 59-UT exon. The 1B exon upstream of Irga1 is found associated only with ESTs (e.g. AK149583.1 and AK149556.1) containing the ORF of Irga2, indicated by the oblique transcription orientation arrow. The ORF of Irga1 itself is interrupted by two frame shifts and cannot generate a full-length Irga1 protein. Furthermore, Irga1 is found in databases exclusively associated with incorrectly-spliced ESTs (e.g. BI658674, BG915086). The transcriptional orientation indicator for Irga2 shows no terminal arrowhead because although the 1B exon of Irga2 is clearly identified by homology no transcripts have been found containing this exon. Irga2 is found exclusively associated with exon 1 of Irga1. Irga5 was described as a pseudogene earlier [10]. The long exon was identified by homology to exon 2 of other IRGA genes but contains no full-length ORF. No ESTs have been described from Irga5 and no 59-UT exon 1 could be identified by homology to either exon 1A or 1B. Irga7 encodes a full- length ORF in exon 2, and the 59-UT was readily identifiable by homology. However no ESTs encoding Irga7 have been found and no transcripts corresponding to Irga7 were detected in IFN-stimulated L929 fibroblasts (Bekpen, 2005). Thus Irga7 is probably a pseudogene in the C57BL/6 mouse and in L929 (C3H/An strain) fibroblasts. doi:10.1371/journal.pone.0006787.g005 Expression of Irga6 in isolated hepatocytes To confirm the expression of Irga6 in hepatocytes, a number of in vitro established cell lines of hepatic origin were assayed for constitutive Irga6 expression. Very low levels of Irga6 were detected, while in all cases Irga6 could be induced with IFNc (Fig. 6A and data not shown). Thus the elevated constitutive expression of Irga6 found in vivo appeared to reflect a differentiated property of hepatocytes that, in common with other differentiated functions of these cells, is lost on in vitro culture [19–21]. This could be directly confirmed by cultivation of primary hepatocytes isolated from mouse liver. The Western blot shown in Fig. 6A (upper panel) shows that expression of Irga6 in isolated primary hepatocytes is higher than the calnexin control signal. This intensity of expression is maintained up to 6 h of culture but thereafter drops markedly. The decline in expression at 24 h can, however, be prevented and indeed reversed if the hepatocytes are cultured for this period with IFNc. The expression of Irga6 in primary hepatocytes reached a minimum by 2 days of culture and remained essentially stable at this low level for up to 5 days thereafter (Fig. 6A, lower panel). The residual expression level after 2 or more days of hepatocyte culture was only slightly higher than that seen in an unstimulated line of hepatocyte-derived tissue culture cells, TIB73. Other IRG proteins were also detected above background in primary PLoS ONE | www.plosone.org 7 August 2009 | Volume 4 | Issue 8 | e6787