5334 Nucleic Acids Research, 2009, Vol. 37, No. 16 speciﬁcally detect cDNA from Ly49dB6 or Ly49dNOD in Southern blot analysis. For Nkg2d, the same primers used for RT–PCR (previous section) were used and the pro- ducts were identiﬁed after Southern blotting with an Nkg2d-speciﬁc oligo probe. Up to ﬁve sets of independent PCRs were performed on each single cell. RESULTS Lack of detectable activating Ly49 transcripts in T-cells Unlike the inhibitory Ly49 receptors, the activating Ly49 receptors of the B6 strain (Ly49D and H) are not expressed on the surface of T and NKT cells (29,30). This absence of surface expression on T and NKT is most likely due to the negligible amounts of the activating adaptor protein DAP12 that seems to be required for cell surface expression of the B6 activating Ly49 receptors (31,32). However, there are reports of the association of Ly49D with the activating adaptor protein CD3z and DAP10 (33,34). We also did not detect Ly49R (an activat- ing receptor in 129 mice) or Ly49D and R surface expres- sion on 129SvEvTac or 129SvS6/B6 F1 hybrid fresh ex vivo splenic T-cells (DX5 , CD3e+) respectively by ﬂow cytometry (data not shown). To determine if transcripts for the activating receptors exist in T-cells, fresh ex vivo splenic T-cells were sorted with high purity and RT–PCR with Ly49d and Ly49h-speciﬁc primers was performed on the isolated RNA (Figure 1A). Transcripts from both Ly49d and Ly49h were detected in whole spleen but very little to none in splenic T-cells, which correlates with the lack of surface expression of these receptors on T-cells. We detected similar amounts of the Figure 1. Transcription of activating Ly49 genes. (A) RT–PCR on whole spleen and sorted splenic T-cells was performed for actin (25 cycles), the inhibitory Ly49g and activating Ly49d and h as well as Nkg2d (30 cycles) with gene-speciﬁc primers. (B) 50RACE of Ly49h on cDNA from whole spleen. Vertical downward-pointing arrows show the transcription start sites assayed by 50RACE. The numbers on top of the arrows show the number of sequenced clones beginning at a given nucleotide position. The exon 1/intron 1 boundary is also indicated. inhibitory Ly49g and Nkg2d cDNA in both splenic lym- phocytes and sorted T-cells. 5’-region DNA methylation of Ly49d and Ly49h correlates with state of expression Ly49 genes have multiple promoters but most are tran- scribed from a region called Pro-2, which is thought to be the main promoter in mature NK cells (19,35). The transcriptional start site of Ly49d has been mapped to the Pro-2 region (35). To determine the promoter of origin for Ly49h, 50RACE was performed on whole spleen RNA with gene-speciﬁc primers. The Ly49h tran- scripts detected originated from Pro-2. However, unlike other Ly49 genes (35) we did not detect transcriptional start site variability for Ly49h (Figure 1B). Based on the 50RACE results, the region equivalent to the Pro-2 of the inhibitory Ly49 genes is the main area of transcriptional start for Ly49h. We examined the DNA methylation status of the CpG dinucleotides of this region that we shall refer to as the 50-region of Ly49h from here onwards. There is a cluster of six CpGs within the proximal 50-region of Ly49h with three CpGs located upstream and three located downstream of the transcription start site (Figure 2A). Sodium bisulﬁte sequencing revealed heavy methylation of this region in FACS-sorted Ly49H-negative (1F8 ) splenic NK and T-cells but not in Ly49H-positive (1F8+, 5E6 ) NK cells (Figure 2B). As with the inhibitory Ly49a gene (22), the CpG sites downstream of the tran- scriptional start site are most heavily methylated in Ly49H-negative cells. However, unlike for Ly49a and Ly49c which display a ‘half-and-half’ mono-allelic methy- lation pattern, the Pro2 region was hypo-methylated in all clones sequenced in the Ly49H+ population. The bisulﬁte sequencing results were also conﬁrmed by COBRA (Figure 2C). There are very few CpG dinucleotides in the 50-region of Ly49d. Hence, we only used COBRA to analyze two CpG dinucletides, one upstream and the other down- stream of the transcription start site (Figure 2D). TaqaI and BmgBI restriction endonucleases were used to assay the upstream and downstream CpGs, respectively. Ly49D expressing and non-expressing NK cells of fresh ex vivo spleen were FACS sorted and analyzed for DNA methylation at these two CpGs. The results are the combination of four individual PCRs per each sorted population. Ly49D negative NK cells show moderate amounts of DNA methylation at both assayed CpGs where as the Ly49D positive NK cells show almost no DNA methylation at either site (Figure 2E) reﬂecting a DNA methylation pattern similar to that observed for the Ly49h 50-region in Ly49H positive NK cells (Figure 2B and C). 5’-region DNA methylation of Ly49d and Ly49r does not correlate with stochastic mono-allelic receptor expression in 129S6/B6 F1 hybrid Mono-allelic gene expression has not been shown for the activating Ly49 genes and we did not observe the bimodal