Identification and methylation analysis of Nap1l5 locus. (A) Map of locus at novel imprinted gene Nap1l5. DPC#24–1 identified the locus and is indicated by a solid box; the CpG island, by a hatched box. The Nap1l5 transcript is contiguous with the genomic DNA; EST AB041556 at Nap1l5 is shown as a black line, and the transcription orientation is indicated by the arrowhead. EST matches (BE984795, BE987942) neighboring AB041556 are indicated as black lines and form part of the same transcription unit as determined by RT-PCR (data not shown). The probe used for methylation analysis by Southern blot hybridization is indicated as a gray bar, and the BsmI fragment analyzed is shown. The RT-PCR product used for imprinted expression analysis by using aBstZI RFLP (Fig. 2B) is shown as a white box, and fragment lengths are given. Hin6I and HpaII sites are given as vertical lines; in some instances, individual sites could not be resolved. (B) Methylation analysis of region containingNap1l5 in kidney DNA from T77H UPD (PatDp.dist6 and MatDp.dist6) adult mice. The DNA was digested with BsmI alone (−) or in combination with Hin6I (Hi), HpaII (Hp), or MspI (M). Wild-type (WT) controls are shown alongside their PatDp.dist6 and MatDp.dist6 littermates. MatDp.dist6 DNA is highly methylated in comparison to PatD.dist6 DNA, indicating that the region is a maternally methylated DMR.
