Figure 2.

Allele-specific utilization of H13 polyA sites. (A) Enlarged schematic showing the fourth intron of H13 including Mcts2, the differentially methylated CpG island, and the H13d polyA site. Exons are black rectangles, and splice patterns are indicated with arrows depicting the direction of transcription. The fourth exon of H13a–c is shown entirely contained within the 3′UTR of H13d. The 3′ RACE primer used in B anneals within this region. (B) 3′ RACE was performed using a gene-specific primer within the fourth exon of H13a–c, in material from F1 hybrids of the C57BL/6J (B6) and Mus musculus castaneus (cast) substrains. The major products utilized either the H13a (1.1 kb) or H13d (0.6 kb) polyA sites. Products corresponding to H13b and H13c are not seen, presumably due to their lower abundance and larger size reducing the efficiency of amplification. Additional splice variants of H13a are seen in whole newborn brain but not in ES cells. In all figures, the maternal strain is given first for hybrid material. (C) RACE products were sequenced over regions containing SNPs between B6 and cast alleles. For whole-brain material, three biological replicates were performed with similar results (data not shown). (D) RT–PCR products covering exons 1–3 (Fig. 1A, probe #3) originate from both parental alleles. No SNP was identified between B6 and cast, so B6 × Mus spretus (spret) material was used. The black box indicates the hybrid sequence trace showing biallelic expression.