a. PIP (percentage identity plot) diagram for human and murine DNMT1. The horizontal axis represents the human genomic sequence and has been annotated to show the position of the novel exon 1o, which unlike the previously recognized DNMT1 exons, shows no sequence conservation relative to mouse. Tall black rectangles indicate the positions of the known human DNMT1 exons. Low grey and white rectangles indicate regions with CpG-island like features (CpG/GpC respectively ≥ 0.75 and ≥ 0.60). b. Alternative first exon (1o) of human DNMT1. The exon is in upper case. Underlining indicates the PCR primers used for genomic sequence analysis (flanking) and double underlining for RT-PCR analysis of oocyte material. c. Detection of DNMT1 expression in cDNA from human oocytes and embryos. The panel labelled DNMT1o shows PCR reactions using the upstream primer indicated in panel b, unique to the novel isoform of human DNMT1. In the DNMT1s panel, an upstream primer in the previously defined exon 1 was used. The ZP3 panel shows control reactions that detect the mRNA for zona pellucida protein ZP3, which is expressed at all stages of oogenesis. Lanes 1–2, primordial follicles (P); 3, mix of primordial and early primary; 4, mix of early primary and primary (1°); 5, primary; 6, secondary (2°); 7–9, ovulated oocytes; 10, 2-cell embryos (2); 11–12, 4-cell embryos (4); 13–16, blastocysts; 17, negative control (0).
Hayward et al. BMC Genetics 2003 4:2 doi:10.1186/1471-2156-4-2