T-associated maternal effect (Tme) is the only known maternal-effect mutation in the mouse. The defect is nuclear-encoded and embryos that inherit a deletion of the Tme locus from their mother die at day 15 of gestation. There are many genomically imprinted regions known in the mouse genome but so far no imprinted genes have been cloned. The Tme locus is absent in two chromosome-17 deletion mutants, Thp and the tLub2, and its position has been localized using these deletions to a 1-cM region. We report here that the genes for insulin-like growth factor type-2 receptor (Igf2r) and mitochondrial superoxide dismutase-2 (Sod-2) are absent from both deletions. Probes for these genes and for plasminogen (Plg) and T-complex peptide 1 (Tcp-1) were used in pulsed-field gel mapping to show that Tme must lie within a region of 800-1,100 kb. We also demonstrate that embryos express Igf2r only from the maternal chromosome, and that Tcp-1, Plg and Sod-2 are expressed from both chromosomes. Therefore Igf2r is imprinted and closely linked or identical to Tme.

T he potential importance of DNA methylation for specifying epigenetic inheritance in eukaryotic cells was recognized soon after the discovery of the role that methylation plays in the modification and restriction of bacterial and bacteriophage DNA (1-5). In eukaryotic cells, inheritance of the methylated state usually involves 5-methylcytosine and predominantly depends on enzymatic recognition of CpG and CNG motifs. Base-pairing rules (6) ensure that these motifs are symmetrically located on complementary strands of DNA (for example, CpG͞CpG dyads), thus providing the opportunity for the inheritance of cytosine methylation after DNA replication (7). In mammals, maintaining a methylated state of CpG cytosines is an important component of X-chromosome inactivation and genomic imprinting (8-10). The failure to maintain a methylated or an unmethylated state of key cytosines can lead to ''epimutations''; such changes may alter cell and developmental pathways, resulting in new phenotypes (11-14) including disease (15-17). The mechanisms and fidelity of epigenetic inheritance are thus of crucial biological and medical importance.A central issue in epigenetics concerns the mechanism by which a locus maintains a stable epigenetic state through many cell divisions. It appears that epigenetic mechanisms that use 5-methylcytosine within CpG dinucleotides have moderate to high fidelities of maintaining a methylated state of cytosine, after a transitory hemimethylation state during DNA replication (9, 18-23). Hemimethylated sites are also transitional states in developmental processes; active demethylation or de novo methylation may sometimes be involved in gene reactivation or inactivation (24-26). In a study to assess the dynamics of DNA methylation, Riggs and colleagues (9, 27), estimated the fidelity of maintenance methylation (E m ) within partially methylated CpG islands to be Ͼ0.99 per methylated cytosine per cell division; de novo methylation efficiency (E d ) for unmethylated cytosines was estimated to be 0.05 per site per generation. This study, carried out with clones of tissue-culture cells in which methylation was perturbed with 5-azacytidine, also provides a useful mathematical model of the kinetics of DNA methylation (9).Current inferences on epigenetic fidelities and transitional methylation states are based on data for single methylation sites or on patterns of methylation derived from populations of complementary strands. A major experimental limitation has been the difficulty in obtaining methylation patterns from the two complementary strands of an individual DNA molecule. If such a method were available, patterns of methylation fidelity, and detection of both gain and loss of methylation, could be studied relatively directly.We have developed ''hairpin-bisulfite PCR'' for this purpose of analyzing patterns of cytosine methylation on complementary strands of individual DNA molecules. This method uses a hairpin linker, targeted and ligated to restriction-enzyme-cleaved genomic DNA, to maintain attachment o...

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