Figure 1.

Imprinting of the human GRB10 gene. A, Monoallelic expression of human GRB10 in fetal brain. The genomic sequence of a normal fetal tissue heterozygous for a G/A polymorphism in exon 3 (top) and the corresponding cDNA sequence (bottom). Note that only the “A” allele is expressed in this sample. B, Determination of the parental origin of the human chromosome 7 in somatic cell hybrids GM10791 and GM13302, which carry a whole chromosome 7 and a derivative chromosome der(7)t(7;17)(q36;q22), respectively: (1) both hybrids harbor PEG1, as evidenced by positive amplification with primer pair 5′-caatagtccacttcttacc-3′ and 5′-ggccgagatcttttaatc-3′ (Riesewijk et al. 1998), corresponding to exon 6 of the human PEG1 genomic sequence; (2) expression of the imprinted PEG1 isoform 1 in GM10791, mUPD7 lymphoblastoid cell line GM11496, GM13302, and a paternal upd(7) (pUPD7) lymphoblastoid cell line (Pan et al. 1998) analyzed by RT-PCR with GRB83 and GRB44, where PEG1 is expressed in GM13302 and pUPD7 but not in GM10791 or mUPD7 and the housekeeping gene, HPRT, is used as a control; (3) methylation status of the promoter of human PEG1 in each cell line, where genomic DNA, treated with bisulfite, is amplified with PEGMF-PEGMR pair and PEG1UF-PEG1UR, specific for the methylated and the unmethylated allele, respectively, and the promoter is methylated in GM10791 and in mUPD7 but is unmethylated in GM13302 and pUPD7. Collectively, the human chromosome 7 is paternally derived in GM13302 and maternally derived in GM10791. C, Expression of GRB10 in GM10791 and GM13302, where (1) both hybrids harbor GRB10, as evidenced by positive amplification, with primer pair GRB7 and GRB8, and (2) GRB10 is expressed in GM10791, which carries a maternally derived chromosome 7, but not in GM13302, which carries a paternally derived chromosome 7.