Figure 2.

(A) Diagram of the gene-targeting vector, the mouse Pcmt-1 gene, and a targeted Pcmt-1 allele. Targeted alleles are identified by Southern blot analysis of EcoRI-cleaved genomic DNA, using a 5′ external probe. (B) Genotyping of knockout mice. The targeted Pcmt-1 allele yields a 6.6-kb EcoRI fragment, whereas the wild-type fragment is 5.0 kb. (C) Absence of protein l-isoaspartate(d-aspartate) O-methyltransferase expression in Pcmt-1 knockout mice. A Western blot analysis of total cellular proteins from various tissues revealed that the 26-kDa methyltransferase protein was present only in wild-type mice; an unidentified cross-reacting protein of ≈60 kDa was present in both wild-type and knockout tissues. (D) Absence of protein l-isoaspartate(d-aspartate) O-methyltransferase activity in Pcmt-1 knockout mice. Cytosol from various tissues was assayed for methyltransferase activity. Tissues from knockout mice contained negligible enzyme activity compared with those from wild-type mice. Error bars represent SEM.