CpG Methylation Modifies the Genetic Stability of Cloned Repeat Sequences

Characteristics of unstable sequences. The plasmid name, sequence of the repeat units, and the total number of repeats are indicated. The purity of the repeat configuration is also indicated. For the fragile X (FRAXA) clones, the + indicates an AGG interruption (Pearson et al. 1998). The α3′HVR repeat is highly homogenous, with some repeat units differing at the 3′ end (Jarman et al. 1986). Only the major repeat unit variants are shown for the DYZ1 repeat (Nakahori et al. 1986). The cloning orientation of the insert relative to the unidirectional plasmid replication origin is indicated by + and −, reflecting the stable and the unstable orientation, respectively, in bacteria. The repeat sequence shown represents the template strand for leading strand synthesis at the replication fork in the stable orientation (+). Both the pure (CGG)53 and the α3′HVR could be cloned in only one orientation (Jarman et al. 1986; Pearson et al. 1998). Because the sequence in both strands of the (GC)n repeat are identical, cloning orientation and hence replication direction was not relevant; NA refers to not applicable. The effects of in vivo methylation on sequence stability are summarized in the rightmost column. Stability was determined from more than five experiments for each sequence by product analysis after multiple controlled subculturings (for a representative example see Fig. 1). Stabilization refers to fewer deletion events and smaller sizes of deletion, whereas reduced stability refers to increased deletion events and larger deletion sizes. The “exp.” Indicates that methylation permitted the stable propagation of tract lengths larger than starting material, which may be expansion products (see text).