Recombination substrate and products. A. The duplication at the Aprt locus in GSAA5 cells. The upstream copy of Aprt contains a mutated EcoRV site (EV mut) in exon 2, as well as a truncated exon 5, rendering it nonfunctional. The downstream copy contains the normal EcoRV site (EV) and is functional, making GSAA5 cells APRT⁺. The herpes virus thymidine kinase (TK) and the guanine phosphoribosyl transferase (gpt) genes served as additional selective markers useful in generating the GSAA5 cell line and for characterizing the recombination products. B. Illustrations of the types of APRT^- colonies that arise in GSAA5 cells. After selection against APRT function, it is possible to recover clones that underwent HR (conversions or crossovers), mutation of the wild-type copy of the Aprt gene (indicated by the * above exon 3), or rearrangement of the Aprt gene (shown here as a deletion of a portion of the Aprt gene). Each of these events can be distinguished by a combination of Southern blot and PCR analysis (see Materials and Methods).

Methylation analysis of the Aprt locus. A. Analysis of global methylation in GSAA5 cells. Genomic DNA was isolated from untreated cells and from cells treated with 5-aza-CdR, digested with MspI (M) or its methyl-sensitive isoschizomer HpaII (H), and displayed by electrophoresis on an agarose gel. Decreased methylation in treated cells is shown by the more complete digestion of DNA by HpaII. A 1-kb marker ladder is shown on the left. B. Restriction maps and probe used for analysis of methylation at the Aprt locus. The complete recombination substrate is shown on top, with an expanded view of the 3′ copy of Aprt shown below. The methylated HpaII (H) site, which is indicated by the filled circle in the downstream copy of Aprt, is not present in the upstream copy. The probe hybridizes to the 12.5- and 4.0-kb BamHI (B) fragments, which contain the upstream and downstream copies of Aprt, respectively. C. Representative Southern blots showing the structure and methylation status of a crossover and two conversions. All samples were digested with BamHI (B) alone or in combination with MspI (M) or HpaII (H). Sizes of the digestion products (in kb) are shown on the right. D. Relative Aprt mRNA levels after 0 and 0.5μM 5-aza-CdR treatment are shown. Error bars represent one standard deviation.