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242 G. KEITH EJ AL

1H1M 2H2M 3H3M 4H4M SHSM6H6M7H7M 8H8M9H9M10H10M P

Figurę 1 Southern biot hybridization with a Dig-dUTP™-labelled ras complementary DNA probe (P). The DNAs were digested with Hpa II (H) or Msp I (M). The lanes correspond to testis DNA from control mice (/: before treatment, 2: 5 + 10 days, 3: 15 + 60 days, 4: 90 + 120 days) and from treated mice (5: 5 days, 6: 10 days, 7: 15 days, 8: 60 days, 9:90 days, 10: 120 days). Arrows from top to bot-tom: -4.8 kbp (c-Ha-ros protooncogene), ~3.7 kbp (v-Ha-rar transgene), -700 bp (v-Ha-rar rcstric-tion fragment), -470 bp (c-Ha-ray restriction fragment), -230 bp (v-Ha-rar restriction fragment), -60 bp (v-Ha-rar restriction fragment).

and 3.7 Kbp for the v-Ha-ras transgene) due probabły to methylation of the dCpdG base pairs in the ras genes.

The testicular DNA digestion pattems in the mouse were similar for both control (lanes 1—4) and 2-butoxyethanol-exposed mice (lanes 5-10) regardless of the elapsed time. The same was found in DNA from spleen, brain, kidney and liver (data not shown). According to these results, one could conclude that the methylation status of the Ha-raś genes was not affected by a subchronic administration of 2-butoxyethanol. In fact, this Chemical did not act as a demethylating agent. In addition, these results demon-strate that in testis and spleen, which are both organs with high mitosis rates, 2-bu-toxyethanol does not alter the activity of the maintenance dCpdG methylase. Indeed, during DNA replication the maintenance dCpdG methylase recognizes the hemimethy-lated DNA, and methylates the newly formed strand. If this enzyme was to be affected or inhibited, demethylated DNA and gene expression should result. In the subchronic mouse experiments that are shown here, 2-butoxyethanol did not induce any demethy-lation of the Ha-ras genes. Conversely, 2-butoxyethanol did not induce 5-mdC for-mation as shown by the Msp I digestion pattems for both genes (Figurę 1). The intensity