DNA methylation of Ac sequences in maize and in transgenic tobacco

--Thomas Ott, Birgit Nelsen-Salz, Hans-Peter Döring

From previous studies with the transposable element Ac in transgenic tobacco cells (Coupland et al., EMBO J. 7:3653, 1988; Coupland et al., Proc. Natl. Acad. Sci. USA 89:9385, 1989) it is known that both subterminal regions of Ac carry cis-acting sequences which are required for the transposability of the element and which are likely to be recognized by the Ac-encoded protein. Within the 5' subterminal region the hexamer motif AAACGG is present in nine copies in either orientation. In several transgenic tobacco cultures carrying one or two copies of a transposable Ac or Ds element we examined the 5' subterminal region of Ac (position 70-220) for the presence of 5-methylcytosine. We used the recently developed ligation-mediated polymerase chain reaction-aided genomic sequencing (Mueller and Wold, Science 246:780, 1989; Pfeifer et al., Science 246:810, 1989) with minor modifications. This method gave good results with 10µg of genomic tobacco DNA for each cleavage reaction according to Maxam and Gilbert. We analysed 60 cytosine positions of both DNA strands and detected no 5-methylcytosine. Thus, DNA methylation is not needed for the transposability of Ac and Ds in transgenic tobacco plants. These findings confirm other results with similar transgenic plants on the lack of de novo methylation at most of a lot of different restriction sties of the Ac sequence (Nelsen-Salz and Döring, H-P., Mol. Gen. Genet. 223:87, 1990).

Maize DNA containing the inactive Ac element of the wx-m9Ds-cy allele (Schwartz and Dennis, Mol. Gen. Genet. 205:476, 1986) was cleaved with SalI and size-fractionated on a sucrose gradient. The 5-6kb fraction which contains the Ac element was used for genomic sequencing. We sequenced one DNA strand and showed that the hexamer motifs between position 120-160 as well as the three cytosines at or next to the BamHI site at position 181 are unmethylated. Southern analysis with maize DNA containing the same allele and restricted with BamHI showed that the majority of the Ac molecules were cleaveable. This finding suggests that BamHI was unable to cleave at completion, or alternatively, that a small fraction of the Ac sequences are methylated and had gone undetected upon genomic sequencing.

If we compare the DNA methylation patterns of the maize lines wx-m9Ac/wx and wx-m9Ds-cy/wx by means of the methylation-sensitive restriction enzymes, we find pronounced differences in DNA methylation of the GC-rich, untranslated leader sequence of the Ac transcription unit. The active Ac element of the wx-m9 allele is unmethylated at the AvaI site (position 86) and the MluI site (position 434). At least one of the EcoRII sites (position 332.340), one of the RsrII sites (position 546.610.657), one of the SacII sites (position 609.656.740), and one of the EcoRII sites (position 840.880) are unmethylated. The latter sites are so densely spaced that it could not be determined which of the sites was unmethylated. Therefore it is well possible that all 12 restriction sites are unmethylated. The inactive Ac element of the wx-m9Ds-cy allele is heavily methylated at all 12 sites. Only a minute fraction of the inactive Ac molecules are unmethylated at position 332 or 340. The Ac sequences of both alleles are completely methylated at position 938 (EcoRII). There is also no significant difference between both strains with respect to methylation in e transcribed region at positions 1320, 3556, and 3844 (PvuII, AvaI, PvuII). In all cases it is only a fraction of the Ac sequences which is unmethylated. We also examined these restriction sites in the wx-m9Ds allele which is a 194bp deletion derivative of the Ac element. In this allele the methylation pattern was almost identical to the pattern which we found in the Ac element of the wx-m9 allele. It was shown previously (Kunze et al., Mol. Gen. Genet. 214:325, 1988) that a specific transcript is produced in the wx-m9 Ac and the wx-m9Ds alleles, whereas no transcript is found for the wx-m9Ds-cy allele. If DNA methylation is involved in the control of Ac activity the decisive cytosines are located in the GC-rich untranslated leader sequence of the Ac transcription unit.

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