Analysis of the methylation state of the transposase binding sites in the termini of Ac
-- Lihua Wang, Manfred Heinlein, Peter Starlinger and Reinhard Kunze

The DNA binding-affinity of the Ac transposase (TPase) protein to the subterminal AAACGG target motifs may be increased or decreased by C-methylation on one or the other of the two DNA strands. This differential recognition of hemimethylated target motifs by TPase could provide a mechanism for the coupling of the transposition reaction to replication.

A prerequisite for such a mechanism is that the TPase target sites are methylated in vivo. This can only be tested by genomic sequencing as the AAACGG motifs are not accessible by restriction endonucleases. We have adopted the recently developed bisulfite genomic sequencing procedure, during which all non-methylated cytosine residues are deaminated into uracil, whereas 5-methyl-cytosines are not converted. This method allows the sequence determination of many individual genomic DNA molecules (Frommer et al., PNAS 89:1827-1831, 1992).

We have begun to investigate the methylation status of the Ac termini in the wx-m9 allele. Genomic DNA was extracted from dry kernels with one wx-m9::Ac dose in the endosperm. As a control for the reaction conditions, plasmid pJAC (which contains the Ac element from the wx-m7::Ac allele) was grown either in a dcm+ or dcm- E. coli strain and mixed in single copy gene-concentration to the genomic maize DNA. Four pairs of primers were designed to amplify the Ac 5´-end. Primer pair 1 will give rise to a PCR band with unreacted genomic wx-m9 DNA. Pair 2 will produce a PCR band with unreacted pJAC. Primer pair 3 was designed to yield a PCR product with bisulfite-reacted genomic wx-m9 DNA, and pair 4 will produce an amplification product exclusively on bisulfite-reacted pJAC DNA.

We have meanwhile established appropriate bisulfite reaction conditions. PCR products of the expected lengths were obtained using the bisulfite reacted genomic DNA/pJAC-mixture as template and the two primer pairs specific for bisulfite reacted DNA, whereas no products were obtained with the primers specific for unreacted DNA.

The PCR products were cloned into pUC19 and sequenced. The pJAC DNA served as a control for the reaction conditions. As expected, all cytosine residues in the dcm- pJAC DNA had been converted, whereas in dcm+ pJAC DNA the two cytosines located in dcm-recognition sites were not converted.

The bisulfite-converted upper DNA strand of the Ac 5´-end could also be amplified in one step and the resulting PCR band was cloned into pUC19. We have obtained preliminary data indicating that the degree of methylation of individual Ac molecules at the 5´-end differs. About half of all Ac molecules contain no methylated cytosines in the 5´-terminal 250 nucleotides. The other half is methylated to varying extents. In the majority of these molecules, more than 50% of the cytosines between Ac positions 26 - 90 are methylated. However, only very few, if any at all, of the more internally located cytosines are methylated. This predominantly unmethylated region encompasses the most prominent blocks of AAACGG-repeats, the TPase binding sites. We are presently extending our studies by analysing the methylation states of the lower DNA strand at the Ac 5´-end and both DNA strands at the 3´-end. 

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