DNA samples from different Lycopersicon esculentum (cultivar UC82) were isolated and used to detect the presence of 5- methylcytosine (5mC) residues in and around a variety of gene locations. We have detected novel methylation states of particular loci by hybridizing specific cDNA probes (Bernatzky and Tanksley 1986) in Southern analyses to DNA treated with the restriction enzyme HpaII. This enzyme is unable to cleave if either of the cytosines in its recognition sequence (CCGG) are methylated. Of six loci screened, four were differentially methylated in DNA isolated from different plant organs (Table 1). From these experiments we observed that not only does DNA methylation differ between plant organs, but differential DNA methylation is observed within the same organ. Altogether, 12 loci were examined using Southern analyses, and all of the loci were located on HpaII/MspI DNA fragments much larger than would be predicted for enzymes which recognize a four base sequence (Table 2). These results indicate that the tomato genome is methylated, methylated sequences are distributed throughout the genome and include regions adjacent to or encompassing coding sequences, and finally that the methylation status of genes may differ as a function of development.
Table 1. Distribution of signal in variable Hpa II fragments in DNA isolated from tomato organs. The DNA fragments observed on autoradiographs developed following hybridization of Southern blots with the indicated cDNA probes were quantified using a scanning laser densitometer. The total signal in each lane was set to 100% and the distribution of the signal among multiple fragments in each lane is indicated.
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% signal
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locus fragmen leaf root seed seedling
size (kb)
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CD25A 5.9 89 100 86 94
4.0 11 - 14 6
CD8 3.2 93 100 100 100
2.3 7 - - -
CD28 7.5 22 - 16 29
7.0 58 93 77 64
5.8 20 7 7 7
CD13A 1.9 58 65 85 81
1.0 27 - 15 17
0.7 15 35 - 2
CD3 15.0 100 100 100 100
CD39 4.5 100 100 100 100
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Table 2. Sizes of HpaII and MspI fragments encompassing cDNA loci in tomato
leaf DNA. The number in parentheses following the cDNA locus,
indicates the chromosomal location of the locus, ND indicates the
digestion was not performed.
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Fragment Sizes (kb) Fragment Sizes (kb)
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Locus HpaI MspI Locus HpaI MspI
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CD12 (1) 10.5 2.5 CD25A (6 5.9 5.0
4.0 3.0
CD28 (1) 7.5 6.8
7.0 1.1 CD14 (6) 10.0 1.0
5.8 7.2
5.5
CD35 (2) >20.0 4.8
4.4 CD40 (8) 3.8 3.8
3.2 3.2 3.2
0.65
CD7 (8) 5.8
CD13A (3) 1.9 3.1 ND
1.0 ND 2.9
0.7
CD8 (9) 3.2 ND
CD3 (3) 15.0 ND 2.3
CD39 (4) 4.5 ND CD2 (12) 9.0 2.5
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Literature cited:Bernatzky, R.B. and S.D. Tanksley. 1986 Genetics 112:887-898