Genomic organization, sequence interrelationship, and physical localization using in situ hybridization of two...

Two tandemly repeated DNA sequences, the 81-bp family and pOS218, have been isolated from a Sau3AI Olea europaea ssp. sativa partial genomic library. Sequencing of the 81-bp element showed the monomer to be between 78 and 84 bases long and to contain 51-58% adenine and thymidine residues. Comparison between the monomers revealed heterogeneity of the sequence primary structure. The clone pOS218 is 218 bases long, and sequence comparison between the two elements revealed that an internal region of the pOS218 repeated DNA sequence had 79% homology to the 81 bp repeat sequence. A breakage-reunion mechanism, involving the CAAAA sequence, could be responsible for the derivation of pOS218 from the 81 bp family element. By using double target in situ hybridization, co-localization of the two sequences on Olea chromosomes was observed. The sequences were present at DAPI stained heterochromatic regions, as major or minor sites having a subtelomeric or interstitial location. Methylation studies using two sets of isoschizomers, Sau3AI-MboI and MspI-HpaII, demonstrated that most cytosine residues in the GATC sites and the internal cytosine in the CCGG sites of both elements were methylated in O. europaea ssp. sativa. No major difference in methylation was apparent between DNA extracted from young leaves or from callus of O. europaea ssp. sativa. Both elements are also present in Olea chrysophylla, Olea oleaster, and Olea africana, but are absent from other Oleaceae genera, including Phillyrea, Forsythia, Ligustrum, Parasyringa, and Jasminum.