Interstitial telomeric sequence blocks in constitutive pericentromeric heterochromatin from Pyrgomorpha conica (Orthoptera) are enriched in constitutive alkali-labile sites

Abstract

The long interstitial telomeric repeat sequence (ITRS) blocks located in the pericentromeric chromosomal regions of most of Chinese hamster chromosomes behave as hot spots for spontaneous and induced chromosome breakage and recombination. The DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) procedure demonstrated that these ITRS are extremely sensitive to alkaline unwinding, being enriched in constitutive alkali-labile sites (ALS). To determine whether this chromatin modification occurs in other genomes with large ITRS that are not phylogenetically related to mammalian species, the grasshopper Pyrgomorpha conica was analyzed. We chose this species because, with conventional FISH, their chromosomes yield extremely small telomeric signals when probed with the (TTAGG) n polynucleotide, but large ITRS blocks as part of their pericentromeric constitutive heterochromatin. A high density of constitutive ALS was evidenced in the ITRS when intact meiotic cells or somatic cells were subjected to the DBD-FISH technique and probed with the specific telomeric DNA. DBD-FISH with simultaneous hybridization using telomeric and whole genome DNA probes showed that the ITRS tend to colocalize with areas of stronger signal from the whole genome probe. Nevertheless, the signal from the whole genome was more widespread than that from the ITRS, thus providing evidence that a high frequency of constitutive ALS was present in more than one DNA sequence type. Furthermore, stretched DNA fibers processed with DBD-FISH, revealed a distribution of telomeric sequences alternating interspersed with other possible highly repetitive DNA sequences. The abundance of ALS varied from one meiotic stage to another. Interestingly, most of the breakage and meiotic recombination in males takes place close to the constitutive heterochromatin, particularly enriched in ALS. These results provide further evidence of a particular, and possible universal, chromatin structure enriched in constitutive ALS at constitutive heterochromatic regions.