Occurrence of DNA sequence differences in C-heterochromatin of Eulemur coronatus and Eulemur macaco, as revealed by fluorescence resonance energy transfer

In the genus Eulemur (Malagasy lemurs) karyotype diversification has occurred mainly through Robertsonian mechanisms of chromosome fusion (Rumpler et al., 1976). Eulemur coronatus is the sole species to have the largest genome size, due to a very large amount of C-heterochromatin, mostly located at the pericentromeric regions of the largest chromosomes (Warter and Rumpler, 1985). This increase in C-heterochromatin was thought to be due to DNA amplification (Ronchetti et al., 1993). The aim of this work was to investigate whether the large C-heterochromatin of Eulemur coronatus might have derived by amplification of the smaller C-heterochromatin of Eulemur macaco, a closely related species with smaller genome size. To obtain information on the overall base composition of the total genomes, on the relative interspersion of AT and GC base paris along the DNA molecule and on the structural differences in C-heterochromatin, we used a quantitative cyto-chemical approach, based on fluorescence resonance energy transfer (FRET) between DNA-specific fluorochromes (i.e. the AT-specific Hoechst 33258, and the non base-specific dye, propidium iodide). Micro-spectrofluorometry and image analysis were used to investigate both the overall FRET efficiency and its spatial distribution along the chromosome arms. FRET efficiency values of the DNA in C-heterochromatin were significantly different in the two Eulemur species, indicating a different qualitative composition of repetitive DNA. This suggests that the repetitive DNA of Eulemur coronatus cannot have originated by amplification in toto of the repetitive DNA sequences of Eulemur macaco.