Cytochemical and immunocytochemical characterization of kinetochores in the holocentric chromosomes of Graphosoma italicum.

In the present paper we have analyzed the trilaminar kinetochores present in the mitotic holocentric chromosomes of the heteropteran Graphosoma italicum by means of different cytochemical and immunocytochemical techniques at the ultrastructural level. The results obtained after EDTA and osmium tetroxide/p-phenylenediamine, both cytochemical methods for preferential detection of ribonucleoproteins, show that the inner and outer kinetochore layers are selectively contrasted. These results agree with those previously reported for the kinetochores of monocentric chromosomes. Additionally, using cytochemical techniques such as ethanolic phosphotungstic acid staining and silver impregnation, which recognize proteins, we report a different response of the inner kinetochore layer compared with those obtained in the middle and outer layers. Finally, our immunoelectron microscopic results employing monoclonal antibodies against DNA indicate the absence of this component from the kinetochore plates in holocentric chromosomes. Although the trilaminar appearance of kinetochores in holocentric chromosomes is very similar to that of the localized kinetochores in mammals, our observations imply the existence of significant differences on the macromolecular organization of both types of kinetochores.