STRUCTURE AND DYNAMIC ORGANIZATION OF CENTROMERES PREKINETOCHORES IN THE NUCLEUS OF MAMMALIAN-CELLS/

Although considerable research has been focused on understanding the s tructure and molecular organization of the centromere-kinetochore comp lex of mitotic chromosomes, few reports have dealt with the centromere (prekinetochore) in the interphase nucleus. In the present study, we utilized anti-centromere antibodies from the serum of patients with th e autoimmune disease, scleroderma CREST (calcinosis, Raynaud's phenome non, esophageal dismotility, sclerodactyly, telangiectasia), as probes to investigate the structure and morphogenesis of the centromere in i nterphase nuclei of three cell lines using laser scanning confocal mic roscopy and immunoelectron microscopy. Of particular interest were the chromosomes of the Indian muntjac (2n=6 in females and 2n=7 in males) , whose large centromeres are thought to have evolved through the tand em fusion of smaller centromeres of a Chinese muntjac-like progenitor species (2n=46), The various forms and patterns of centromeres observe d in the nucleus correlated with stages in the cell cycle as determine d by bromodeoxyuridine labeling and apparently represent stages in pre replication, replication and maturation. Immunoelectron microscopic st udies using CREST antisera indicated that the high order structure of chromatin associated with each prekinetochore undergoes a regular unfo lding-refolding cycle, displaying small beadlike subunits tandemly arr anged along a linear thread of centromeric DNA, much like that reporte d for mitotic chromosomes. Individual centromeres/prekinetochores form a stable association with the 9-13 nm core filaments of the nucleoske letal network in the nucleus that later become the chromosome scaffold of mitotic chromosomes, Our findings provide morphological support fo r the hypothesis that the spatial arrangements of individual centromer es within the nucleus may have influenced centromeric translocations a nd fusions during chromosome evolution, Therefore, the centromere-kine tochore complex, best known for its essential role in partitioning chr omosomes in mitosis and meiosis, may also function in chromosome movem ents and associations in interphase.