Human centromeres and neocentromeres show identical distribution patterns of > 20 functionally important kinetochore-associated proteins

Using combined immunofluorescence and fluorescence in situ hybridization (F ISH) analysis we have extensively characterized the proteins associating wi th two different homologue human neocentromeres at interphase and prometaph ase/metaphase, and compared these directly with those found with normal hum an centromeres. Antisera to CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, INCENP, CLIP-170, dynein, dynactin subunits p150(Glued) and Arp1, MCAK, Tsg24, p55 CDC, HZW10, HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase II and a murine HP1 homologue, M31, were used in immunofluorescence experiments in c onjunction with FISH employing specific DNA probes to clearly identify neoc entromeric DNA. We found that except for the total absence of CENP-B bindin g, neocentromeres are indistinguishable from their alpha satellite-containi ng counterparts in terms of protein composition and distribution. This sugg ests that the DNA base of a potential centromeric locus is of minimal impor tance in determining the overall structure of a functional kinetochore and that, once seeded, the events leading to functional kinetochore formation o ccur independently of primary DNA sequence.