Human Molecular Genetics, 2000, Vol. 9, No. 2 175-185
© 2000 Oxford University Press
Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins.
The Murdoch Institute, Royal Children’s Hospital, Flemington Road, Parkville 3052, Australia and 1Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195, USA
Using combined immunofluorescence and fluorescence in situ hybridization (FISH) analysis we have extensively characterized the proteins associating with two different homologue human neocentromeres at interphase and prometaphase/metaphase, and compared these directly with those found with normal human centromeres. Antisera to CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, INCENP, CLIP-170, dynein, dynactin subunits p150Glued and Arp1, MCAK, Tsg24, p55CDC, HZW10, HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase II and a murine HP1 homologue, M31, were used in immunofluorescence experiments in conjunction with FISH employing specific DNA probes to clearly identify neocentromeric DNA. We found that except for the total absence of CENP-B binding, neocentromeres are indistinguishable from their alpha satellite-containing counterparts in terms of protein composition and distribution. This suggests that the DNA base of a potential centromeric locus is of minimal importance in determining the overall structure of a functional kinetochore and that, once seeded, the events leading to functional kinetochore formation occur independently of primary DNA sequence.
+ To whom correspondence should be addressed. Tel: +61 3 93455045; Fax: +61 3 93481391; Email: choo@cryptic.rch.unimelb.edu.au
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