Human centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins.
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 p150 (Glued) and Arp1, MCAK, Tsg24, p55CDC, HZW10, HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase II and a murine HP1 homologue, M31, were used in immuno-fluorescence 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.
Saffery
R
R
The Murdoch Institute, Royal Children's Hospital, Flemington Road, Parkville 3052, Australia.
Irvine
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DV
Griffiths
B
B
Kalitsis
P
P
Wordeman
L
L
Choo
K H
KH
D000705:Anaphase / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle / Q000235:genetics; D002461:Cell Line, Transformed; D002503:Centromere / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D002879:Chromosomes, Human, Pair 10 / Q000378:metabolism; D002890:Chromosomes, Human, Pair 20 / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism* / Q000502:physiology; D008677:Metaphase / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D014407:Tumor Cells, Cultured
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D007106:Immune Sera; D008869:Microtubule-Associated Proteins