Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds.
We describe the generation of 11 monoclonal antibodies that bind to the centromere/kinetochore region of human mitotic chromosomes. These antibodies were raised against mitotic chromosome scaffolds and screened for centromere/kinetochore binding by indirect immunofluorescence against purified chromosomes. Immunoblot analyses with these antibodies revealed that all of the antigens are greater than 200 kD and are components of nuclei, chromosomes, and/or chromosome scaffolds. Comparison of the immunolocalization of the antigens with that observed for the centromere-associated protein CENP-B revealed that each of these centromere/kinetochore proteins lies more peripherally to the DNA than does CENP-B. In cells normally progressing through the cell cycle, these antigens displayed four distinct patterns of centromere/kinetochore association, corresponding to a minimum of four novel centromere/kinetochore-associated proteins.
Compton
D A
DA
Department of Biological Chemistry Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Yen
T J
TJ
Cleveland
D W
DW
D000818:Animals; D001324:Autoantigens; D002453:Cell Cycle; D002460:Cell Line; D002503:Centromere / Q000648:ultrastructure*; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis*; D002877:Chromosomes, Human / Q000648:ultrastructure*; D004268:DNA-Binding Proteins; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells / Q000166:cytology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008938:Mitosis
D001324:Autoantigens; C497926:CENPB protein, human; C497927:Cenpb protein, mouse; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins