Comparative sequence analysis of histone H3-like proteins used in this study. (a) Cartoon depicting nucleosomal core regions and unaligned N-terminal tails. Boxes representing the N-terminal tails are shaded differentially to depict the absence of sequence similarity between H3-like proteins. (b) Phylogenetic tree for the core region. Proteins used in this study are underlined. (c) Boxshade multiple alignment for the core region of the proteins used in this study.

Detection of a Cid epitope at centromeric constrictions and in an interphase nucleus by using anti-Cid antiserum (1:5,000). (a) Kc167 cells. (b) Larval neuroblasts. Cid signal is shown in green 4′,6-diamidino-2-phenylindole staining in gray.

Plasmid constructs used for transient transfection. In each case, the full protein-coding sequence is fused to a 6-aa spacer followed by the N terminus of GFP (27).

Localization of H3-like protein-GFP fusions to centromeres of Drosophila. Kc167 cells were transfected with GFP fusion constructs driven by the cid promoter (a and d–k) or by basal (b) or induced (c) expression from the Hsp70 promoter. (a) Localization of Cid-GFP to the centromeres in a metaphase spread and to intense foci in interphase nuclei. Similar foci are seen in many interphase nuclei when Cid-GFP is expressed from the Hsp70 promoter (b and c) and are 10-fold more intense (185 ± 61 vs. 2,085 ± 979 pixel intensity) when this promoter has been induced (c). (d) Euchromatic labeling by cid-driven H3-GFP. Localization of GFP fusions on representative X chromosomes from mitotic figures with Drosophila H3 (e), Drosophila H2B (f), Drosophila Cid (g), human CENP-A (h), S. cerevisiae Cse4p (i), C. elegans HCP-3 (SWISS-PROT YMH3_CAEEL) (j), and C. elegans D6H3 (k) (GenBank accession no. AAB37052). GFP fluorescence was consistently lower with the H3-like heterolog-GFP fusion proteins than with the Cid- or the histone-GFP fusions. The X centromere is indicated with an arrowhead. The X chromosome is acrocentric, and the heterochromatin coheres well in metaphase spreads; thus, the position of the centromere can be identified by morphology alone. Identification of centromeres in metaphase spreads was confirmed by detection of the POLO kinetochore epitope. GFP signal is shown in green, the POLO epitope is shown in red (a, d, e, and g), and 4′,6-diamidino-2-phenylindole staining is shown in gray. Coincidence of GFP and POLO is yellow.

Colocalization of H3-like proteins with centromeres in human interphase nuclei. HeLa cells were transfected with GFP fusions of D. melanogaster H3 (a–c), C. elegans HCP-3 (d–f), or S. cerevisiae Cse4p (g–i) driven by the constitutive cytomegalovirus promoter. Cells were fixed and stained with ACA serum against centromeres (red); the GFP signal is shown in green. Representative interphase nuclei are shown. We chose the 60 brightest GFP spots in d and g; 70% of these spots colocalized with the ≈60 ACA spots (f and i). Arrows point to representative small spots and arrowheads point to large spots.

Colocalization of HCP-3-GFP with heterochromatin in human metaphase chromosomes. See legend to Fig. 5. 4′,6-Diamidino-2-phenylindole staining of DNA is shown in blue. Insets show two chromosomes with strong pericentric HCP-3-GFP signals (Left) and one chromosome with centromeric as well as telomeric HCP-3-GFP localization (Right).