The DNA sequence was 67.3% similar to that of OsCENH3 reported previously (Nagaki et al., 2004). and the DNA sequence was decided. The DNA sequence was 67.3% similar to that of OsCENH3 reported previously (Nagaki et al., 2004). Subsequently, we designed a 5 quick amplification of cDNA ends (RACE) primer (LnCENH3-5RACE) to amplify the 5 end of the cDNA. The 5 RACE PCR using the primer and mRNA from as a template amplified a 620-bp fragment, which was subsequently cloned and sequenced. The 3 end of the cDNA, 700 bp in length, was also amplified using the primer LnCENH3-3RACE based on the 5 RACE sequence. A full-length LnCENH3 cDNA sequence was determined by overlapping the 5 RACE and 3 RACE sequences and aligned with other plant CENH3s (see Supplemental Figure 1 online). All of the CENH3 cDNAs were conceptually translated into amino Sodium phenylbutyrate acid sequences, and the deduced sequences were Sodium phenylbutyrate aligned to determine conserved and variable regions. The core region was well conserved among LnCENH3 and other CENH3s and also with rice canonical histone H3 (Figure 1, bottom). Four helices were found in the core region of LnCENH3, as reported in other species (Vermaak et al., Rabbit Polyclonal to BL-CAM (phospho-Tyr807) 2002; Black et al., 2004), and N-helix and 3-helix were more conserved than 1-helix and 2-helix (Figure 1, bottom). On the other hand, the N-terminal ends were highly variable (Figure 1, top). Nevertheless, the first six amino residues were completely conserved among the CENH3s listed (Figure 1). This finding suggested that the region possesses an important function in plant CENH3s. In the remaining amino tail region, LnCENH3 had greater similarity to Arabidopsis HTR12 than to cereal CENH3s (Figure 1, top). Open in a separate window Figure 1. Amino Acid Sequence Alignment of LnCENH3 and CENH3s from Other Plant Species and Canonical Histone H3 from Rice. Identical, similar, and weakly similar amino acids are indicated by asterisks, colons, and dots, respectively. A red box indicates the amino acid residues used for raising a peptide antibody against LnCENH3. Blue boxes correspond to the -helices in human canonical histone H3, reported previously (Black et al., 2004). An anti-LnCENH3 antibody was raised against a synthetic peptide containing the N-terminal 20 amino acid Sodium phenylbutyrate residues of the deduced LnCENH3 protein. To determine the antibody specificity, a protein gel blot assay was performed using the affinity-purified Sodium phenylbutyrate antibody on a protein extract from mature leaves of (Figure 2). Although the molecular mass deduced from the LnCENH3 amino acids was 18.7 kD, the antibody detected a major 23-kD band and a minor 36-kD band on the blot (Figure 2). Because histones are highly basic and are known to show anomalous migration during protein electrophoresis (Talbert et al., 2002), the major band observed possibly corresponds to the LnCENH3 protein, whereas the minor band could represent a posttranslationally modified form. Open in a separate window Figure 2. Protein Gel Blot Analysis Using the Anti-LnCENH3 Antibody. Protein extracts were isolated from young leaves of and to characterize the holocentric chromosomes, immunostaining was conducted using the purified antibody and cultured cells. The antibody detected the Sodium phenylbutyrate LnCENH3 protein in all cultured cells investigated (Figures 3 and ?and4;4; see Supplemental Figure 2 online). However, the strength of the LnCENH3 signal differed at different cell cycle stages (Figures 3A to 3C; see Supplemental Figure 2A online). At interphase, many heterochromatic regions, which were deeply stained with 4,6-diamidino-2-phenylindole (DAPI), appeared in nuclei (Figures 3A, bottom left, and ?and3F,3F, right). Compared with the immunosignals at metaphase (Figures 3B and 3C, top), the signals at interphase were quite weak (Figures 3B and.