Isolation of a Species-Specific Satellite DNA with a Novel CENP-B-like Box from the North African Rodent Lemniscomys barbarus

A species-specific satellite DNA (Lb-MspISAT) was isolated from the North African rodent Lemniscomys barbarus. This DNA is highly homogeneous in the sequence of different repeats and shows no internal repetitions. Filter and in situ hybridizations demonstrated that it is tandemly repeated at the centromeres of all chromosomes of the complement. A 19-bp CENP-B-like motif was found in Lb-MspISAT which conserves 12 of the 17-bp of the human CENP-B box, but only 5 of the 9-bp of the canonical sequence that is necessary to bind the CENP-B protein. Compared with the human CENP-B box, nucleotide substitutions and insertions increase the palindromic structure of this motif. The possibilities that it may be involved in centromeric function or in homogenization of the Lb-MspISAT sequence are discussed.     

A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.     

Isolation of chromosome-associated proteins from Drosophila melanogaster that bind a human centromeric DNA sequence

The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is though to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.     

Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein which recognizes a 17-bp sequence (CENP-B box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere domains of its chromosomes. To elucidate the AGM CENP-B-CENP-B box interaction, we have analyzed the gene structure, expression, biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was established to be highly homologous to that of human and mouse CENP-B. In particular, the DNA binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. Immunoblotting and DNA mobility shift analyses revealed CENP-B to be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhibited the same DNA binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antibodies, a centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B boxes with the core recognition sequence or CENP-B box variants) in centromeric satellite DNA.     

Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.     

Presence and abundance of CENP-B box sequences in great ape subsets of primate-specific α-satellite DNA

CENP-B, a highly conserved centromere-associated protein, binds to -satellite DNA, the centromeric satellite of primate chromosomes, at a 17-bp sequence, the CENP-B box. By fluorescence in situ hybridization (FISH) with an oligomer specific for the CENP-B box sequence, we have demonstrated the abundance of CENP-B boxes on all chromosomes (except the Y) of humans, chimpanzee, pygmy chimpanzee, gorilla, and orangutan. This sequence motif was not detected in the genomes of other primates, including gibbons, Old and New World monkeys, and prosimians. Our results indicate that the CENP-B box containing subtype of -satellite DNA may have emerged recently in the evolution of the large-bodied hominoids, after divergence of the phylogenetic lines leading to gibbons and apes; the box is thus on the order of 15–25 million years of age. The rapid process of dispersal and fixation of the CENP-B box sequence throughout the human and great ape genomes is thought to be a consequence of concerted evolution of -satellite subsets on both homologous and nonhomologous chromosomes.Correspondence to: T. Haaf     

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B.     

Nap1 regulates proper CENP-B binding to nucleosomes

CENP-B is a widely conserved centromeric satellite DNA-binding protein, which specifically binds to a 17-bp DNA sequence known as the CENP-B box. CENP-B functions positively in the de novo assembly of centromeric nucleosomes, containing the centromere-specific histone H3 variant, CENP-A. At the same time, CENP-B also prevents undesired assembly of the CENP-A nucleosome through heterochromatin formation on satellite DNA integrated into ectopic sites. Therefore, improper CENP-B binding to chromosomes could be harmful. However, no CENP-B eviction mechanism has yet been reported. In the present study, we found that human Nap1, an acidic histone chaperone, inhibited the non-specific binding of CENP-B to nucleosomes and apparently stimulated CENP-B binding to its cognate CENP-B box DNA in nucleosomes. In human cells, the CENP-B eviction activity of Nap1 was confirmed in model experiments, in which the CENP-B binding to a human artificial chromosome or an ectopic chromosome locus bearing CENP-B boxes was significantly decreased when Nap1 was tethered near the CENP-B box sequence. In contrast, another acidic histone chaperone, sNASP, did not promote CENP-B eviction in vitro and in vivo and did not stimulate specific CENP-B binding to CENP-A nucleosomes in vitro. We therefore propose a novel mechanism of CENP-B regulation by Nap1.     

A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity

The alphoid DNA-CENP-B (centromere protein B) complex is the first sequence-specific DNA/protein complex detected in the centromeric region of human chromosomes. In the reaction, CENP-B recognizes a 17-bp sequence (CENP-B box) and assembles two alphoid DNA molecules into a complex, which is designated complex A (Muro, Y., H. Masumoto, K. Yoda, N. Nozaki, M. Ohashi, and T. Okazaki. 1992. J. Cell Biol. 116:585-596). Since CENP-B gene is conserved in mammalian species and CENP-B boxes are found also in mouse centromere satellite DNA (minor satellite), this sequence-specific DNA-protein interaction may be important for some kind of common centromere function. In this study we have characterized the structure of CENP-B and CENP-B-alphoid DNA complex. We have shown by chemical cross-linking that CENP-B formed a dimer, and have estimated by molecular weight determination the composition of complex A to be a CENP-B dimer and two molecules of alphoid DNA. The DNA binding domain has been delimited within the NH2-terminal 125-amino acid region containing four potential alpha-helices using truncated CENP-B made in Escherichia coli cells. We have shown that CENP-B had sites highly sensitive to proteases and that the DNA binding domain was separable from the dimerizing activity by the proteolytic cleavage at 20 kD from the COOH terminus of the molecule. Thus, CENP-B may organize a higher order structure in the centromere by juxtaposing two CENP-B boxes in the alphoid DNA repeat through both the DNA-protein and protein-protein interactions.     

Stable complex formation of CENP-B with the CENP-A nucleosome

CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.     

CENP-B is a highly conserved mammalian centromere protein with homology to the helix-loop-helix family of proteins

CENP-B is a centromere associated protein originally identified in human cells as an 80 kDa autoantigen recognized by sera from patients with anti-centromere antibodies (ACA). Recent evidence indicates that CENP-B interacts with centromeric heterochromatin in human chromosomes and may bind to a specific subset of human alphoid satellite DNA. CENP-B has not been unambiguously identified in non-primates and could, in principal, be a primate-specific alphoid DNA binding protein. In this work, a human genomic DNA segment containing the CENP-B gene was isolated and subjected to DNA sequence analysis. In vitro expression identified the site for translation initiation of CENP-B, demonstrating that it is encoded by an intronless open reading frame (ORF) in human DNA. A homologous mouse gene was also isolated and characterized. It was found to possess a high degree of homology with the human gene, containing an intronless ORF coding for a 599 residue polypeptide with 96% sequence similarity to human CENP-B. 5 and 3 flanking and untranslated sequences were conserved at a level of 94.6% and 82.7%, respectively, suggesting that the regulatory properties of CENP-B may be conserved as well. CENP-B mRNA was detected in mouse cells and tissues and an immunoreactive nuclear protein identical in size to human CENP-B was detected in mouse 3T3 cells using human ACA. Analysis of the sequence of CENP-B revealed a segment of significant similarity to a DNA binding motif identified for the helix-loop-helix (HLH) family of DNA binding proteins. These data demonstrate that CENP-B is a highly conserved mammalian protein that may be a member of the HLH protein family and suggest that it plays a role in a conserved aspect of centromere structure or function.     

Human centromere protein B induces translational positioning of nucleosomes on alpha-satellite sequences

The human centromere proteins A (CENP-A) and B (CENP-B) are the fundamental centromere components of chromosomes. CENP-A is the centromere-specific histone H3 variant, and CENP-B specifically binds a 17-base pair sequence (the CENP-B box), which appears within every other alpha-satellite DNA repeat. In the present study, we demonstrated centromere-specific nucleosome formation in vitro with recombinant proteins, including histones H2A, H2B, H4, CENP-A, and the DNA-binding domain of CENP-B. The CENP-A nucleosome wraps 147 base pairs of the alpha-satellite sequence within its nucleosome core particle, like the canonical H3 nucleosome. Surprisingly, CENP-B binds to nucleosomal DNA when the CENP-B box is wrapped within the nucleosome core particle and induces translational positioning of the nucleosome without affecting its rotational setting. This CENP-B-induced translational positioning only occurs when the CENP-B box sequence is settled in the proper rotational setting with respect to the histone octamer surface. Therefore, CENP-B may be a determinant for translational positioning of the centromere-specific nucleosomes through its binding to the nucleosomal CENP-B box.     

Organization of the 1.9-kb repeat unit RCE1 in the centromeric region of rice chromosomes

This paper presents the first report on the structure of a 14-kb centromere sequence in a cereal genome that includes 1.9-kb direct repeats. The cereal centromeric sequence (CCS1) conserved in some Gramineae species contains a 17-bp motif similar to the CENP-B box, which serves as the binding site for the centromere-specific protein CENP-B in human. To isolate centromeric units from rice (Oryza sativa L.), we performed PCR using the CENP-B box-like sequences (CBLS) as primers. A 264-bp clone was amplified by this method, and called RCS1516. It appeared to be a novel member of the CCS1 family, sharing about 60% identity with the CCS1 sequences of other cereals. Then, a 14-kb genomic clone, λRCB11, carrying the RCS1516 sequence was isolated and sequenced. It was found to contain three copies of a 1.9-kb direct repeat, RCE1, separated by 5.1- and 1.7-kb. A 300-bp sequence at the 3′ end of RCE1 is highly conserved in all three copies (>90%) and is almost identical to the RCS1516 sequence including the CBLS motif. The copy number of RCE1 was estimated to range from 10² to 10³ in the haploid genome of rice. Cloned RCE1 units were used for fluorescent in situ hybridization (FISH) analysis, and signals were observed on almost every primary constriction of rice chromosomes. Thus it was concluded that RCE1 is a significant component of the rice centromere. The λRCB11 clone contained at least four A/T-rich regions, which are candidate for matrix attachment regions (MARs), in the sequences between the RCE1 repeats. Other elements that are homologous to the short centromeric repetitive sequences pSau3A9 and pRG5, detected in both sorghum and rice, were also found in the clone. Received: 9 June 1998 / Accepted: 16 September 1998     

Molecular cytogenetic analysis of the highly repetitive DNA in the genome of Apodemus argenteus, with comments on the phylogenetic relationships in the genus Apodemus.

The DNA of Apodemus argenteus was digested with DraI, and the resultant DraI fragment of highly repetitive DNA was isolated and analyzed by DNA filter hybridization, cloning, sequencing, and fluorescence in situ hybridization (FISH). Southern blot hybridization and nucleotide sequencing revealed that most of the DraI fragment consisted of a 230-bp repeating unit and contained no sex-chromosome-specific nucleotide sequences. The DraI fragment included the CENP-B box-like sequence, with a strong homology to the human CENP-B box sequence. FISH revealed that the DraI fragment was specific to all pericentromeric C-band-positive regions, as well as to the C-block of the X chromosome. No hybridization signals were obtained from A. speciosus, A. peninsulae peninsulae, A.p. giliacus, A. agrarius, A. sylvaticus, A. semotus, or Mus musculus when the DraI fragment was used as probe. Peptide nucleic acid (PNA)-FISH using the CENP-B box-like sequence in the DraI fragments as probe suggested that this nucleotide sequence was also specific to all pericentromeric C-heterochromatic regions of A. argenteus chromosomes. Zoo-blot hybridization using DraI-digested genomic DNA from three species of Apodemus (namely, A. argenteus, A. speciosus, and A. peninsulae) and from Mus musculus strongly suggested that the consensus DraI fragment contained nucleotide sequences that were species-specific for A. argenteus. These results also suggest that A. argenteus is phylogenetically distant from other Apodemus species examined, as well as the possibility that the DraI fragment might be related directly to the delayed quinacrine mustard fluorescence of many pericentromeric C-heterochromatic regions of the chromosomes in A. argenteus.     

A helix-turn-helix structure unit in human centromere protein B (CENP-B).

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.     

Cytogenetic and molecular evaluation of centromere-associated DNA sequences from a marsupial (Macropodidae: Macropus rufogriseus) X chromosome

The constitution of the centromeric portions of the sex chromosomes of the red-necked wallaby, Macropus rufogriseus (family Macropodidae, subfamily Macropodinae), was investigated to develop an overview of the sequence composition of centromeres in a marsupial genome that harbors large amounts of centric and pericentric heterochromatin. The large, C-band-positive centromeric region of the X chromosome was microdissected and the isolated DNA was microcloned. Further sequence and cytogenetic analyses of three representative clones show that all chromosomes in this species carry a 178-bp satellite sequence containing a CENP-B DNA binding domain (CENP-B box) shown herein to selectively bind marsupial CENP-B protein. Two other repeats isolated in this study localize specifically to the sex chromosomes yet differ in copy number and intrachromosomal distribution. Immunocytohistochemistry assays with anti-CENP-E, anti-CREST, anti-CENP-B, and anti-trimethyl-H3K9 antibodies defined a restricted point localization of the outer kinetochore at the functional centromere within an enlarged pericentric and heterochromatic region. The distribution of these repeated sequences within the karyotype of this species, coupled with the apparent high copy number of these sequences, indicates a capacity for retention of large amounts of centromere-associated DNA in the genome of M. rufogriseus.     

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.     

The centromeric satellite of the wedge sole (Dicologoglossa cuneata, Pleuronectiformes) is composed mainly of a sequence motif conserved in other vertebrate centromeric DNAs

Here, a new satellite-DNA family is isolated and characterized from wedge sole, Dicologoglossa cuneata Moreau, 1881 (Pleuronectiformes), a fish having a small genome. This satellite-DNA family of sequences was isolated by conventional cloning after digestion of genomic DNA with the DraI restriction enzyme. Repeat units are 171 bp in length with a high AT content (63%). Several runs of consecutive adenines and thymines were found, and concomitantly computer analyses revealed that these regions are prone to acquire stable sequence-directed curvature. Especially remarkable is that the DraI sequences are composed almost entirely of the repetition of up to fourteen 9-bp motifs (T/C)GTC(A/C)AAAA similar to other vertebrate centromeric satellite-DNA sequences. In fact, we demonstrate the origin of this satellite through duplication of this motif plus the addition of a stretch of cytosines. The centromeric location and the presence in this satellite-DNA sequence of not only different vertebrate motifs (CENP-B box, pJalpha) but also others such as the CDEIII motif of Saccharomyces cerevisiae reveal a possible role in centromere function. All these characteristics provide important information on the origin, function, and the evolution of the centromeric satellite DNAs in wedge sole.     

CENP-B is a conserved gene among vegetal species

To explore the CENP-B centromere protein in beans, carrots, onions and potatoes, total RNA was isolated and reverse transcribed by PCR, and the cDNA encoding the CENP-B amino terminus domain amplified using CENP-B oligonucleotides. Blots containing PCR products were hybridized with a nick-translated pG/CNPB probe containing a complete human CENP-B gene. In all the plant species, anti-CENP-B antibodies recognized an 80-kDa protein. A 360-bp sequence encoding for the amino terminus region of the CENP-B protein was amplified by PCR in all the species and the nick translated pG/CNPB probe hybridized with the PCR products. Apparently the CENP-B centromere protein or an equivalent protein is widely distributed in the vegetal kingdom.