The cenpB gene is not essential in mice

Centromere protein B (CENP-B) is a centromeric DNA-binding protein that binds to alpha-satellite DNA at the 17 bp CENP-B box sequence. The binding of CENP-B, along with other proteins, to alpha-satellite DNA sequences at the centromere, is thought to package the DNA into heterochromatin subjacent to the kinetochore of mitotic chromosomes. To determine the importance of CENP-B to kinetochore assembly and function, we generated a mouse null for the cenpB gene. The deletion removed part of the promoter and the entire coding sequence except for the carboxyl-terminal 35 amino acids of the CENP-B polypeptide. Mice heterozygous or homozygous for the cenpB null mutation are viable and healthy, with no apparent defect in growth and morphology. We have established mouse embryo fibroblasts from heterozygous and homozygous cenpB null littermates. Microscopic analysis, using immunofluorescence and electron microscopy of the cultured cells, indicated that the centromere-kinetochore complex was intact and identical to control cells. Mitosis was identical in fibroblasts derived from cenpB wild-type, heterozygous and null animals. Our studies demonstrate that CENP-B is not required for the assembly of heterochromatin or the kinetochore, or for completion of mitosis.     

A neocentromere on human chromosome 3 without detectable alpha-satellite DNA forms morphologically normal kinetochores

A neocentromere at 3q26 was observed in a father and his daughter on a chromosome 3 with deleted centromeric region. No alpha-satellite DNA was detectable at the 3q26 neocentromere, but it was weakly positive with anticentromere (CREST) antibodies. Electron microscopy showed that the neocentromere formed microtubule-associated kinetochores with normal morphology and of the same size as the kinetochores of other large chromosomes. The deleted centromere formed a small linear marker chromosome that reacted strongly with anticentromere antibodies, but showed reduced kinetochore size. The 3q26 neokinetochore was stable under adverse growth conditions, which often caused kinetochore loss in the original 3-centromere on the small marker.     

Sequence analysis of an 80 kb human neocentromere

We previously described the cloning of an 80 kb DNA corresponding to the core protein-binding domain of a human chromosome 10-derived neocentromere. Here we report the complete sequence of this DNA (designated NC DNA) and its detailed structural analysis. The sequence is devoid of human centromeric alpha-satellite DNA and the pericentric beta- and gamma-satellites, the ATRS and 48 bp repeat DNA. One copy of a sequence that is related to the CENPB box motif is present, and a number of copies of other pericentric sequences including pJalpha and classical satellites I and III are present but both their relative sparsity and non-tandem organization suggest that each sequence, on its own, is unlikely to mimic any role the sequence may have in the normal centromere. The DNA-binding motifs of the architectural and regulatory proteins HMGI and topoII have a normal abundance and random distribution, implying that these sequences are not key functional elements. The total A + T content of the sequence is not notably different from that of the human genome, but an abundance of AT-rich islands and a biased distribution of these islands within the NC sequence are clearlydiscernible and may be functionally significant. Substantial amounts of transposable elements and low copy number tandem repeats, including several that are highly AT- and purine-rich, are also present and may act as functional elements. One of the AT-rich tandemrepeats (AT28) may form interesting structures and is described in detail. The defined features show only a loose resemblance to the structures of known centromeres, highlighting the possibility that, rather than a conserved primary sequence, it is the overallcomposition and distribution patterns of various unknown functional elements, or any 'ordinary' DNA under appropriate epigenetic influences, that determine centromere formation and function. This is the firstdetailed analysis of a neocentromere DNA and provides a basis for comparison against future sequences.     

The centromere enhancer mediates centromere activation in Schizosaccharomyces pombe

The centromere enhancer is a functionally important DNA region within the Schizosaccharomyces pombe centromeric K-type repeat. We have previously shown that addition of the enhancer and cen2 centromeric central core to a circular minichromosome is sufficient to impart appreciable centromere function. A more detailed analysis of the enhancer shows that it is dispensable for centromere function in a cen1-derived minichromosome containing the central core and the remainder of the K-type repeat, indicating that the critical centromeric K-type repeat, like the central core, is characterized by functional redundancy. The centromeric enhancer is required, however, for a central core-carrying minichromosome to exhibit immediate centromere activity when the circular DNA is introduced via transformation into S. pombe. This immediate activation is probably a consequence of a centromere-targeted epigenetic system that governs the chromatin architecture of the region. Moreover, our studies show that two entirely different DNA sequences, consisting of elements derived from two native centromeres, can display centromere function. An S. pombe CENP-B-like protein, Abp1p/Cbp1p, which is required for proper chromosome segregation in vivo, binds in vitro to sites within and adjacent to the modular centromere enhancer, as well as within the centromeric central cores. These results provide direct evidence in fission yeast of a model, similar to one proposed for mammalian systems, whereby no specific sequence is necessary for centromere function but certain classes of sequences are competent to build the appropriate chromatin foundation upon which the centromere/kinetochore can be formed and activated.     

NMR structure of the extended Myb cognate sequence and modeling studies on specific DNA-Myb complexes

The recognition sequence of the Myb protein has been recently described to be pyAACKGHH (where py = T/C, K = G/T, and H = A/C/T), modifying the earlier identification as pyAACKG [Ording, E., et al. (1994) Eur. J. Biochem. 222, 113-120]. We had earlier determined the solution structure of the minimal cognate sequence TAACGG, choosing py = T and K = G, embeded in a 12-mer DNA duplex by NMR and related computational techniques [Radha, P. K., et al. (1995) Biochemistry 34, 5913-5912]. To understand the structural significance of the above modification and the role of the variability in the recognition sequence, we have investigated here the solution structure of a different DNA segment, d-ACAACTGCAGTTGT, which contains the extended Myb cognate site, CAACTGCA. The three-dimensional structure of the 14-mer duplex has been determined from NMR data by relaxation matrix and restrained molecular dynamics calculations. The structure of the above cognate sequence in the 14-mer duplex has been compared with that of the cognate sequence, TAACGG, in the 12-mer duplex and also with that in the NMR structure of the Myb DNA binding domain (R2R3)-DNA complex determined by Ogata et al. recently [Ogata, K., et al. (1994) Cell 79, 639-648]. The comparison highlighted differences in several structural parameters for the cognate sites in the DNA segments. Modeling studies by taking out the protein from the complex and presenting it with 12-mer and 14-mer DNA structures indicated that the protein induces structural alterations to drive the cognate site to a reasonably conserved structure. The extent of similarity of the derived structures was, however, dependent on the base sequences. Base changes in the minimal cognate sequence in the 12-mer-protein complex and in the 14-mer-protein complex so as to match the sequence of Ogata et al. produced a more conserved structure of the complex. A reverse exercise, in which the Ogata DNA in the complex was mutated to match the 12-mer and 14-mer minimal cognate sequences, complemented the above observations of the subtle sequence dependence of the structure in the complex. On the other hand, base changes in the extension did not influence the DNA-protein complex structure significantly. We also observed that the structural changes in the protein were very minor when different DNA sequences or different DNA structures were presented to it. These observations would be of interest from the point of view of DNA-Myb recognition.     

Serial determinations of cerebral water content by magnetic resonance imaging after an infusion of hypertonic saline

We have determined the genomic structure and organization of the mouse Cenpa and Cenpc genes. CENPA is a member of the histone H3-like proteins and is thought to replace histone H3 in centromeric nucleosomes. CENPC is a DNA-binding protein that is located at the inner kinetochore plate of active mammalian centromeres. The Cenpa cDNA encodes a 134-amino-acid product that is 70% identical and 84% similar to its human homolog. The mouse Cenpa gene is approximately 8 kb in length and contains five exons. Sequence analysis of the 5' DNA sequence of the gene revealed two consensus CAAT boxes, a putative TFIID-binding site, an Sp1-binding domain, and two cell cycle regulatory motifs, but no consensus TATA element. The mouse Cenpc gene spans 60 kb and contains 19 exons that range in size from 44 to 602 bp. Sequence analysis of the C+G-rich promoter region showed the presence of known promoter elements, including a CpG island, a CAAT box, and several GC boxes, but the absence of a consensus TATA element.     

Comparison of 18FDG-PET with 131iodine and 99mTc-sestamibi scintigraphy in differentiated thyroid cancer

Based on sequence analyses of 17 complete centromeric DNA monomers from ten different deer species, a model is proposed for the genesis, evolution, and genomic organization of cervid satellite I DNA. All cervid satellite I DNA arose from the initial amplification of a 31-bp DNA sequence. These 31-bp subrepeats were organized in a hierarchical fashion as 0.8-kb monomers in plesiometacarpalia deer and 1-kb monomers in telemetacarpalia deer. The higher-order repeat nature of cervid centromeric satellite DNA monomers accounts for their high intragenomic and intraspecific sequence conservation. Such high intraspecific sequence conservation validates the use of a single cervid satellite I DNA monomer from each deer species for interspecific sequence comparisons to elucidate phylogenetic relationships. Also, a specific 0.18-kb tandem duplication was observed in all 1-kb monomers, implying that 1-kb cervid satellite I DNA monomers arose from an unequal crossover event between two similar 0.8-kb ancestral DNA sequences.     

Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin

Tandemly repeated DNA families appear to undergo concerted evolution, such that repeat units within a species have a higher degree of sequence similarity than repeat units from even closely related species. While intraspecies homogenization of repeat units can be explained satisfactorily by repeated rounds of genetic exchange processes such as unequal crossing over and/or gene conversion, the parameters controlling these processes remain largely unknown. Alpha satellite DNA is a noncoding tandemly repeated DNA family found at the centromeres of all human and primate chromosomes. We have used sequence analysis to investigate the molecular basis of 13 variant alpha satellite repeat units, allowing comparison of multiple independent recombination events in closely related DNA sequences. The distribution of these events within the 171-bp monomer is nonrandom and clusters in a distinct 20- to 25-bp region, suggesting possible effects of primary sequence and/or chromatin structure. The position of these recombination events may be associated with the location within the higher-order repeat unit of the binding site for the centromere-specific protein CENP-B. These studies have implications for the molecular nature of genetic recombination, mechanisms of concerted evolution, and higher-order structure of centromeric heterochromatin.     

A method for measurement of angiotensin II in tissues and its application to rat kidney

Two distinct satellite DNAs, amounting to 25% of the total DNA, were isolated from the nuclei of the red-necked wallaby, Macropus rufogriseus. The physical properties of native, single-stranded and reassociated molecules were studied in buoyant-density gradient centrifugation. The homogeneity of each satellite fraction was examined using melting characteristics of native and reassociated DNA, and renaturation kinetics. These data suggest that sequence heterogeneity exists in both fractions. Each satellite fraction was found by in situ hybridization to be localized in heterochromatin of interphase nuclei and in the centromeric regions of metaphase chromosomes. The chromosomal distributions of the two satellite DNAs differentiate the sex chromosomes, which have sequences of only one satellite, from the autosomes which have sequences of both satellites in the centromeric heterochromatin. Giemsa C-banding techniques also showed a differentiation of the centromeric regions of sex chromosomes from those of the autosomes.     

Activation of latent HIV-1 by Mycobacterium tuberculosis and its purified protein derivative in alveolar macrophages from HIV-infected individuals in vitro

A chromosome-specific satellite DNA from the South American fish species Leporinus obtusidens has been isolated and characterized. Sequence analysis and Southern hybridization studies indicate that the cloned 483-bp fragment is 60% AT rich and appears to comprise two diverged monomers. A highly variable low-copy number polymorphism was detected and, thus, this satellite DNA may serve as a valuable genetic marker. Using a Southern blot approach, the cloned satellite DNA cross-hybridized strongly to the DNA of Leporinus elongatus but failed to detect homologous sequences in the genomes of other closely related Leporinus species and higher vertebrates. Using fluorescence in situ hybridization to mitotic metaphase spreads of L. obtusidens and L. elongatus, this satellite DNA was located to the (peri)centromeric region of one single chromosome pair in both species. As the cloned satellite DNA sequence clearly evolved along a chromosomal lineage and is highly variable, it may serve as a very useful marker in further genetic, molecular and cytogenetic studies of the genus Leporinus.     

Hybridization of chromosome 18 alpha-satellite DNA probe to chromosome 22

Fluorescence in situ hybridization (FISH) of uncultured chorionic villus diploid cells with a chromosome 18 alpha-satellite DNA probe (D18Z1) revealed a third small signal in addition to two large signals. FISH analysis of diploid metaphase cells from cultured chorionic villus cells and from maternal lymphocytes revealed that the third signal resulted from hybridization to the centromere of chromosome 22. This is the first report of a variant involving D18Z1 detected by FISH and of hybridization of alpha-satellite from a sub-metacentric chromosome to the centromere of an acrocentric chromosome. We propose that this inherited variant resulted from insertion of chromosome 18 specific alpha-satellite DNA sequences into the centromeric region of chromosome 22.     

Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

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.     

Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions

A set of oat-maize chromosome addition lines with individual maize (Zea mays L.) chromosomes present in plants with a complete oat (Avena sativa L.) chromosome complement provides a unique opportunity to analyze the organization of centromeric regions of each maize chromosome. A DNA sequence, MCS1a, described previously as a maize centromere-associated sequence, was used as a probe to isolate cosmid clones from a genomic library made of DNA purified from a maize chromosome 9 addition line. Analysis of six cosmid clones containing centromeric DNA segments revealed a complex organization. The MCS1a sequence was found to comprise a portion of the long terminal repeats of a retrotransposon-like repeated element, termed CentA. Two of the six cosmid clones contained regions composed of a newly identified family of tandem repeats, termed CentC. Copies of CentA and tandem arrays of CentC are interspersed with other repetitive elements, including the previously identified maize retroelements Huck and Prem2. Fluorescence in situ hybridization revealed that CentC and CentA elements are limited to the centromeric region of each maize chromosome. The retroelements Huck and Prem2 are dispersed along all maize chromosomes, although Huck elements are present in an increased concentration around centromeric regions. Significant variation in the size of the blocks of CentC and in the copy number of CentA elements, as well as restriction fragment length variations were detected within the centromeric region of each maize chromosome studied. The different proportions and arrangements of these elements and likely others provide each centromeric region with a unique overall structure.     

CeRep25B forms chromosome-specific minisatellite arrays in Caenorhabditis elegans

With the completion of the Genome Sequencing Project, it is now possible to rapidly and accurately determine the frequency and position of a particular repeat sequence in the Caenorhabditis elegans genome. Several repeat sequences with a variety of characteristics have been examined and with few exceptions they show a near-random distribution throughout the genome. We characterized several genes near the left end of Chromosome III in the C. elegans genome, and found a 24-bp minisatellite repeat sequence present in the introns of two unrelated genes. This prompted a search of the databank for other occurrences of this sequence. Multiple copy arrays of this repeat are all located on the same autosome and fall in two clusters: one near the left end, and one in the central region separated by approximately 10 Mb. There are >200 copies of this repeat on the chromosome. This euchromatic repeat sequence seems unrelated to gene expression, is absent from homologous sites in a related species, is unstable in Escherichia coli, and is polymorphic between different wild isolates of C. elegans. Most CeRep25B units in the array match the consensus sequence very well, suggesting that either this repeat originated quite recently or its sequence is functionally constrained. Although chromosome-specific repeat sequences have been reported previously in many organisms, such sequences are usually structural and heterochromatic (e.g., centromeric alpha-satellite) or on the mammalian sex chromosomes. This report describes the first confirmed instance from a whole genome sequencing project of an autosomal euchromatic chromosome-specific minisatellite repeat.     

Hearing the voices of women in war through continuous quality improvement

Double color in situ hybridization was used to determine the specificity of cloned alpha-satellite cosmid DNA sequences belonging to satellite DNA sequences shared by chromosomes 13 and 21. It was determined that cosmid clone cos56D8 is more specific to chromosome 13, cosmid clone cos37E is more specific to chromosome 21 and cosmid clones cosA5130 is specific to both chromosomes 13 and 21. These new cosmid probes could bw useful in the molecular-cytogenetic analysis of trisomic cells at Patau or Down syndromes as well as in analysis of marker chromosomes originated from the chromosomes 13 and 21.     

Fragmentation of centromeric DNA and prevention of homologous chromosome separation in male mouse meiosis in vivo by the topoisomerase II inhibitor etoposide

The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe; and (ii) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction.     

Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.