Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes.

We have partially isolated the kinetochore and associated centromeric structures from mammalian metaphase chromosomes. Human autoantibodies from scleroderma CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) patients were used as immunofluorescent probes to monitor fractionation. The procedure includes digestion of total chromosomal DNA with micrococcal nuclease, dehistonization with heparin, and dissociation of the remaining material with detergent and urea. We used a density gradient (metrizamide) to obtain an enriched fraction of stained material (kinetochore). When examined by electron microscopy, the kinetochore fraction is seen to contain numerous small immunoperoxidase-positive masses which are morphologically similar to the centromere/kinetochore region of intact metaphase chromosomes. The particulate fraction that contains kinetochore components represents less than 5% of total chromosomal proteins and contains less than 1% of total DNA. Two polypeptides of 18 and 80 kD were identified as kinetochore antigens by immunoblotting with CREST antiserum. In this paper we discuss the distribution of these kinetochore polypeptides with the associated centromeric chromatin.

High-resolution mapping of satellite DNA using biotin-labeled DNA probes.

We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity-purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.

CENP-B: a major human centromere protein located beneath the kinetochore.

The family of three structurally related autoantigens CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD) are the best characterized components of the human centromere, and they have been widely assumed to be components of the kinetochore. Kinetochore components are currently of great interest since this structure, which has long been known to be the site of microtubule attachment to the chromosome, is now believed to be a site of force production for anaphase chromosome movement. In the present study we have mapped the distribution of CENP-B in mitotic chromosomes by immunoelectron microscopy using two monospecific polyclonal antibodies together with a newly developed series of ultra-small 1-nm colloidal gold probes. We were surprised to find that greater than 95% of CENP-B is distributed throughout the centromeric heterochromatin beneath the kinetochore. This strongly supports other emerging evidence that CENP-B is specifically associated with alpha-satellite heterochromatin. Although in certain instances CENP-B can be seen to be concentrated immediately adjacent to the lower surface of the kinetochore, the outer plate remains virtually unlabeled. Similar analysis with a human autoimmune serum that recognizes all three CENP antigens reveals an additional unsuspected feature of kinetochore structure. In addition to recognizing antigens in the centromeric heterochromatin, the autoantiserum recognizes a concentration of antigens lateral to the kinetochore. This difference in staining pattern may reflect the presence of a "collar" of chromatin rich in CENP-C and/or CENP-A encircling the kinetochore plates.

Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae.

We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selectively excised from the chromatin DNA with restriction endonucleases. CEN3 DNA is organized in chromatin such that a 220-250-bp region encompassing the elements of centromere homology is resistant to nuclease digestion. Restriction enzyme linkers encoding the Bam HI-recognition site were ligated to a 289 base pair DNA segment that spans the 220-250-bp protected core (Bloom et al., 1984). Replacement of this CEN3-Bam HI linker cassette into a chromosome or plasmid results in formation of a complete structural and functional centromeric unit. A centromere core complex that retains its protected chromatin conformation can be selectively excised from intact nuclei by restriction with the enzyme Bam HI. The centromeric protein-DNA complex is therefore not dependent upon the intact torsional constrains on linear chromosomes for its structural integrity. Isolation of this complex provides a novel approach to characterizing authentic centromeric proteins bound to DNA in their native state.

Ribonucleoprotein staining of centrioles and kinetochores in newt lung cell spindles.

The distribution of ribonucleoprotein (RNP) within the mitotic spindle of newt lung epithelial cells was studied with the high voltage electron microscope (HVEM) using Bernhard's uranyl-EDTA-lead staining of thick sections in conjunction with the ribonuclease digestion of fixed cells. The results indicate that aside from ribosomes, the major RNP-containing components of the spindle are the kinetochores and centrioles, both of which stain electron-opaque after EDTA treatment. In both cases, the electron-opaque material associated with these microtubule organizing centers (MTOC's) can be removed by RNAse digestion and cold perchloric acid (PCA) extraction under conditions which leave the spindle microtubules (Mts) centrioles, and kinetochores intact. The staining reaction is not abolished by cold PCA extraction alone or by substituting other positively charged proteins (i.e., cytochrome c or lysozyme) for RNAse. The RNP component of the kinetochore is closely associated with the bases of the kinetochore microtubules. The RNP component of the centriole can be seen to surround the microtubules of the triplet blades. No evidence was found to indicate the presence of RNP in the pericentriolar material. The possible function of both kinetochore and centriolar RNP is discussed.

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus.

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.

Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae.

Saccharomyces cerevisiae centromeres have a characteristic 120-base-pair region consisting of three distinct centromere DNA sequence elements (CDEI, CDEII, and CDEIII). We have generated a series of 26 CEN mutations in vitro (including 22 point mutations, 3 insertions, and 1 deletion) and tested their effects on mitotic chromosome segregation by using a new vector system. The yeast transformation vector pYCF5 was constructed to introduce wild-type and mutant CEN DNAs onto large, linear chromosome fragments which are mitotically stable and nonessential. Six point mutations in CDEI show increased rates of chromosome loss events per cell division of 2- to 10-fold. Twenty mutations in CDEIII exhibit chromosome loss rates that vary from wild type (10(-4)) to nonfunctional (greater than 10(-1)). These results directly identify nucleotides within CDEI and CDEIII that are required for the specification of a functional centromere and show that the degree of conservation of an individual base does not necessarily reflect its importance in mitotic CEN function.

High frequency DNA rearrangements associated with mouse centromeric satellite DNA.

A DNA transformed mouse cell line, generated by the microinjection of a pBR322 plasmid containing the herpes thymidine kinase (tk) gene, was observed to exhibit a high frequency of DNA rearrangement at the site of exogenous DNA integration. The instability in this cell line does not appear to be mediated by the tk inserts or the immediately adjacent mouse DNA, but instead may be a consequence of the larger host environment at the chromosomal site of tk insertion. Results obtained from restriction analysis, in situ chromosome hybridizations, and cesium chloride density-gradient fractionations indicate that the tk inserts are organized as a single cluster of direct and inverted repeats embedded within pericentromeric satellite DNA. To determine the molecular identity of the flanking host sequences, one of the mouse-tk junction fragments was cloned, and subsequent restriction and sequence analyses revealed that this DNA fragment consists almost entirely of classical mouse satellite DNA. On the basis of these observations, we suggest that the instability in this cell line may reflect the endogenous instability or fluidity of satellite DNA.

Continuous reorganization leads to extensive polymorphism in a monkey centromeric satellite.

Previously we reported the existence of a highly polymorphic satellite, deca-satellite, in the African green monkey genome; deca-satellite probe anneals to complex sets of repeated restriction endonuclease fragments that differ from individual to individual in the monkey population. Here we present experiments aimed at clarifying the structure and organization of deca-satellite sequences and investigating the mechanisms that generate the polymorphisms. Deca-satellite represents less than 1% of the monkey genome but the percentage varies from one monkey to another. The core sequence 5'-C-C-G-G within the ten base-pair deca-satellite repeat unit is well conserved and the central 5'-C-G is sometimes but not always methylated. Restriction endonuclease analysis with BamHI and EcoRI defines separate satellite domains that have evolved in an independent manner. In situ hybridization shows deca-satellite to be located at the centromeric regions of some but not all monkey chromosomes. This location is independently confirmed by a high frequency, in monkey libraries, of segments containing junctions between deca-satellite and alpha-satellite, the main monkey centromeric satellite. The total number of metaphase chromosomes that show centromeric grains after in situ hybridization with a deca-satellite probe varies from one monkey to another. Moreover, in situ hybridization to endoreduplicated diplochromosomes showed that deca-satellite is occasionally distributed asymmetrically on one or the other of the two pairs of sister chromatids in one diplochromosome. This indicates that major reorganization of the satellite can occur frequently in somatic cells. We discuss several possible mechanisms by which deca-satellite sequences could be either amplified or deleted during a single replicative cycle. Also, on the basis of the marked fluidity of deca-satellite abundance and organization and other well-known attributes of centromeric satellites, we suggest that the existence and maintenance of centromeric satellite rests on the role of the tandem repeats themselves and not on any particular nucleotide sequence, repeat length or organization.

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome.

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.

Immunofluorescence analysis of sucrose-induced changes in spindle morphology.

Metaphase and anaphase PtK1 cells show spindle elongation without concomitant chromosome motion when treated with culture medium containing 0.5 M sucrose. Electron microscopy has shown sucrose-induced changes in microtubule (MT) organization, changes in trilaminar kinetochore structure, and specific kinetochore-MT associations which may account for these results. In this paper we employ double-label immunofluorescence techniques using antibodies against tubulin and the kinetochore to analyze changes in spindle microtubule and kinetochore distribution produced by sucrose treatment. Cells treated from prometaphase through anaphase with 0.5 M sucrose from 10 min to 2 h showed spindle elongation and a distinct rearrangement of spindle microtubules into bundles, with a pronounced increase in length of interpolar microtubule bundles. In sucrose-treated mitotic cells kinetochores remained as antigenically distinct structures, similar to those found in untreated interphase cells. Kinetochore determinants remained positioned within a diffuse chromatin mass, but the orientation of sister kinetochores to opposite spindle poles was lost. Instead, kinetochore pairs were found in lateral association with microtubule bundles, with several pairs of determinants associated with a single bundle in many instances. Cells released from 0.5 M sucrose treatment showed a return of the spindle to a pretreatment arrangement for both the microtubules and kinetochore determinants.

Molecular evolution of centromere-associated nucleotide sequences in two species of canids.

The major centromeric satellite nt sequences present in the domestic dog (Canis familiaris) and in the grey fox (Urocyon cineroargenteus) have been examined. The dog satellite monomer is 737 bp long and contains 51% G + C; the grey fox satellite monomer is 880 b long and contains 54% G + C. The two satellites share three regions of 78, 92 and 314 bp with 70-80% sequence similarity. Sequence data from 16 monomers of dog satellite and 19 monomers of grey fox satellite demonstrate that the substitution spectra are different in the two canid species. For example, substitutions involving G or C residues are much more common in the grey fox satellite than in the domestic dog satellite despite their similar G + C contents.

Slit-scan flow cytometry of mammalian chromosomes.

A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded values represents the distribution of fluorescence along the chromosome and is stored in the memory of a waveform recorder. The total fluorescence of each chromosome is also measured and recorded. Preliminary studies show that doublets of 1.83 micrometers diameter microspheres flow with their long axes parallel to the direction of flow and that the two microspheres are resolved in the slit-scan profile. Ethidium bromide stained Muntjac and Chinese hamster chromosomes have also been slit-scanned. Centromeres were resolved in many of the Nos. 1 and 2 Chinese hamster chromosomes and the Nos. 1 and X + 3 Muntjac chromosomes.

Investigation of a continuous murine cell line RVP3 by C-staining of chromosomes.

A continuous murine cell line RVP3 was karyologically examined, using staining for centromeric heterochromatin. All cells of this line contained microchromosomes with centromeric heterochromatin. The origin of the microchromosomes is discussed.

Structure of repeated sequences in the centromeric region of the human Y chromosome.

Alphoid satellite DNA is a family of sequences with an approximately 170 bp periodicity which is found near the centromere of all human chromosomes. The structure of the human Y-chromosome alphoid DNA has been studied in two somatic cell hybrids, 3E7 and 853 (Tyler-Smith & Brown, 1987). The 170 bp alphoid subunits are tandemly repeated and are organized into units approximately 5.7 kb long. A few variant units on the 3E7 Y chromosome contain two extra 170 bp subunits and are approximately 6.0 kb long; the variant units are present in two clusters at least 90 kb apart on the chromosome. On each Y chromosome there is a single major block of alphoid DNA: on the 3E7 Y chromosome it is approximately 440 kb long and on the 853 Y chromosome it is approximately 540 kb long. A long-range restriction map of the 853 block has been constructed covering approximately 1.1 mb of DNA. The distribution of restriction sites suggests that the sequences on one side of the alphoid block may be typical euchromatic DNA, while the sequences on the other side may be another satellite sequence.

Partial inversion of the secondary constriction of chromosome 9. Does it exist?

Pericentric inversion of chromosome 9, a common abnormality, has been much studied because of its possible genetic effect. Apart from total inversion, in which the whole heterochromatic segment of chromosome 9 appears to be situated on the short arm, some authors describe partial inversion, in which the heterochromatin is found partly on the long arm and partly on the short arm. Our study indicates that firstly, the heterochromatic segment of chromosome 9 is composed of two biochemically different subunits: the heterochromatin of the centromere itself and the heterochromatin of the secondary constriction. Secondly, it suggests that partial inversion of the secondary constriction of chromosome 9 is an unusual event, as the majority of published cases can be interpreted as the result of an increase in the centromeric heterochromatin without alteration of the secondary constriction.