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.     

Assembly of correct kinetochore architecture in Xenopus laevis egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one employs direct assembly of chromatin from sperm nuclei in CSF-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step, followed by re-establishing of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: the amounts of outer kinetochore proteins such as Bub1, BubR1 and Dynactin subunit p150glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. In contrast, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results argue that transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

Disruption of the typical chromatin structure in a 2500 base-pair region at the 5' end of the actively transcribed ovalbumin gene.

We examined the chromatin organizations of approximately 3 kb of DNA in the 5'-end flanking region of the ovalbumin gene in chicken erythrocyte and oviduct cell nuclei. With specific DNA probes and an indirect end-labeling technique, we analysed the pattern of the DNA fragments obtained after micrococcal nuclease digestion and generated comparative maps of the nuclease cuts. This region of the chicken genome displays a "typical" chromatin arrangement in erythrocyte nuclei, with nucleosomes apparently positioned at random. In contrast, in oviduct nuclei, the same region has an "altered" chromatin structure, and lacks a typical nucleosomal array. The existence of specifically positioned proteins and of alterations in the DNA secondary structure in this region of the oviduct chromatin is suggested by comparison of the nuclease cleavage maps which reveals specific changes: disappearance of nuclease cuts present in "naked" and erythrocyte chromatin DNAs, and appearance of new cuts absent from these DNAs.     

The kinetochore is part of the metaphase chromosome scaffold

We used antisera from patients with the CREST syndrome of scleroderma (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) to show that an antigenic component of the kinetochore present in metaphase chromosomes is also present in nonhistone chromosome scaffolds isolated following extensive digestion of the DNA and extraction of the bulk of chromosomal protein. All sera from 12 scleroderma CREST patients previously shown by immunofluorescence microscopy to have circulating antikinetochore antibodies recognise a protein of Mr 77,000 (CREST-77) in an immunoblotting assay. 9 of the 12 sera also recognise an antigen of Mr 110,000 (CREST-110). These proteins are present in isolated chromosomes and nonhistone scaffolds derived from them by two different procedures. Sera of five scleroderma CREST patients who are antikinetochore negative (by immunofluorescence) bind to neither protein in immunoblots. These data suggest that CREST-77 (and possibly CREST-110) is a component of the human kinetochore, and that the kinetochore is an integral part of the mitotic chromosome scaffolding.     

Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum

Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Assembly of correct kinetochore architecture in Xenopus egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying the formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one uses a direct chromatin assembly from sperm nuclei in cytostatic factor (CSF)-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step with subsequent reestablishment of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: The amounts of outer kinetochore proteins such as Bub1, BubR1, and Dynactin subunit p150^glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. On the contrary, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results indicate that the transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

Chemical subdomains within the kinetochore domain of isolated CHO mitotic chromosomes

We have used indirect immunofluorescence in combination with correlative EM to subdivide the mammalian kinetochore into two domains based on the localization of specific antigens. We demonstrate here that the fibrous corona on the distal face of the kinetochore plate contains tubulin (previously shown by Mitchison, T. J., and M. W. Kirschner. 1985. J. Cell Biol. 101:755-765) and the minus end-directed, ATP-dependent microtubule motor protein, dynein; whereas a 50-kD CREST antigen is located internal to these components in the kinetochore. Tubulin and dynein can be extracted from the kinetochore by 150 mM KI, leaving other, as yet uncharacterized, components of the kinetochore corona intact. Microtubules and tubulin subunits will associate with kinetochores in vitro after extraction with 150 mM KI, suggesting that other functionally significant, corona-associated molecules remain unextracted. Our results suggest that the corona region of the kinetochore contains the machinery for chromosome translocation along microtubules.     

Monoclonal antibodies to tissue-specific chromatin proteins

Antisera raised in mice to chromatins from different tissues of the chicken reacted preferentially with the chromatin type that was used for immunization. This tissue specificity was also evident in the spectrum of monoclonal antibodies generated when mice were immunized with erythrocyte chromatin. Three erythroid-specific antigens and one antigen that was present in a number of chicken tissues were characterized in further detail. These antigens, which comprised less than 0.1% of the erythrocyte chromatin proteins, were nuclear localized although three were also detected in the cytoplasm. Two of the erythroid-specific antigens existed as multiple polypeptides in isolated chromatin. The multiple chromatin forms of one antigen were derived from a precursor protein that was selectively cleaved within 1 min after erythrocyte lysis. Analysis of this antigen in extracts from erythrocytes and reticulocytes indicated that the cleavage of the precursor protein was developmentally regulated in vivo.     

Compound kinetochores of the Indian muntjac

The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) are unique among mammals due to their low diploid number (2N=6, 7) and large size. It has been proposed that the karyotype of this small Asiatic deer evolved from a related deer the Chinese muntjac (Muntiacus reevesi) with a diploid chromosome number of 2n= 46 consisting of small telocentric chromosomes. In this study we utilized a kinetochore-specific antiserum derived from human patients with the autoimmune disease scleroderma CREST as an immunofluorescent probe to examine kinetochores of the two muntjac species. Since CREST antiserum binds to kinetochores of mitotic chromosomes as well as prekinetochores in interphase nuclei, it was possible to identify and compare kinetochore morphology throughout the cell cycle. Our observations indicated that the kinetochores of the Indian muntjac are composed of a linear beadlike array of smaller subunits that become revealed during interphase. The kinetochores of the Chinese muntjac consisted of minute fluorescent dots located at the tips of the 46 telocentric chromosomes. During interphase, however, the kinetochores of the Chinese muntjac clustered into small aggregates reminiscent of the beadlike arrays seen in the Indian muntjac. Morphometric measurements of fluorescence indicated an equivalent amount of stained material in the two species. Our observations indicate that the kinetochores of the Indian muntjac are compound structures composed of linear arrays of smaller units the size of the individual kinetochores seen on metaphase chromosomes of the Chinese muntjac. Our study supports the notion that the kinetochores of the Indian muntjac evolved by linear fusion of unit kinetochores of the Chinese muntjac. Moreover, it is concluded that the evolution of compound kinetochores may have been facilitated by the nonrandom aggregation of interphase kinetochores in the nuclei of the ancestral species.     

Synthesis of azidotubulin: a photoaffinity label for tubulin-binding proteins

A photoaffinity label for the identification of tubulin-binding proteins was synthesized from phosphocellulose-purified bovine brain tubulin and (N-hydroxysuccinimidyl)-4-azidosalicylic acid. The azidotubulin derivative retained the ability to undergo temperature-dependent microtubule assembly and disassembly. When incubated with purified tau protein, the azidotubulin and tau formed cross-linked complexes upon photoactivation. When 125I-labeled azidotubulin was used to photoaffinity label tubulin-binding proteins within the kinetochore of isolated mammalian chromosomes, a 130-kDa band was identified on autoradiographs of SDS-polyacrylamide gels of the 125I-labeled azidotubulin/chromosome preparations. The 130-kDa complex was isolated by antitubulin affinity chromatography and analyzed by immunoblotting using both antitubulin and kinetochore-specific sera obtained from human patients with the autoimmune disease scleroderma CREST. The immunoblots demonstrated that the 130-kDa band that was observed on autoradiographs was a complex of a subunit of the tubulin dimer and an 80-kDa CREST-specific kinetochore protein. The binding of azidotubulin to the 80-kDa kinetochore protein was significantly decreased when chromosomes were treated with a mixture of 9 parts underivatized tubulin to 1 part azidotubulin prior to photolysis. The formation of the 130-kDa azidotubulin/kinetochore protein complex was not inhibited by pretreating the chromosomes with CREST serum prior to incubation with azidotubulin. Azidotubulin should be a useful probe for the identification and characterization of tubulin-binding proteins.     

Reactivation of DNA replication in erythrocyte nuclei by Xenopus egg extract involves energy-dependent chromatin decondensation and changes in histone phosphorylation.

Reactivation of chicken erythrocyte nuclei for DNA replication in Xenopus egg extracts involves two phases of chromatin remodelling: a fast decondensation leading to a small volume increase and chromatin dispersion occurring within a few minutes (termed stage I decondensation), followed by a slower membrane-dependent decondensation and enlargement of up to 40-fold from the initial volume (stage II decondensation). Chromatin decondensation as measured by nuclear swelling and micrococcal nuclease digestion required ATP. We observed a characteristic change in the phosphorylation pattern of erythrocyte proteins upon incubation in egg extract. While histones H5, H2A, and H4 became selectively phosphorylated during decondensation, the phosphorylation of histone H3 and of several nonhistone proteins was prevented. Furthermore, histone H5 was selectively released from erythrocyte nuclei in an energy-dependent reaction. These molecular changes already occurred during stage I decondensation and they persisted during stage II decondensation. DNA replication was confined to nuclei of stage II decondensation which incorporated lamin LIII from the egg extract. These results show that initiation of DNA replication in chicken erythrocytes requires in addition to ATP-dependent chromatin remodelling (stage I), further changes in chromatin structure that correlates with lamin LIII incorporation, and stage II decondensation.     

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.     

Immunospecificity of nuclear antigens in chicken erythroid cells

Summary Specific antisera were produced against chicken reticulocyte dehistonized chromatin. The antisera reacts strongly with chicken reticulocyte chromatin, but only marginally with chicken erythrocyte chromatin. There is no reticulocyte antigen detected in chicken liver. Reticulocyte maturation is accompanied by a gradual decrease in the chromatin immunological activity and template capacity. The reduction of immunological activity is due to the change of chromatin conformation during erythrocyte maturation. Dehistonization and sonication of erythrocyte chromatin raises the erythrocyte chromatin immunological activity to levels similar to those of reticulocyte chromatin. The erythrocyte nuclear antigens are class specific, not being found in frog erythroid cell or murine Friend leukemia cell chromatins.     

Chicken erythrocyte chromatin and nuclear envelope antigens

Chromatin and inner layer nuclear envelope were isolated from chicken erythrocyte nuclei. Two antisera against dehistonized chromatin and nuclear envelope of chicken erythrocytes were obtained. Using the antiserum against dehistonized chromatin of erythrocytes we found: the presence of the antigens at approximate mol. wts of 56,000 and 77,000 tightly bound with DNA and characteristic of only erythrocyte chromatin; localized antigens at approximate mol. wts of 63,000, 68,000 and 92,000 tightly bound with DNA and common only for chromatin and nuclear envelope of chicken erythrocytes; heterogeneity of the antigens tightly bound with DNA. Using the antiserum against inner layer nuclear envelope we did not find antigens specific only for nuclear envelope and absent in erythrocyte chromatin. Some of the antigens were present in the control preparations of chicken liver chromatin and may be regarded as being species specific.     

A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones

We have detected and begun to characterize a 17-kD centromere-specific protein, CENP-A (Earnshaw, W. C., and N. Rothfield, 1985, Chromosoma., 91:313-321). Sera from several humans with CREST scleroderma autoimmune disease (CREST: calcinosis, Raynaud's phenomenon, esophageal dsymotility, sclerodactyly, and telangiectasia) bind this protein in immunoblot assays of HeLa whole cell or nuclear extracts. We have affinity purified the anti-17-kD centromere protein (anti-CENP-A) specific antibodies from immunoblots of HeLa nuclear protein. The antibodies react with epitopes present on CENP-A derived from humans but apparently do not recognize specific epitopes in either rat or chicken nuclei. Only human nuclear protein is CENP-A positive by immunoblot. Furthermore, human cells show localization of anti-CENP-A antibody to centromeres by immunofluorescence microscopy, whereas rat cells do not. On extraction from the nucleus, CENP-A copurifies with core histones and with nucleosome core particles. We conclude that this centromere-specific protein is a histone-like component of chromatin. The data suggest that CENP-A functions as a centromere-specific core histone.