Assignment of the centrosomal protein 110 gene (Cep110) to mouse chromosome bands 2B-C1 by in situ hybridization.
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Animals↗
Biomedical subjects
J B Rattner
Publications and source records attributed to J B Rattner.
ASE-1: an autoantigen in systemic lupus erythematosus.
ASE-1 is a 55 kDa nucleolar autoantigen. We show that autoantibodies to this antigen occur at a higher frequency in the sera of patients with SLE than in other systemic rheumatic diseases and that the specificity of ASE-1 as a serum marker of SLE increases as the number of epitopes recognized by the sera increases. Autoantibodies to ASE-1 were temporally associated with autoantibodies to HsEg5 but were not found in conjunction with other known serum markers of SLE. The frequency of antibodies to ASE-1 epitopes in a SLE cohort was approximately the same as anti-dsDNA. However, anti-dsDNA is associated with renal involvement, whereas ASE-1 reactivity shows an association with a history of serositis. We conclude that ASE-1 is correlated with serositis and that ASE-1 should be added to a list of autoantigens that are considered important serological features of SLE.
Adult↗
Mapping and characterization of the functional domains of the nucleolar protein RNA helicase II/Gu.
RNA helicase II/Gu (RH-II/Gu) is a nucleolar RNA helicase of the DEAD-box superfamily. In this study, the functional domains of RH-II/Gu molecule were mapped by fusing the protein or its deletion mutants with a green fluorescence protein and subsequently transfecting or microinjecting the recombinant constructs into HeLa cells. In addition to the identification of a nuclear localization signal (NLS) in the N-terminus and a nucleolar targeting signal in the central helicase domain, a hidden NLS and a nucleolar targeting signal were found in the C-terminal arginine/glycine-rich domain. RH-II/Gu colocalized with fibrillarin, a component of the dense fibrillar region of the nucleolus. Overexpression of the entire RH-II/Gu protein or specific domains of the protein in HeLa cells did not interfere with the normal distribution of fibrillarin. However, when the helicase domain was truncated, the distribution pattern of fibrillarin was distorted. Microinjection of the wild-type RH-II/Gu cDNA into the nucleus of HeLa cells did not disrupt normal cell growth. However, when cells were injected with mutant DNA, only a small percentage of HeLa cells progressed through the cell cycle. Analysis of centrosomes in transfected cells demonstrated that most of the mutant-expressing cells were arrested early in the cell cycle. The results suggest that each of the structural domains of RH-II/Gu is necessary for cell growth and cell cycle progression.
Binding Sites↗
Tissue transfers: substrates for cytology and cytochemistry of animal tissues.
The Tissue Transfer Technique (TTT) is a novel method of sampling animal tissue that can be used to study tissue morphology, chemistry and physiology. This review provides an overview of the technique and demonstrates its use to detect the tissue distribution of specific epitopes, lectin binding sites and nucleic acids as well as its application as an organ monolayer in culture. These applications are compared and contrasted with standard histological techniques including the "Tissue Printing Technique" developed to sample plant tissue.
Animals↗
Autoantibodies to a group of centrosomal proteins in human autoimmune sera reactive with the centrosome.
OBJECTIVE: Human autoantibodies reacting with protein components of the microtubule organizing center of the cell, the centrosome, are rare and have not been extensively studied. We therefore investigated the number, type, and frequency of autoantibodies reactive with centrosomal proteins in a cohort of human sera. METHODS: To establish the type of autoantibodies found in autoimmune sera reactive with the centrosome, we used a prototype human serum, which was chosen for its intense reactivity with the centrosome throughout the cell cycle, to screen a HeLa complementary DNA (cDNA) (expression) library. Positive cDNA clones were sequenced and classified as encoding either known centrosomal autoantigens, known centrosomal proteins but unknown as human autoantigens, or previously unknown centrosomal antigens. To investigate whether these centrosomal autoantibody classes were characteristic of centrosomal-reactive sera, sera from 21 subjects with centrosomal reactivity by indirect immunofluorescence were characterized by Western blotting for reactivity to recombinant protein from each of the classes of centrosomal antigens. Clinical features were studied by retrospective chart review. RESULTS: In each of the sera, autoantibodies that recognize a group of centrosomal proteins were identified. This group included known centrosomal autoantigens (pericentrin and pericentriolar material 1 [PCM-1]), the human homolog of a known mouse centrosomal protein, ninein, which was previously unknown as a human autoantigen, and a novel centrosomal protein (Cep250). Autoantibodies to PCM-1 were the least common (8 of 21 subjects; 38%) while those to ninein, Cep250, and pericentrin occurred at roughly equal frequencies (17 subjects [81%], 17 subjects [81%], and 19 subjects [90%], respectively). There was no apparent correlation between serum autoantibody reactivity and the clinical diagnosis. CONCLUSION: Each of the autoimmune sera contained autoantibodies that reacted with a group of centrosomal proteins. We found that the centrosomal component ninein, first identified in mice, has a human homolog that is an autoantigen. Also, anticentrosomal sera contained antibodies to previously undetected centrosomal components. One of these novel antigens was identified and was designated Cep250. Thus, a characteristic of sera reactive with the centrosome is that they contain antibodies to a group of centrosomal proteins.
Adolescent↗
Autoantibodies to components of the mitotic apparatus.
Autoantibodies directed to a variety of cellular antigens and organelles are a feature of autoimmune diseases. They have proven useful in a clinical setting to establish diagnosis, estimate prognosis, follow disease progression, alter therapy, and initiate new investigations. Cellular and molecular biologists have used autoantibodies as probes to identify molecules involved in key cellular processes. One of the most interesting sets of autoantibodies are those that target antigens within the mitotic apparatus (MA). The MA includes chromosomes, spindle microtubules and centrosomes. The identification, localization, function, and clinical relevance of MA autoantigens is the focus of this review.
Autoantibodies↗
Expanding the role of HsEg5 within the mitotic and post-mitotic phases of the cell cycle.
The BimC family of kinesin like proteins are involved in spindle dynamics in a wide variety of organisms. The human member of this family, HsEg5, has been implicated in centrosome separation during prophase/prometaphase and in the organization of in vitro mitotic asters. HsEg5 displays a complex distribution during mitosis, associating with the centrosomes, spindle microtubules, specific regions of the intracellular bridge and a microtubule bundle that forms in association with the post-mitotic migration of the centrosome. In an effort to determine the function of HsEg5 during late mitotic events and refine its proposed function during early mitotic centrosome separation, we microinjected antibodies specific to HsEg5 into HeLa cells during various stages of mitosis. In the presence of HsEg5 antibodies we find that the microtubule arrays responsible for both pre- and post-mitotic centrosome movement never form. Similarly, the microtubule bundle within the intracellular bridge becomes prematurely altered following karyokinesis resulting in the loss of the microtubule array at either end of the bridge. In addition, some peri-centrosomal material at the spindle poles becomes fragmented and the distribution of the spindle protein NuMA becomes more concentrated at the minus ends of the spindle microtubules. Our study also provides direct evidence that there is a link between post-mitotic centrosome migration and Golgi complex positioning and reformation following mitosis. We conclude that HsEg5 plays a recurrent role in establishing and/or determining the stability of specific microtubule arrays that form during cell division and that this role may encompass the ability of HsEg5 to influence the distribution of other protein components associated with cell division
Cell Cycle↗
The relationship of ASE-1 and NOR-90 in autoimmune sera.
OBJECTIVE: The nucleolar proteins ASE-1 and NOR-90 can become confused because they have similar cytological and Western blot features. We investigated the frequency and relationship between these 2 proteins and identified clinical features of patients with ASE-1 antibodies. METHODS: The characteristics of ASE-1 and NOR-90 are shown by indirect immunofluorescence (IIF) and Western blot data. The sera are characterized by their ability to immunoprecipitate the in vitro transcription and translation (TnT) product of either the ASE-1 or NOR-90 cDNA. Clinical features were obtained by retrospective chart review. RESULTS: Of the 15 sera identified as potentially NOR-90 positive by IIF and Western blot 8/15 (53%) were able to immunoprecipitate a NOR-90 TnT product. Of the remaining 7 sera, 4 (57%) were only able to immunoprecipitate an ASE-1 TnT product. Four (57%) of the remaining 7 sera were able to immunoprecipitate an ASE-1 TnT product. In a second cohort of confirmed NOR-90 positive sera, 2/8 (25%) were able to immunoprecipitate an ASE-1 TnT product. In total, ASE-1 autoantibodies were found in 6/16 (37.5%) of confirmed NOR-90 sera from both cohorts. There were no common clinical features found in seven ASE-1 positive patients; however, 3 (43%) had a malignancy and 3 (43%) had slowly progressive systemic sclerosis. CONCLUSION: Autoantibodies to ASE-1 and NOR-90 can occur alone or together in autoimmune sera. Due to their similar IIF and Western blot profile the only way to correctly characterize these sera is by immunoprecipitation of the appropriate TnT product.
Autoantibodies↗
Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix.
Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes.
Animals↗
ASE-1: a novel protein of the fibrillar centres of the nucleolus and nucleolus organizer region of mitotic chromosomes.
A novel nucleolar component has been identified and cloned using a human autoimmune serum. This antigen, as inferred from the cDNA sequence, is an Mr 55000 protein. Immuno blot analysis, however, of both the native protein and the in vitro translation products of the cDNA showed that they migrate on SDS-PAGE at an apparent molecular mass of 90000 A BLAST search using the cDNA sequence indicated that it is in an antisense orientation to and overlaps the gene of the DNA repair enzyme ERCC-1. An open reading frame, without a translational start site, had been observed by others in this region of the chromosome 19 (19q13.3) and the putative protein was termed ASE-1 (Anti-Sense to ERCC-1). Our cDNA is a full-length equivalent of that open reading frame. ASE-1 was found to contain two domains that are present in a number of nucleolar specific proteins originating from a variety of organisms: a glycine-, arginine- and phenylalanine-rich putative nucleotide interaction domain and an alternating basic/acidic region. Indirect immunofluorescence analysis using antibodies generated to cloned regions of ASE-1 indicated that this protein occurs at the fibrillar centres of the nucleolus in interphase, the putative sites of rDNA transcription, and during cell division it is localized to the nucleolus organizer regions of the chromosomes. ASE-1 co-localises with the RNA polymerase I transcription initiation factor UBF/NOR-90 throughout all stages of the cell cycle and these two proteins associate with each other in vitro.
Amino Acid Sequence↗
High frequency of neoplasia in patients with autoantibodies to centromere protein CENP-F.
OBJECTIVE: To study the clinical features of patients with autoantibodies to centromere protein CENP-F and the frequency of CENP-F autoantibodies in patients with various diseases. DESIGN: Retrospective clinical and serologic study. METHODS: Thirty-six patients with anti-CENP-F were identified by a characteristic pattern of indirect immunofluorescence (IIF) on HEp-2 cells. Fifty patients with melanoma, 50 with breast cancer, 10 with lung cancer, 354 with systemic sclerosis, 120 with systemic lupus erythematosus and 50 with rheumatoid arthritis were also studied. Recombinant proteins were produced from 5 CENP-F cDNA clones representing amino acids 2192-3317 (p-F1), 5561-7126 (p-F2), 5892-6883 (p-F3), 7538-10,116 (p-F4) and 9242-10,096 (p-F5). The presence of CENP-F antigen was studied in a breast carcinoma cell line, cryosections of breast carcinoma, normal breast tissue and tonsils. RESULTS: Twenty-two of 36 patients with CENP-F antibodies had neoplasms; breast (9/22) and lung (5/22) cancer were the most common diagnoses. Thirty-three sera were available for further study; when tested for reactivity to the recombinant peptides, the sera of 21 of 21 patients with neoplasms and 5 of 12 patients with other diseases bound the C-terminal p-F4 peptide. When the terminal third of the p-F4 peptide (p-F5) was studied, a significant difference in pattern of reactivity was not detected. By comparison, the frequency of reactivity with peptides representing other domains of CENP-F was less than that with p-F4 (p-F2 > p-F3 > p-F1). CENP-F autoantibodies were not found in any of the control sera from patients with systemic lupus erythematosus, rheumatoid arthritis or systemic sclerosis or in unselected sera from various malignancies. CENP-F antigens were identified in breast carcinoma tissue but were rarely observed in normal tissues. CONCLUSIONS: A high proportion of individuals with CENP-F antibodies have neoplasia, and there is a bias among their sera for reactivity with determinants in the carboxy terminal domain of CENP-F. CENP-F antigens appear to be highly expressed in malignant tissues.
Adult↗
Changes in the nuclear matrix of chicken erythrocytes that accompany maturation.
The protein composition and structure of nuclear matrices isolated from adult chicken immature and mature erythrocytes were analysed. Visualization of nuclear matrices by electron microscopy showed that immature-erythrocyte nuclear matrices had internal structures, while most mature-erythrocyte nuclear matrices did not. Both mature- and immature-erythrocyte nuclear matrices were surrounded by a fibrous network of intermediate filaments. Two-dimensional gel electrophoretic analysis of proteins obtained from fractionated nuclear matrices led to the assignment of the proteins as components of the nuclear porelamina, internal matrix, or cytoskeleton. Common and different proteins belonging to one of the three groups were identified in nuclear matrices of immature and mature erythrocytes. Investigation of the partitioning of histone deacetylase activity, an enzyme associated with the internal matrix, among the erythroid nuclear matrix fractions provided evidence that mature- and immature-erythrocyte nuclear matrices have internal structures. However, the activity of histone deacetylase and level of internal matrix proteins from mature-erythrocyte nuclear matrices were less than those from immature-erythrocyte matrices. The low levels of nuclear RNA and internal matrix proteins may account for lack of visual evidence for an internal matrix in mature erythrocytes.
Animals↗
The relationship of HsEg5 and the actin cytoskeleton to centrosome separation.
Although centrosome separation is essential to the formation of a bipolar spindle, it can proceed along several different pathways. This raises questions as to the similarity between the mechanism(s) underlying these various forms of separation. To address this question we reinvestigated centrosome separation in HeLa cells using a variety of techniques. We present a refined description of the two major pathways of centrosome separation found in HeLa cells and demonstrate that each of these pathways has its own timing, protein requirements, morphological characteristics, and relationship to spindle assembly. The first pathway, which occurs in prophase cells, is dependent on an intact actin cytoskeleton, and when this pathway is completed prior to nuclear envelope breakdown, the microtubules associated with this process do not become part of the spindle. Thus, centrosome separation and spindle pole organization can occur as two separate events. The second centrosome separation pathway is found in cells in which separation occurs concurrent with prometaphase. In this case, centrosome separation and the formation of the mitotic spindle are integrated together and an intact actin cytoskeleton is not required. The relationship between these multiple pathways of centrosome separation and the distribution of the human kinesin-like protein HsEg5 was also investigated. This protein was found associated with all centrosomal microtubules present during both prophase and prometaphase centrosome separation, as well as with prophase centrosomes displaying independent movement in Cytochalasin-D treated cells. In addition, we demonstrate that this protein is associated with post-mitotic centrosome movement which involves a single centrosome. Thus, HsEg5 is a feature of individual centrosome function and does not require anti-parallel microtubule arrays.
Actins↗
The centromere kinesin-like protein, CENP-E. An autoantigen in systemic sclerosis.
OBJECTIVE: Autoantibodies directed against centromere proteins (CENPs) are a serologic feature in some patients with systemic sclerosis (SSc). Previous studies have focused on autoantibodies to CENPs A, B, and C. CENP-E is a recently described 312-kd protein that also localizes to the centromere. Therefore, we studied the presence of autoantibodies to recombinant CENP-E in patients with SSc. METHODS: Sixty sera from patients with the SSc spectrum of diseases were screened for the presence of autoantibodies against CENP-E, by indirect immunofluorescence and immunoblotting using recombinant CENP-E protein. HLA class II alleles were determined by DNA oligotyping. RESULTS: Among the SSc sera, 15 of 60 (25%) demonstrated antibody reactivity with recombinant CENP-E, and 14 of these 15 sera (93%) had antibodies directed against another CENP. Anti-CENP-E was seen in 13 of 30 sera with anti-CENP (43%). All patients with anti-CENP-E had a limited form of SSc, known as the CREST variant (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). When patients with anti-CENPs A, B, or C were compared with patients with anti-CENP-E, no unique clinical features in the anti-CENP-E positive group were identified. Ninety-three percent of the patients with anti-CENP-E had HLA-DQB1 alleles that had polar amino acids at position 26 (primarily DQB1*05), similar to patients with other CENP autoantibodies. CONCLUSION: Antibodies to CENP-E are common in patients with SSc, and are seen in higher frequency in sera from patients with a limited form, or CREST variant, of the disease.
Autoantibodies↗
The spindle kinesin-like protein HsEg5 is an autoantigen in systemic lupus erythematosus.
OBJECTIVE: Autoantibodies directed against the mitotic spindle apparatus (MSA) have been shown to target an antigen referred to as NuMA (nuclear mitotic apparatus). In this study, we identified a second MSA antigen as the spindle kinesin-like protein HsEg5. We studied the frequency of antibodies to HsEg5 in human sera that demonstrate the MSA pattern of staining, the frequency of autoantibodies to HsEg5 in patients with systemic lupus erythematosus (SLE), and the clinical features of patients with antibodies to HsEg5. METHODS: A prototype serum from an SLE patient was used to isolate a 4.8-kilobase complementary DNA (cDNA) from a HeLa cDNA library. Western blot, immunoprecipitation, and sequence analysis revealed that the antigen was an approximately 130-kd protein, HsEg5. The frequency of autoantibodies to recombinant HsEg5 in 51 sera that demonstrated an MSA pattern of staining on HEp-2 and HeLa cells was detected by immunoblotting 2 constructs of the cDNA. The clinical features of patients with antibodies directed against HsEg5 was obtained by retrospective chart review. RESULTS: The antigen responsible for the MSA-35 pattern was identified as the human kinesin-like protein HsEg5. Seven of 51 sera (14%) that demonstrated an MSA pattern of staining reacted with recombinant HsEg5. Six of 7 of the HsEg5-positive patients (86%) had SLE, and 1 had Sjögren's syndrome. The indirect immunofluorescent staining pattern of sera that reacted with HsEg5 could be distinguished from the other sera that reacted with NuMA. In an unselected cohort of 52 SLE patients, 3 (6%) had autoantibodies reactive with the recombinant HsEg5. CONCLUSION: Autoantibodies to MSA fall into 2 major classes: those reactive with NuMA and those reactive with HsEg5. Autoantibodies to HsEg5 are found in a lower frequency than NuMA in sera that demonstrate the MSA pattern of staining and appear to be specifically associated with SLE. HsEg5 can be distinguished from NuMA by indirect immunofluorescence and Western blotting.
Adult↗
Enhanced glomerular retrieval for renal biopsies.
A substantial number of glomeruli were found to become dislodged from renal biopsy cores during the biopsy procedure. These "lost" glomeruli can be retrieved and immobilized on membrane substrates; the morphology of these membrane-immobilized glomeruli is of diagnostic quality. Hence, this technique of glomerular retrieval offers an opportunity to maximize the number of glomeruli obtained at biopsy and also makes available additional material for diagnostic studies, physiology studies, and archiving.
Animals↗
Topoisomerase II alpha is associated with the mammalian centromere in a cell cycle- and species-specific manner and is required for proper centromere/kinetochore structure.
A study of the distribution of Topoisomerase II alpha (Topo II) in cells of six tissue culture cell lines, human (HeLa), mouse (L929), rat, Indian muntjac, rat kangaroo (PTK-2), and wallaby revealed the following features: (1) There is a cell cycle association of a specific population of Topo II with the centromere. (2) The centromere is distinguished from the remainder of the chromosome by the intensity of its Topo II reactivity. (3) The first appearance of a detectable population of Topo II at the centromere varies between species but is correlated with the onset of centromeric heterochromatin condensation. (4) Detectable centromeric Topo II declines at the completion of cell division. (5) The distribution pattern of Topo II within the centromere is species- and stage-specific and is conserved only within the kinetochore domain. In addition, we report that the Topo II inhibitor ICRF-193 can prevent the normal accumulation of Topo II at the centromere. This results in the disruption of chromatin condensation sub-adjacent to the kinetochore as well as the perturbation of kinetochore structure. Taken together, our studies indicate that the distribution of Topo II at the centromere is unlike that reported for the remainder of the chromosome and is essential for proper formation of centromere/kinetochore structure.
Animals↗
CENP-F is a protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly degraded after mitosis.
Centromere protein-F (CENP-F) is mammalian kinetochore protein that was recently identified by an autoimmune serum (Rattner, J. B., A. Rao, M. J. Fritzler, D. W. Valencia, and T. J. Yen. Cell Motil. Cytoskeleton. 26:214-226). We report here the human cDNA sequence of CENP-F, along with its expression and localization patterns at different stages of the HeLa cell cycle. CENP-F is protein of the nuclear matrix that gradually accumulates during the cell cycle until it reaches peak levels in G2 and M phase cells and is rapidly degraded upon completion of mitosis. CENP-F is first detected at the prekinetochore complex during late G2, and is clearly detectable as paired foci that correspond to all the centromeres by prophase. During mitosis, CENP-F is associated with kinetochores from prometaphase until early anaphase and is then detected at the spindle midzone throughout the remainder of anaphase. By telophase, CENP-F is concentrated within the intracellular bridge at either side of the mid-body. The predicted structure of the 367-kD CENP-F protein consists of two 1,600-amino acid-long coil domains that flank a central flexible core. A putative P-loop nucleotide binding site (ADIPTGKT) is located within the globular carboxy terminus. The structural features deduced from our sequence studies and the spatial and temperal distribution of CENP-F revealed in our cytological and biochemical studies suggest that it may play a role in several mitotic events.
Amino Acid Sequence↗