Chromosome nondisjunction and loss induced by protons and X rays in primary human fibroblasts: role of centromeres in aneuploidy

To study the origin of micronuclei induced in human primary fibroblasts by low-energy protons (7.7 and 28.5 keV/microm) and X rays, we have developed a combined antikinetochore-antibody (CREST) and FISH staining with pancentromeric probes. This technique allowed us to analyze the integrity of the kinetochore and centromeric DNA structures and to assess their role in induced aneuploidy. The effect of LET on radiation-induced chromosome nondisjunction was studied in binucleated cells with centromeric-specific DNA probes for chromosomes 7 and 11. Our results indicate that, though more than 90% of radiation-induced micronuclei were CREST(-)/FISH(-), 28.5 keV/microm protons and X rays were also able to induce statistically significant increases in the number of micronuclei that were CREST(-)/FISH(+) and CREST(+)/FISH(+), respectively. One interpretation of these results could be that the protons induced chromosome loss by kinetochore detachment or by breakage in the centromeric DNA region, whereas X rays induced aneuploidy through a non-DNA damage mechanism. Nondisjunction appears to be a far more important mechanism leading to radiation-induced aneuploidy. Irrespective of the higher frequency of micronuclei induced by 28.5 keV/microm protons, the frequency of chromosome loss was markedly higher for X rays than for 28.5 keV/microm protons, strengthening the hypothesis that non-DNA targets, such as components of the mitotic spindle apparatus, may be involved in aberrations in chromosome segregation after X irradiation.     

无血清培养上调N2a细胞钙反应性反式激活因子的表达

目的探讨钙反应性反式激活因子(calcium-responsive transactivator,CREST)是否与神经细胞的分化有关。方法选用在促分化因素作用下具有分化为神经元能力的小鼠神经母细胞瘤(N2a)细胞,用无血清培养(血清撤除)诱导N2a细胞分化。接种和培养N2a细胞12～24h后,将其分为对照组和无血清诱导组,对照组继续在含5%胎牛血清(FBS)的培养基中培养,无血清诱导组在不含FBS的培养基中培养。采用免疫印迹技术、免疫荧光技术和RT-PCR检测无血清诱导分化对N2a细胞CREST蛋白和mRNA表达的影响。结果在含5%胎牛血清(FBS)培养基培养的对照N2a细胞中,CREST蛋白水平在各时间点均无明显变化,CREST mRNA水平仅在培养48h后略有升高。而在无血清诱导分化的N2a细胞中,CREST蛋白表达水平在无血清诱导12h时无明显变化,但在诱导24h后明显升高,诱导48h后进一步升高;RT-PCR检测显示,CREST mRNA在无血清诱导12h后即开始升高,诱导24h和48h则升高更为明显。结论无血清诱导分化的N2a细胞中CREST的表达上调,提示CREST可能参与神经细胞的分化。     

CREST maps somatic structural variation in cancer genomes with base-pair resolution

We developed 'clipping reveals structure' (CREST), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80%, demonstrating that CREST had a high predictive accuracy.     

昆明山海棠在微核实验中非整倍体毒性的研究

本文报道以小鼠着丝粒次要卫星DNA探针FISH和抗着丝粒CREST染色,研究了可疑的非整倍体毒剂昆明山棠(THH)诱导的小鼠NIH3T3细胞微核(MN)的着丝粒组成情况,结果THH(10-60μg/ml)诱导的62.1-68.4%的MN为FISH阳性, 35.9-42.2%的MN为CREST阳性,较精确的FISH结果显示THH具有较强的非整倍体诱发效应,同时认为次要卫星DNA探针比CREST染色更适合于MN的着丝粒检测。     

丙烯酰胺非整倍体诱发效应的荧光原位杂交和CREST染色的研究

本文以小鼠着丝粒次要卫星DNA探针FISH和抗着丝粒CREST染色,研究了可疑的非整倍体毒剂丙烯酰胺(AA)诱导的小鼠NIH3T3细胞微核(MN)的着丝粒组成情况和小鼠骨髓染色体畸变(CA)情况。结果发现AA在100—400μg/ml诱导的MN约52.7％—71.6％为FISH阳性,60.5％—68.2％的MN为CR-EST阳性,两种结果均显示AA具有较强的非整倍体诱发效应。小鼠骨髓CA的FISH表明,AA既能诱导染色体结构畸变,又能诱导非整倍体形成,而以非整倍体诱发效应更为明显。     

Organizing the Carotid Revascularization Endarterectomy versus Stenting Trial (CREST): National Institutes of Health, Health Care Financing Administration, and industry funding

The Carotid Revascularization Endarterectomy versus Stenting Trial (CREST) is a prospective, randomized, multicenter clinical trial of carotid endarterectomy (CEA) versus carotid artery stenting (CAS) as prevention for stroke in patients with symptomatic stenosis greater than or equal to 50%. CREST is sponsored by the US National Institute of Neurological Disorders and Stroke (NINDS) of the US National Institutes of Health (NIH), with additional support by a device manufacturer, and will provide data to the US Food and Drug Administration (FDA) for evaluation of a stent device. Because of budget constraints for CREST, Health Care Financing Administration (HCFA) reimbursement for hospital costs incurred by CREST patients will be essential. The involvement of academic scientists, industry, and three separate government agencies (NIH, FDA, HCFA) has presented many challenges in conducting the trial. A review of the pathways followed to meet these challenges may be helpful to others seeking to facilitate sharing of the costs and burdens of conducting innovative clinical research.     

Kinetochore components recognized by human autoantibodies are present on mononucleosomes.

We have developed a competitive enzyme-linked immunosorbent assay for solubilized kinetochore components, using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia) scleroderma autoimmune antibodies specific for these kinetochore elements. Using this quantitative assay, we found interphase persistent or "pre-kinetochore" components in low- and moderately high-salt (375 mM salt) extracts of micrococcal nuclease-digested rat liver and chicken erythrocyte nuclei. The release of antigen activity from nuclei under these conditions has been correlated with loss of pre-kinetochore foci as determined by immunofluorescence microscopy. Combined biochemical and competition assay analysis of chicken erythrocyte nuclear extracts indicates that pre-kinetochore components are tightly bound to chromatin of mononucleosome size. The conclusions based on competition assay data are supported by a direct binding assay, which confirms that antigens recognized by CREST sera are present on chromatin. These results raise the possibility that the kinetochore-specific chromosomal antigen(s) we have detected substitutes for "standard" mononucleosome components, such as histone H1. Furthermore, they suggest approaches to the isolation of kinetochore-specific DNA sequences from higher eucaryotes.     

Flow cytometry measurements of human chromosome kinetochore labeling

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.     

A microchromosome derived from chromosome 11 in a patient with the CREST syndrome of scleroderma.

A patient with the CREST syndrome of scleroderma was found to carry a mosaicism for a supernumerary microchromosome. The microchromosome was approximately 1 micron in size and present in over half of the lymphocyte metaphases examined. It bound centromeric proteins specifically recognized by CREST autoimmune sera (including the patient's serum). In situ hybridization with a panel of chromosome-specific alpha-satellite probes showed that the microchromosome was derived from chromosome 11, most or all of its chromatin consisting of the chromosome 11 subset of alpha-satellite DNA. It had no detectable telomeric sequences. Microchromosomes observed by electron microscopy had no visible free ends. The chromatin looked exactly the same as it did in normal chromosomes. Although we have no direct evidence for a circular structure, we conclude that the microchromosome originated by an interstitial deletion including the alpha-satellite DNA sequences and subsequent ring formation. The newly formed chromosomal element proved to be relatively stable somatically and was transmitted through meiosis. Since it possesses at least some structural and functional features of a centromeric region, the microchromosome can be thought of as an isolated centromere.     

Identification of centromere proteins in different mammalian cells

The characterization of centromeric proteins is facilitated using anti-centromere antibodies present in the sera of patients with the CREST variant of scleroderma. We have employed these sera to determine whether or not those proteins are present in different mammalian species, as well as to study their tissue distribution. Here, we describe the immunofluorescent pattern and the proteins recognized by CREST sera in dividing and resting cells from mouse, rat, swine, hamster, rabbit, and man. In nuclear preparations from cultured cells, thymocytes and spermatozoa from these species, the antigens recognized by CREST sera are proteins of 18 to 20 kDa in all species tested, except in rat. Additionally, two peptides of 80 and 140 kDa were observed in human preparations. In contrast, a 50 kDa peptide is the primary protein detected by the sera in rat nuclei.     

Centromeric association of a microchromosome

Summary A supernumerary microchromosome measuring 0.5–1 m found in over half of the metaphases of a CREST scleroderma patient and his daughter has been characterized by various cytogenetic techniques. The microchromosome consisted of constitutive heterochromatin and contained nuclear antigens reacting with specific anti-kinetochore antibodies. The most remarkable property of the microchromosome was its non-random position: it was closely associated with the centromere of any of the normal chromosomes in the majority of the metaphases. Furthermore, an inordinately high rate of Y chromosome aneuploidy was found in the CREST scleroderma patient. The origin and structure of the microchromosome, its possible connection with the CREST variant of scleroderma, and the phenomenon of centromeric association are discussed.     

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.     

Immunocytochemical demonstration of kinetochores in human sperm heads

CREST sera have been used to identify kinetochores in mature mammalian sperm heads. It is necessary to decondense the sperm heads artificially to permit access of the reagents before the kinetochores can be demonstrated immunocytochemically. The distribution of kinetochores in the sperm heads appears to be random. These results show that the kinetochore antigen recognized by the CREST sera used here is retained during spermiogenesis and is passed on to the zygote at fertilization.     

Identification of the mouse beta'-COP Golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse chromosome 9

In an immunological screening of a mouse testicular cDNA library with a human CREST serum we isolated five overlapping cDNA clones encoding the mouse homolog of a Golgi coatomer complex protein (accession number AF043120), designated beta'-COP in bovine and p102 in humans. We generated antibodies against this protein which specifically recognize the Golgi apparatus of mouse spermatocytes. FISH analyses assigned the beta'-COP gene Copb2 to mouse Chromosome 9, region E3-F1. Our results demonstrate that CREST sera can contain antibody components against Golgi proteins as well as against nuclear proteins.     

Ciliary microtubule capping structures contain a mammalian kinetochore antigen

Structures that cap the plus ends of microtubules may be involved in the regulation of their assembly and disassembly. Growing and disassembling microtubules in the mitotic apparatus are capped by kinetochores and ciliary and flagellar microtubules are capped by the central microtubule cap and distal filaments. To compare the ciliary caps with kinetochores, isolated Tetrahymena cilia were stained with CREST (Calcinosis/phenomenon esophageal dysmotility, sclerodactyly, telangiectasia) antisera known to stain kinetochores. Immunofluorescence microscopy revealed that a CREST antiserum stained the distal tips of cilia that contained capping structures but did not stain axonemes that lacked capping structures. Both Coomassie blue-stained gels and Western blots probed with CREST antiserum revealed that a 97-kD antigen copurifies with the capping structures. Affinity-purified antibodies to the 97-kD ciliary protein stained the tips of cap-containing Tetrahymena cilia and the kinetochores in HeLa, Chinese hamster ovary, and Indian muntjak cells. These results suggest that at least one polypeptide found in the kinetochore is present in ciliary microtubule capping structures and that there may be a structural and/or functional homology between these structures that cap the plus ends of microtubules.     

Antikinetochore antibodies interfere with prometaphase but not anaphase chromosome movement in living PtK2 cells.

Injection of CREST antikinetochore antiserum (AKA) containing antibodies to the kinetochore into living prometaphase PtK2 cells decreased chromosome velocity to near zero. Injection of either phosphate-buffered saline or CREST antiserum without antikinetochore antibodies (antikinetochore negative: AKN) had no effect on prometaphase oscillations. AKA antiserum injected into anaphase cells at the beginning of chromatid separation had no effect on anaphase chromosome velocity, spindle elongation, or cytokinesis. Visible binding of antikinetochore antibodies in prometaphase cells at room temperature occurred between 5 and 15 minutes after injection. Anaphase cells injected at the beginning of chromatid separation had bound antibody at the end of anaphase. AKA antiserum recognizes in Western blots proteins associated with the primary constriction: CENP-B, -C, and -D, as reported by other workers. The control antiserum, AKN, does not recognize these proteins. These results imply that the antigens recognized by CREST antibodies are important for chromosome movement. Whether or not these antigens are themselves motor molecules cannot be addressed by the present data. In addition, the results suggest that these antigens are not involved in an important way in anaphase movement.     

CREST – Classification Resources for Environmental Sequence Tags

Sequencing of taxonomic or phylogenetic markers is becoming a fast and efficient method for studying environmental microbial communities. This has resulted in a steadily growing collection of marker sequences, most notably of the small-subunit (SSU) ribosomal RNA gene, and an increased understanding of microbial phylogeny, diversity and community composition patterns. However, to utilize these large datasets together with new sequencing technologies, a reliable and flexible system for taxonomic classification is critical. We developed CREST (Classification Resources for Environmental Sequence Tags), a set of resources and tools for generating and utilizing custom taxonomies and reference datasets for classification of environmental sequences. CREST uses an alignment-based classification method with the lowest common ancestor algorithm. It also uses explicit rank similarity criteria to reduce false positives and identify novel taxa. We implemented this method in a web server, a command line tool and the graphical user interfaced program MEGAN. Further, we provide the SSU rRNA reference database and taxonomy SilvaMod, derived from the publicly available SILVA SSURef, for classification of sequences from bacteria, archaea and eukaryotes. Using cross-validation and environmental datasets, we compared the performance of CREST and SilvaMod to the RDP Classifier. We also utilized Greengenes as a reference database, both with CREST and the RDP Classifier. These analyses indicate that CREST performs better than alignment-free methods with higher recall rate (sensitivity) as well as precision, and with the ability to accurately identify most sequences from novel taxa. Classification using SilvaMod performed better than with Greengenes, particularly when applied to environmental sequences. CREST is freely available under a GNU General Public License (v3) from http://apps.cbu.uib.no/crest and http://lcaclassifier.googlecode.com.