Random arrangement of chromosomes inUvularia (Liliaceae)

Chromosome arrangement in interphase has been inferred from an analysis of the relative positions of the chromosomes and the chromosome arms in untreated haploid pollen grain metaphases ofUvularia grandiflora. The distances between centromeres forming the smallest possible circle were measured in 43 metaphases. The relative positions of the chromosomes did not differ significantly from randomness. Neither did similar-sized chromosome arms show any tendency to be next to each other. The results thus disagree both with the hypothesis ofComings (1968) that each chromosome occupies a definite position in the interphase nucleus and with the claim ofBennett (1982) that similar-sized chromosome arms lie next to each other.     

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126±36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 m thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20–30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.     

Genistein abolishes nucleoside uptake by cardiac fibroblasts

Genistein, a soy isoflavone, is reported to exert significant beneficial action in several human disorders, which has generated immense interest in the mechanisms underlying its effects on diverse cellular processes. It has anti-proliferative action on many cell types, an effect generally attributed to tyrosine kinase inhibition. In this study, genistein was found to cause total inhibition of [³H]-thymidine incorporation into DNA and a modest reduction in [³H]-proline incorporation into protein in primary cultures of cardiac fibroblasts. The decrease in [³H]-thymidine incorporation was not associated with a decrease in cell proliferation but correlated exactly with low intracellular levels of [³H]-thymidine. Genistein dramatically reduced [³H]-thymidine but not [³H]-proline uptake by these cells in which the equilibrative nucleoside transporter may be the major route of nucleoside uptake. The effect was irreversible and was demonstrable in pulmonary fibroblasts as well. The findings suggest that nucleoside uptake mechanisms may be a novel target of genistein action in cardiac fibroblasts and point to serious limitations in using genistein to assess the role of tyrosine kinase in cell proliferation by the standard technique of [³H]-thymidine incorporation.     

The pattern of DNA replication in the chromosomes of Puschkinia libanotica

The uptake of H³-thymidine into the chromosomes of Puschkinia libanotica has been studied in plants possessing or lacking a heterochromatic B chromosome. The pattern of H³-thymidine uptake by the A chromosomes at the end of the S phase is similar in plants of both genotypes. Regions around the centromere take up more H³-thymidine at the end of S than do more distal regions. The rate of uptake into the heterochromatin of the B chromosome increases towards the end of S, but there is no evidence that synthesis in the B chromosome carries on after the completion of DNA synthesis in the euchromatic A complement. It is proposed that at the end of the S phase more replicons in the heterochromatin of the B chromosome are engaged in DNA synthesis than in euchromatin.     

Localization of chromosome regions in potoroo nuclei (<Emphasis Type="Italic"> Potorous tridactylus </Emphasis>Marsupialia: Potoroinae)

Chromosome paints of the rat kangaroo ( Aepyprymnus rufuscens, 2n =32) were used to define chromosome regions in the long nosed potoroo ( Potorous tridactylus, 2n =12 female, 13 male) karyotype and localize these regions in three-dimensionally preserved nuclei of the potoroo to test the hypothesis that marsupial chromosomes have a radial distribution. In human nuclei chromosomes are distributed in a proposed radial fashion. Gene-rich chromosomes in the human interphase nucleus are preferentially located in the central area while gene-poor chromosomes are found more at the periphery of the nucleus; this feature is conserved in primates and chicken. Chromosome ordering in nuclei of P. tridactylus is related to their size and centromere position. Its relationship with replication patterns in interphase nuclei and metaphase was studied. In addition it was observed that the nucleus was not a smooth entity but had projections occupied by specific chromosome regions. Edited by: R. Allshire     

Interphase chromosome order: A proposal

A model for the arrangement of chromosomes in interphase nuclei is proposed. The model assumes that interphase chromosomes have a Rabl orientation (a relic telophase arrangement). During interphase and prophase telomeres are attached to the nuclear envelope often in pairs. The association of telomeres, homologous or nonhomologous, is based on similarity of arm lengths and occurs at the time the nuclear envelope reforms. At this stage arm lengths will vary to some extent due to the amount of uncoiling etc. The sequence of chromosomes resulting from telomere-telomere pairing may vary among cells, but the number of arrangements will be restricted by arm length similarities.The ramifications of this model on melotic pairing, the constant attachment of chromosomes to some structure throughout the cell cycle, the distribution of genes within nuclei, and chromosome evolution are raised.     

Interferon inhibition of thymidine incorporation into DNA through effects on thymidine transport and uptake

Replenishment of medium after 72 hr of growth of HeLa-S3 cells in dense suspension cultures increased [³H]-thymidine uptake into cells and incorporation into DNA, with the levels reaching a peak ～ 12 hr following medium change; β interferon inhibits the enhanced uptake of [³H]-thymidine and labeling of DNA in a dose-dependent manner. Some reduction in these processes is observed at a concentration as low as 1 u/ml, and ～ 75% inhibition at 640 u/ml. Kinetic analysis has revealed that the rate of labeling of the acid-soluble pool with [³H]-thymidine, measured either at 22°C, or 37°C, is reduced in interferon-treated (640 u/ml, 24 hr) HeLa-S3 cells. At 22°C, the initial rate of thymidine transport at a high (500 μM) thymidine concentration, determined within the first 30 sec of [³H]-thymidine addition was depressed by 44% in interferon-treated HeLa cells. At 37°C, labeled precursors accumulate in acid-soluble material for ～ 8 min after the addition of [³H]-thymidine, after which an apparent equilibrium level is attained. At this temperature, the rate of thymidine uptake and the apparent equilibrium level attained were depressed by 70% in interferon-treated HeLa cells. The reduced incorporation of [³H]-thymidine into DNA in interferon-treated HeLa-S3 cells can be largely explained by interferon inhibition of thymidine transport and phosphorylation.     

Individual interphase chromosome domains revealed by in situ hybridization

Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.     

Uninterrupted DNA synthesis during S-phase in untransformed diploid hamster fibroblasts

Untransformed Syrian hamster fibroblasts in exponential growth were exposed to a pulse of [³H]-thymidine for 5 min, followed immediately by bromodeoxyuridine, and serial samples were taken up to 16 h. Preparations were autoradiographed and stained for replication banding. No cell with replication bands was found without significant [³H]-thymidine uptake, although the extent of uptake varied between sub-phases of S. Thus there is no indication of a total cessation of synthesis at any period during S-phase.     

SYNTHETIC CAPACITIES OF CHROMOSOME FRAGMENTS CORRELATED WITH THEIR ABILITY TO MAINTAIN NUCLEOLAR MATERIAL

Onion (Allium cepa) and bean (Vicia faba) root tip cells containing many micronuclei, derived from x-ray-induced chromosome fragments, were exposed to H³-thymidine and H³-cytidine to determine the ability of such fragments to undergo DNA and RNA synthesis. Only a few micronuclei in onion and many in bean roots synthesize nucleic acid simultaneously with their main nuclei. A few micronuclei labeled with H³-thymidine undergo mitotic chromosome condensation along with the main nuclei, while the unlabeled ones never do so. The onset of nucleic acid synthesis as well as mitosis in micronuclei appears to be under generalized cellular control. Although all chromosomes and chromosome fragments at telophase give a positive reaction for a silver stainable nucleolar fraction, in the subsequent interphase only some micronuclei, derived from such chromosome fragments, are found to maintain nucleoli; others lose them with time. Those micronuclei which maintain nucleoli, perhaps due to the presence of specific chromosomal regions, are also active in DNA and RNA synthesis. These results are compatible with the concept that nucleoli and associated chromosome regions play an important role in the primary biosynthetic processes of the cell.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF^Slimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF^Slimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF^Slimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.     

Gene expression is highly correlated on the chromosome level in urinary bladder cancer

Objective: Chromosome correlation maps display correlations between gene expression patterns on the same chromosome. Our goal was to map the genes on chromosome regions and to identify correlations through their location on chromosome regions.

Materials and Methods: Following microarray analysis we used Ingenuity Pathway Analysis (IPA) to construct gene networks of the co-deregulated genes in bladder cancer. Chromosome mapping, mathematical modeling and data simulations were performed using the WebGestalt and Matlab^® softwares.

Results: The top deregulated molecules among 129 bladder cancer samples were implicated in the PI3K/AKT signaling, cell cycle, Myc-mediated apoptosis signaling and ERK5 signaling pathways. Their most prominent molecular and cellular functions were related to cell cycle, cell death, gene expression, molecular transport and cellular growth and proliferation. Chromosome correlation maps allowed us to detect significantly co-expressed genes along the chromosomes. We identified strong correlations among tumors of Tα-grade 1, as well as for those of Tα-grade 2, in chromosomes 1, 2, 3, 7, 12 and 19. Chromosomal domains of gene co-expression were revealed for the normal tissues, as well. The expression data were further simulated, exhibiting an excellent fit (0.7 < R² < 0.9). The simulations revealed that along the different samples, genes on same chromosomes are expressed in a similar manner.

Conclusions: Gene expression is highly correlated on the chromosome level. Chromosome correlation maps of gene expression signatures can provide further information on gene regulatory mechanisms. Gene expression data can be simulated using polynomial functions.     

Utility of the modified ATP III defined metabolic syndrome and severe obesity as predictors of insulin resistance in overweight children and adolescents: a cross-sectional study

Background

Inhibition of vascular smooth muscle cell (vSMC) proliferation by oral anti-hyperglycemic agents may have a role to play in the amelioration of vascular disease in diabetes. Thiazolidinediones (TZDs) inhibit vSMC proliferation but it has been reported that they anomalously stimulate [³H]-thymidine incorporation. We investigated three TZDs, two biguanides and two sulfonylureas for their ability of inhibit vSMC proliferation. People with diabetes obviously have fluctuating blood glucose levels thus we determined the effect of media glucose concentration on the inhibitory activity of TZDs in a vSMC preparation that grew considerably more rapidly under high glucose conditions. We further explored the mechanisms by which TZDs increase [³H]-thymidine incorporation.

Methods

VSMC proliferation was investigated by [³H]-thymidine incorporation into DNA and cell counting. Activation and inhibition of thymidine kinase utilized short term [³H]-thymidine uptake. Cell cycle events were analyzed by FACS.

Results

VSMC cells grown for 3 days in DMEM with 5% fetal calf serum under low (5 mM glucose) and high (25 mM glucose) increased in number by 2.5 and 4.7 fold, respectively. Rosiglitazone and pioglitazone showed modest but statistically significantly greater inhibitory activity under high versus low glucose conditions (P < 0.05 and P < 0.001, respectively). We confirmed an earlier report that troglitazone (at low concentrations) causes enhanced incorporation of [³H]-thymidine into DNA but did not increase cell numbers. Troglitazone inhibited serum mediated thymidine kinase induction in a concentration dependent manner. FACS analysis showed that troglitazone and rosiglitazone but not pioglitazone placed a slightly higher percentage of cells in the S phase of a growing culture. Of the biguanides, metformin had no effect on proliferation assessed as [³H]-thymidine incorporation or cell numbers whereas phenformin was inhibitory in both assays albeit at high concentrations. The sulfonylureas chlorpropamide and gliclazide had no inhibitory effect on vSMC proliferation assessed by either [³H]-thymidine incorporation or cell numbers.

Conclusion

TZDs but not sulfonylureas nor biguanides (except phenformin at high concentrations) show favorable vascular actions assessed as inhibition of vSMC proliferation. The activity of rosiglitazone and pioglitazone is enhanced under high glucose conditions. These data provide further in vitro evidence for the potential efficacy of TZDs in preventing multiple cardiovascular diseases. However, the plethora of potentially beneficial actions of TZDs in cell and animal models have not been reflected in the results of major clinical trials and a greater understanding of these complex drugs is required to delineate their ultimate clinical utility in preventing macrovascular disease in diabetes.     

Visualization and Characterization of High-Order Chromatin Fibers under Light Microscope during Interphase and Mitotic Stages in Plants

Using genomic in situ hybridization with genomic DNA, high-order chromatin fibers were successfully exhibited under a light microscope through the cell cycle in barley, rice, maize and field bean. From the interphase to prophase and metaphase of mitosis, the fibers were basically similar. Each was estimated to be around 200 nm in diameter, but the strength of signals was not the same along the fiber length. Through the cell cycle a series of dynamic distribution changes occurred in the fibers. In the interphase, they were unraveled. At the early prophase they were arranged with parallel and mirror symmetry. During late-prophase and metaphase, the fibers were bundled and became different visible chromosomes. The parallel coiling and mirror symmetry structures were visible clearly until the metaphase. In anaphase they disappeared. During telophase, in peripheral regions of congregated chromosome group, borderlines of the chromosomes disappeared and the fibers were unraveled. This demonstrated that mitotic chromosomes are assembled and organized by parallel and adjacent coiling of the fibers and the fibers should be the highest order structure for DNA coiling.     

Unscheduled DNA synthesis after partial UV irradiation of the cell nucleus. Distribution in interphase and metaphase.

Cells of an euploid strain of the Chinese hamster synchronized in the G1 phase were microirradiated in the nucleus with a laser UV microbeam (λ = 257 nm) and pulse-labelled with [³H]thymidine. In autoradiographs of cells fixed immediately after the pulse unscheduled DNA synthesis (UDS) was found restricted to the microirradiated part of the nucleus. The rate of UDS varied with the UV energy applied and the post-irradiation incubation time. In other experiments chromosome preparations were established after an additional chase and a subsequent growth period. In 28 mitotic cells autoradiographic label was found concentrated on a few chromosomes which lay adjacent to each other in one part of the metaphase plate. The distribution of label on the chromosomes could clearly be distinguished from patterns which originate from semi-conservative DNA synthesis within S phase. The label on chromosomes of microirradiated cells thus represents UDS. Our findings support the following ideas on the arrangement of interphase chromosomes: (1) Decondensed interphase chromosomes may occupy rather compact territories. (2) Chromosomes do not necessarily exhibit a close and permanent association with their respective homologues.     

Inhibition of the M r 70,000 S6 kinase pathway by rapamycin results in chromosome malsegregation in yeast and mammalian cells

The antifungal and immunosuppressive drug rapamycin arrests the cell cycle in G1-phase in both yeast and mammalian cells. In mammalian cells, rapamycin selectively inhibits phosphorylation and activation of p70 S6 kinase (p70^S6K), a protein involved in the translation of a subset of mRNAs, without affecting other known kinases. We now report that rapamycin causes chromosome malsegregation in mammalian and yeast cells. Chromosome malsegregation was determined by metaphase chromosome analysis of human lymphocytes and lymphoblasts, detection of CREST-positive micronuclei in human lymphoblasts and Chinese hamster embryonic fibroblast (CHEF) cells, and selection of doubly prototrophic cells in a specially constructed yeast strain. The number of ana-telophases with displaced chromosomes and interphase and mitotic cells with an irregular number of centrosomes was also determined in CHEF cells. In quiescent mammalian cells (human lymphocytes and CHEF cells) induced with growth factor to re-enter the cell cycle, rapamycin was effective when cells were exposed at the time of p70^S6K activation. In yeast, rapamycin was more effective when treatment was started in G1- than in G2-synchronized cells. Cells from ataxia telangiectasia (A-T) patients are characterized by chromosome instability and have recently been found to be resistant to the growth-inhibiting effect of rapamycin. We found that an A-T lymphoblastoid cell line was also resistant to the induction of chromosome malsegregation by rapamycin, but the level of spontaneous aneuploidy was higher than in normal cells. In yeast, the induction of chromosome malsegregation was dependent on the presence of a wild-type TUB2 gene, encoding the β-subunit of tubulin. The finding that rapamycin acts in different cell types and organisms suggests that the drug affects a conserved step important for proper segregation of chromosomes. One or more proteins required for chromosome segregation could be under the control of the rapamycin-sensitive pathway. Received: 3 August 1998 / Accepted: 20 August 1998     

Suppression of thyroid cell growth by serum IgG in Graves' disease.

To determine whether serum immunoglobulin in addition to epidermal growth factor (EGF) augment growth in human thyroid cells, effects of these factors on thyrocytes were tested using IgG derived from 34 patients with Graves' disease and 12 normal subjects. The cell growth was estimated by [3H]-thymidine uptake, cell cycle determined by FACS analysis and the expression of c-fos mRNA in monolayer thyrocytes enzymatically prepared from Graves' thyroid. The addition of IgG taken from patients with Graves' disease inhibited the [3H]-thymidine uptake compared to that taken from control subjects. IgG taken from Graves' disease suppressed EGF-induced increase of S + G2/M phase in cell cycle and the expression of c-fos mRNA, while those taken from normal subjects did not affect at all. [3H]-thymidine uptake was more suppressed by IgG from patients with a smaller-sized goiter than by those with a larger-sized one. There was a negative correlation between the suppression of [3H]-thymidine uptake and levels of TBII (p less than 0.05). There was no correlation between the degree of suppression and the levels of T3, T4, TSAb, TSBAb or MCHA. Thus, in conclusion, IgG derived from sera of Graves' may inhibit the growth of Graves' thyrocytes, leading to the determination of the goiter size.     

Microsurgically-extracted metaphase chromosomes of the Indian muntjac examined with phase contrast and scanning electron microscopy.

Metaphase chromosomes are extracted from Indian muntjac cultured fibroblasts either through the use of microneedles or by the application of a droplet of silicone oil onto the cell surface. Interconnecting fibers among the chromosomes allow the entire diploid complement to be extracted from the cell. The seven muntjac chromosomes are brought to the surface of a glass coverslip for analysis. Each chromosome can be identified on the basis of morphology, and particular chromosomes or chromosome parts can be isolated. Many of the fibers which interconnect the chromosomes may be attributed to adhesions formed between the sticky chromosome surfaces during extraction. However, when interchromosomal contacts are avoided during extraction, the chromosomes are found to be arranged radially with the centromeres near the center and interconnected by fibers. This arrangement is similar to that seen inside muntjac cells at metaphase. Scanning electron microscopy reveals the chromosome surfaces to consist of looping fibers, except for regions near the centromeres and the secondary constrictions. Chromosome fibers at these sites are organized into parallel bundles. Chromosome interconnections are strands composed of multiple fibers which seem to be continuous with chromosome fibers.