Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."     

Nucleosomes in metaphase chromosomes.

Previous studies of the structure of metaphase chromosomes have relied heavily on electron micrography and have revealed the existence of a 10-nm unit fiber that is thought to generate the native 23-30-nm fiber by higher order folding. The structural relationship of these metaphase fibers to the interphase fiber remains obscure. Recent studies on the digestion of interphase chromatin have revealed the existence of a regularly repeating subunit of DNA and histone, the nucleosome that generates the appearance of 10-nm beads connected by a short fiber of DNA seen on electron micrographs. It was therefore of interest to probe the structure of the metaphase chromosome for the presence of nucleosomal subunits. To this end metaphase chromosomes were prepared from colchicine-arrested cultures of mouse L-cells and were subjected to digestion with stayphylococcal nuclease. Comparison of the early and limit digestion products of metaphase chromosomes with those obtained from interphase nuclei indicates that although significant morphologic changes occur within the chromatin fiber during mitosis, the basic subunit structure of the chromatin fiber is retained by the mitotic chromosome.     

Mitotic Chromosome Structure: Reproducibility of Folding and Symmetry between Sister Chromatids

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an ∼1 μm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to ∼300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.     

Chromosome scaffold and structural integrity of mitotic chromosomes

Chromosome scaffold represents a continuous protein substructure revealed in isolated metaphase chromosomes after harsh extraction. According to postulates of the widespread radial loop model the scaffold plays an important role in the formation and maintenance of structural integrity of the mitotic chromosomes. Here, the data concerning the structure and major components of the chromosome scaffold are presented. The experiments suggesting that the scaffold represents a system of discrete linker proteins and the data about high mobility of scaffolding proteins are discussed. Furthermore, the data about higher-level chromatin structures (elementary chromonema and 200–250 nm fibers) and behavior of scaffolding proteins are compared. The results presented agree with the idea that at the present stage it is possible to discriminate chromatin complexes, whose structural integrity is not maintained by the chromosome scaffold.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

Scaffold attachment regions in centromere-associated DNA

Due to indications that kinetochore proteins are an integral part of the protein scaffold component of the chromosome (Earnshaw et al. 1984), we chose to map the distribution of scaffold attachment regions (SARs) at centromeres. Using the SAR mapping assay of Mirkovitch et al., Southern blots were prepared and probed with ³²P-labeled fragments from the human 1.9 kb centromeric α-satellite repeat unit of chromosome 1 or the 1.7 kb centromeric α-satellite repeat unit of chromosome 16. Our results demonstrated the presence of one SAR site per 1.9 kb repeat unit in chromosome 1, and every 1.7 kb repeat unit in chromosome 16, separated by regions of small DNA loops over the length of the α-satellite regions. We also identified several in vitro vertebrate topoisomerase II and cenP-B consensus sequences throughout the chromosome 1 α-satellite region using computer and base ratio analysis, to address the question as to why some α-satellite regions are SAR related and others are not. To provide in situ indications of SAR localization in the human genome, SAR DNA and non-SAR DNA were prepared following lithium 3,5-di-iodosalicylate extraction. Sequences protected from DNAse I digestion by SAR proteins, as compared with unprotected DNA that was digested by the enzyme, was labeled with biotin-UTP, hybridized to chromosomal DNA in situ, and then detected with fluorescein-avidin-DCS. Both SAR and non-SAR DNA selectively labeled virtually all centromeric regions of the human metaphase karyotype. Chromosomal arms were less strongly bound by SAR DNA, with a pattern that followed the chromosomal axis. In the more condensed chromosomes an R-banding pattern was evident. In general, labeling patterns produced by both SAR and non-SAR fractions were similar, as expected from the indications that SAR DNAs are heterogenous in sequence and do not form a specific class of sequences. We conclude that centromeric regions of several, possibly all, human metaphase chromosomes are also regions where the chromosomal axis contains loops, smaller in size than in the arms and where attachment sites are concentrated. This clustering of SARs may be responsible in part for the tight chromatin packing associated with the primary constriction of the centromeric region. Received: 10 October 1995; in revised form: 10 May 1996 / Accepted: 13 May 1996     

Nuclear asynchrony in multinucleate rat kangaroo cells

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.     

A three-dimensional approach to mitotic chromosome structure: evidence for a complex hierarchical organization

We describe findings on the architecture of Drosophila melanogaster mitotic chromosomes, made using a three-dimensional-oriented structural approach. Using high-voltage and conventional transmission electron microscopy combined with axial tomography and digital contrast-enhancement techniques, we have for the first time visualized significant structural detail within minimally perturbed mitotic chromosomes. Chromosomes prepared by several different preparative procedures showed a consistent size hierarchy of discrete chromatin structural domains with cross-sectional diameters of 120, 240, 400-500, and 800-1,000 A. In fully condensed, metaphase-arrested chromosomes, there is evidence for even larger-scale structural organization in the range of 1,300-3,000-A size. The observed intrachromosomal arrangements of these higher-order structural domains show that both the radial loop and sequential helical coiling models of chromosome structure are over-simplifications of the true situation. Finally, our results suggest that the pathway of chromatin condensation through mitosis consists of concurrent changes occurring at several levels of chromatin organization, rather than a strictly sequential folding process.     

Studies on the mitotic chromosome scaffold of Allium sativum

An argentophilic structure is present in the metaphase chromosomes of garlic(Allium sativum),Cytochemical studies indicate that the main component of the structure is non-histone proteins(NHPs).The results of light and electron microscopic observations reveal that the chromosme NHP scaffold is a network which is composed of fibres and granules and distributed throughout the chromosomes.In the NHP network,there are many condensed regions that are connected by redlatively looser regions.The distribution of the condensed regions varies in individual chromosomes.In some of the chromosomes the condensed regions are lognitudinally situsted in the central part of a chromatid while in others these regions appear as coillike transverse bands.At early metaphase.scaffolds of the sister chromatids of a chromosome are linked to each other in the centromeric region,meanwhile,they are connected by scafold materials along the whole length of the chromosome.At late metaphase,however,the connective scaffold materials between the two sister chromatids disappear gradually and the chromatids begin to separate from one another at their ends.but the chromatids are linked together in the centromeric region until anaphase.This connection seems to be related to the special structure of the NHP scaffold formed in the centromeric region.The morphological features and dynamic changes of the chromosome scaffold are discussed.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Physical constraints in the condensation of eukaryotic chromosomes. Local concentration of DNA versus linear packing ratio in higher order chromatin structures

The local concentration of DNA in metaphase chromosomes of different organisms has been determined in several laboratories. The average of these measurements is 0.17 g/mL. In the first level of chromosome condensation, DNA is wrapped around histones forming nucleosomes. This organization limits the DNA concentration in nucleosomes to 0. 3-0.4 g/mL. Furthermore, in the structural models suggested in different laboratories for the 30-40 nm chromatin fiber, the estimated DNA concentration is significantly reduced; it ranges from 0.04 to 0.27 g/mL. The DNA concentration is further reduced when the fiber is folded into the successive higher order structures suggested in different models for metaphase chromosomes; the estimated minimum decrease of DNA concentration represents an additional 40%. These observations suggest that most of the models proposed for the 30-40 nm chromatin fiber are not dense enough for the construction of metaphase chromosomes. In contrast, it is well-known that the linear packing ratio increases dramatically in each level of DNA folding in chromosomes. Thus, the consideration of the linear packing ratio is not enough for the study of chromatin condensation; the constraint resulting from the actual DNA concentration in metaphase chromosomes must be considered for the construction of models for condensed chromatin.     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

番茄的CPD带型和45S rDNA位点的鉴别

采用CPD（PI和DAPI组合）染色对番茄减数分裂粗线期和有丝分裂中期染色体进行了显带分析,随后用两种不同的45S rDNA克隆在相同的分裂相进行了荧光原位杂交定位分析。CPD染色在8条粗线期染色体上显示出了10条红色的CPD带纹,在6对有丝分裂中期染色体上显示出了12条CPD带纹。有丝分裂中期染色体上的CPD带纹与粗线期染色体上显著的带纹具有对应性。用改良的CPD染色程序清晰而稳定地显示出这些特征性的CPD带纹为番茄的染色体,特别是有丝分裂中期染色体提供了新的识别标记。用番茄的一个45S rDNA克隆进行的荧光原位杂交,不仅在位于2号染色体短臂的随体上显示了强的杂交信号,而且在粗线期染色体的5个CPD带区或有丝分裂中期染色体的4对CPD带区显示了弱的杂交信号。然而,用来自小麦的45S rDNA克隆pTa71进行的原位杂交却只在随体上显示了杂交信号。鉴于所用的两个45S rDNA克隆在序列上的差异,推断在番茄基因组中只有随体含有45S rDNA单位的编码区,即番茄只有一对45S rDNA位点。     

Size heterogeneity of telomeric DNA in mouse meiotic chromosomes

Heterogeneity for the length of telomeric DNA sequences has been found among different mitotic chromosomes in several mammalian species. However, there are no studies reporting such heterogeneity in meiotic chromosomes. To analyse this heterogeneity we have performed fluorescence in situ hybridization with a telomeric (C(3)TA(2))(3) peptide nucleic acid (PNA) probe on spread metaphase chromosomes during both male mouse meiotic divisions. Our results show that independently of the meiotic division, telomeric DNA signals were always surrounded by DAPI-stained chromatin, even at centromeric regions. Moreover, we have found heterogeneity for the size of telomeric DNA signals among different chromosomes, between homologues, and even within a given chromosome. We discuss the functional significance of the location of telomeric DNA in condensed meiotic chromosomes, and then the possible origin for the different polymorphisms found.     

Chromosomal localisation of DNA sequences in condensed and dispersed human chromatin.

The DNAs purified from condensed and dispersed human chromatin were used as templates for the in vitro synthesis of ³H-labelled complementary RNAs (cRNAs). These cRNAs were hybridised in situ to preparations of fixed human metaphase chromosomes which had previously been stained with quinacrine and photographed with fluorescent (UV) light. Autoradiographs of the hybridised chromosomes were stained and photographed and the results analysed by comparison of the fluorescence photographs with the autoradiographs. This method allowed positive identification of every chromosomal site of hybridisation and quantitative analysis of grain distribution over a number of metaphase spreads. The cRNA transcribed from condensed chromatin DNA (cRNA_C) hybridised mainly to a limited number of sites close to or including centromeric heterochromatin (C-bands) and also to the brightly fluorescent regions of the Y chromosome. Many of these C-band regions are known to contain satellite DNAs, indicating that the repeated DNA in the condensed chromatin fraction consists largely, if not entirely, of satellite sequences. The cRNA transcribed from dispersed chromatin DNA (cRNA_D) does not contain satellite DNAs and hybridised more generally over the chromosome arms. However, the main sites of hybridisation with cRNA_D included the C-bands in the Y chromosome and autosomes, i.e. those regions which bound cRNA_C. This suggests that nonsatellite repeated DNA sequences may be associated with satellite DNAs in the chromosomes. No general correlation between the distribution of either kind of cRNA and the overall level of quinacrine fluorescence in chromosomes or chromosome arms was detectable, nor could the dispersed fraction be equated with cytological euchromatin, since it hybridised in many sites which appear heterochromatic. However, there was a suggestion that some non-fluorescing Q-bands bound cRNA_D preferentially. The differences which were found between the distribution of the cRNAs from the two chromatin fractions may be associated with differences in genetic activity.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

Clustering of Human Chromosome Fragments on the Mouse Genome by Chromosome-Mediated Gene Transfer

Pieces of metaphase chromosomes prepared from mouse cells containingneo-tagged human chromosome 7 were transferred to mouse cellswith calcium phosphate to isolate G418-resistant clones. FISHanalysis revealed that the majority of them contained humanDNA at a single site on their genome. These transformants containedSTS markers mapped to various regions of chromosome 7. It isthus suggested that pieces of human chromosomes tend to assembleand integrate on the mouse genome.     

多头绒泡菌间期细胞核和中期染色体中的银染蛋白分析*

电镜原位观察结合图象分析研究了多头绒泡菌Physarum polycephalum Schw间期细胞核和中期染色体中银染蛋白的形状、大小和分布。结果看到,银染蛋白主要呈颗粒状存在于间期细胞核和中期染色体中。银粒的大小不一,分布不均匀。间期细胞核中存在众多直径在5~15nm的银粒,其中10nm以上的较大银粒主要分布于核仁,集缩染色质和核基质部分10nm以上银粒不多。中期细胞核内10nm以上的较大银粒主要分布于染色体中。染色体中除含有一些较大银粒外,多数银粒的直径为5~10nm。本文结果提示,构成染色体骨架的嗜银蛋白可能来自间期细胞核的染色质、核基质和核仁。     

Chromosomal localization of a proinsulin transgene in Japanese quail by laser pressure catapulting

Transgenic avian bioreactors produce therapeutic recombinant proteins in egg white. To date, however, methods for transgenic modification of the avian genome or determining transgenic status of individual birds are scarce. The dual, but interrelated, goals of this research were to: (1) develop a method of detecting stable DNA insertion into Japanese quail; and (2) provide a method for gene location on avian chromosomes. We created Teflon-coated coverslip slides to facilitate laser pressure catapulting of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G₂ Japanese quail, containing germline incorporation of proinsulin, were identified by isolation of chromosomes using laser microdissection and laser pressure catapulting. Subsequent amplification of each chromosome identified 2–5 chromosomes with the proinsulin transgene inserted. Nucleotide sequencing of each chromosomal insertion was identical to the proinsulin portion of the original vector. By applying laser pressure catapulting and PCR of individual chromosomes, we were able to determine that the transgene correctly inserted into avian chromosomes and that the majority of the insertions occurred within microchromosomes. Because many potential therapeutic transgenes have similar or nearly identical nucleotide sequence to the host’s native gene, laser microdissection and subsequent analysis may be required for detailed documentation of transgene expression before proceeding with transgenic protein production.