Scaffold morphology in histone-depleted HeLa metaphase chromosomes

In a study of HeLa metaphase chromosomes depleted of histories with 2 M NaCl and spread with cytochrome c, two new types of images of chromosome scaffolds have been observed in the electron microscope. In the first type, scaffolds are very large and fibrous but still display the shape typical of metaphase chromosomes. The regularity and lack of distortion in these scaffolds, despite their openness and seeming fragility, support the notion that the underlying scaffold structure is an interconnected network of fibers. In the second type, fibrous regions and dense regions are juxtaposed in the same chromosome scaffold. These micrographs suggest that the dense appearance of some previously observed scaffolds may be the result of incomplete adherence to the cytochrome c monolayer, leading to collapse and aggregation during dehydration.     

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     

Differences in DNA composition along mammalian metaphase chromosomes

Denaturation of chromosomal DNA in situ can be achieved without disruption of chromosomal morphology by heating slides at 25–90° C in 10–95% formamide in SSC. The extent of denaturation is proportional to formamide concentration and temperature. Reassociation of denatured DNA is prevented with formaldehyde. — The DNA in the paracentromeric constrictions in human chromosomes 1, 9 and 16 denatures earlier than in any other regions, as shown by the red colour with acridine orange. When the temperature or formamide concentration is raised a red and green banding pattern emerges in which regions known to stain brightly with quinacrine mustard are red whereas other regions are green. The last regions to turn red are the short arms of some acrocentric chromosomes. Since A+T-rich DNA denatures before G+C-rich DNA, it is inferred that QM-bright areas are rich in A+T. Similar results are obtained with mouse and Microtus agrestis cells. — Reassociation of chromosomal DNA denatured by heat and formamide occurs if no formaldehyde is used. In human cells, kinetic studies on reassociation indicate that the highest degree of repetition is in the DNA of the distal half of the Y chromosome. Next in degree of repetition are the paracentromeric constrictions, the short arm regions of some of the acrocentric chromosomes, and all the centromeric regions. Highly repetitious DNA is found in all mouse centromeric regions except that of the Y chromosome. Constitutively heterochromatic segments of X and Y and the autosomal centromeric regions of Microtus agrestis also contain repetitious DNA. — It is proposed that differential base content and susceptibility to denaturation of DNA contribute to or at least accompany Q-, G- and R-banding. The degree of C-banding is related to repetitious DNA. The human Y chromosomal DNA is probably A+T-rich and exceptionally repetitious, exhibiting spontaneous reassociation under many experimental conditions.     

Structure of DNA in metaphase chromosomes of mouse fibroblasts

We show that N-1 in adenine of chromosomal DNA is methylated by treatment of metaphase chromosomes with dimethylsulphate while this is not the case in chromatin. The data on methylation are consistent with those obtained from the experiments with S1-nuclease treatment of chromatin and chromosomes. This suggests a disarrangement of DNA secondary structure in the metaphase chromosomes.     

DNA catenation maintains structure of human metaphase chromosomes

Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical ‘X’ shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIα significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation, and destroyed to complete sister chromatid disjunction. In addition to demonstrating the value of microfluidics as a tool for examining chromosome structure, these results lend support to certain models of DNA catenation organization and regulation: in particular, we conclude from our observation of centromere-concentrated catenation that spindle forces could play a driving role in decatenation and that Topoisomerase IIα is differentially regulated at the centromeres, perhaps in conjunction with cohesin.     

The DNA content of Chinese hamster meiotic metaphase chromosomes

DNA values of Chinese hamster male meiotic metaphase chromosomes were measured by slide-based Feulgen cytometry. All the autosomes were distinguishable on the basis of their DNA content. No significant differences were found between the autosomes of the two male animals studied, but a significant difference was found in the DNA content of the sex-chromosome pair between these two animals.     

New sites of methylcytosine-rich DNA detected on metaphase chromosomes

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions.     

Natural size classes in DNA from Chinese hamster metaphase chromosomes

DNA from isolated Chinese hamster ovary (CHO) metaphase chromosomes can be obtained in three different molecular weight classes. The two largest forms have sedimentation coefficients of 80 and 120 S at 7,500 rpm. Based on sedimentation and speed dependence analysis these have molecular weights of 220 million and above 5,000 million, and are thought to be analogs of DNA classes observed in a prior study of human metaphase chromosomes. An extract can be converted to primarily the 80 S form through alkaline pH treatment of metaphase DNA. The third class (45 S DNA) is formed as a result of metaphase chromosome exposure to the nuclease Bal31, and has a mass distribution analogous to the CHO replicon.     

Fluorescence analysis of late DNA replication in human metaphase chromosomes.

Thymidine incorporated as a terminal pulse into chromosomes otherwise substituted with 5-bromodeoxyuridine can be detected by associated bright 33258 Hoechst fluorescence. The location of metaphase chromosome regions identified by this method as last to complete DNA synthesis is consistent with the results of autoradiographic analyses with tritiated thymidine. The very late-replicating regions correspond to a subset of those which appear as bands after chromosomes are stained by quinacrine or modified Giemsa techniques. The high resolution of the 33258 Hoechst fluorescence pattern within individual cells is especially useful for revealing variations in the order of terminal replication. Both homolog asynchrony and fluctuations in the distribution of bright 33258 Hoechst fluorescence within chromosomes from different cells are apparent and localized to individual bands. The results are consistent with the possibility that these bands constitute units of chromosome replication as well as structure.     

Rapid isolation of metaphase chromosomes containing high molecular weight DNA

Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.     

Radiation-induced double-strand scission of the DNA of mammalian metaphase chromosomes

The molecular weight distribution of double-stranded mammalian (hamster) DNA was determined by ultracentrifugation of isolated metaphase chromosomes previously layered onto sucrose gradients containing high salt concentrations to dissociate the protein and nucleic acid components. In untreated controls the distribution (as determined by counting the incorporated radioactivity in the resultant fractions) exhibited a peak at 225 X 10(6) daltons. Inclusion of mercaptoethanol and hydroxylamine into the gradients produced no significant change of these sedimentation patterns. Gamma-radiation-induced reduction in the number and weight average molecular weights was used to calculate a value of 1.05 X 10(11) double-strand breaks/gram rad, equivalent to about 600 eV/break. No significant difference was observed for chromosomes irradiated either before or after isolation from intact mitotic cells. Irradiation in the presence of cystamine resulted in at least a sevenfold reduction in the apparent double-strand scission. The observed sedimentation patterns were compared with those generated by a theoretical computer simulation of radiation-induced degradation which assumed random selection and breakage of molecules. These results suggested that at least 80 to 90 % of the isolated DNA was distributed approximately normally with a mean molecular weight of about 200 X 10(6) daltons and a standard deviation of about 50 X 10(6) daltons.     

Immunoelectronmicroscopy of metaphase chromosomes

Summary A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Immunoelectronmicroscopy of metaphase chromosomes

A method for the preparation of ultrathin sections of metaphase chromosomes is described. This method was applied to human metaphase chromosomes, which were immunocytochemically stained with anti-DNA and anti-ribonucleoprotein antibodies, derived from patients with auto-immune disease. Conventionally prepared metaphase spreads as well as cytocentrifuge preparations of chromosome suspensions were studied. The results indicate that the ultrastructure of chromosomes and the immunoreactivity of chromosomal constituents are influenced by the applied preparation methods. In comparison with whole mount preparations, ultrathin sections of immunostained chromosomes allow higher resolution and more precise localization of immunoreactive sites within the chromosomal structure.     

Scaffold/matrix attachment regions and intrinsic DNA curvature

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 598–606.     

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.     

Photographic equidensitometry of metaphase chromosomes

Summary Photographic equidensitometry, which is a procedure to obtain specific lines or symbols for points of equal optical density, has been utilized in the study of chromosome images. Equidensitometric techniques, using a special contour film, permitted three approaches, namely, production of line equidensities (in the form of families, sequences and contour maps), color equidensities (color conversion of line sequences), and screen equidensities (substituting characteristic symbols for densities). All these techniques have proved very useful to analyze images of metaphase chromosomes and occurrence of spontaneous banding patterns, by showing the precise distribution and relative values of the grey gradient. This report demonstrates the potential of photographic equidensitometrical procedures for chromosome studies, which obviates the need to purchase elaborate equipment.     

Distribution of UV-induced unscheduled DNA synthesis in metaphase chromosomes of Chinese hamster cells

Unscheduled DNA synthesis (USD) occurred in metaphase chromosomes of cultured Chinese hamster cells after ultraviolet light (UV) irradiation. When the chromosomes were labeled by UV-induced USD in metaphase, the number of grains was in proportion to the amount of chromosomal DNA and the grain densities were approximately equal all over the segments of chromosomes.