Description of a chromosome replication unit in individual prematurely condensed human S-phase chromosomes

Mammalian chromosome replication was studied by the aid of premature chromosome condensation (PCC). After induction of PCC the sites of DNA replication appear as “gaps” between condensed chromosomal regions. These condensed particles are unineme before and bineme after DNA replication. The two phases are due mainly to the unineme or bineme nature of the particles. During early S-phase almost all particles are unineme, during late S-phase they are bineme and there is only one transitory stage between these two main stages. Premature chromosome condensation was studied in detail on a specific human chromosome 22 which is marked by its heterochromatin constitution. This led to easy identification of these elements in S-phase PCC (S-PCC) preparations. For each stage of the S-phase there was a reproducible pattern of condensed chromosomal particles making up the whole chromosome. The number of these particles was rather limited and a complementary pattern was found in early versus late S-phase. The pattern of early S-PCC corresponded to the banding pattern of G-banded prometaphase chromosomes; the pattern of late S-PCC, to R-banded prometaphase chromosomes. Thus, “gaps” and condensed particles as observed after PCC induction are obviously homologous to chromosome replication units. Replication of constitutive heterochromatin occurred during the very late S-phase. During this stage PCC induction led to condensation of the heterochromatin into several small, highly fluorescent particles.     

Human cells in suspension 2

Summary When PHA stimulated human lymphoid cells are allowed to proliferate in vitro and chromosomal slides are prepared from the culture after 48,72, 96,120, and 144h of growth, gradual changes in chromosome morphology can be observed after traditional Giemsa staining of the slides. Keeping culture conditions, colcemid exposure, and fixation procedure constant for all samples, it is found that average chromosome length decreases with increasing culture time. A shift from high frequencies of subbanded chromosomes (sample 48 h and sample 72 h) to high frequencies of unbanded and G banded chromosomes (sample 120 h and sample 144 h) takes place simultaneously with the general compaction of the chromosomes. Examination of trypsin-induced G bands as well as examination of untreated G banded chromosomes from all samples clearly indicate that the basic G band pattern is not altered during proliferation and differentiation, although the progressive compaction of the chromosome observed with increasing culture time results in a phenomenon similar to that observed during mitosis, where the compact late metaphase chromosome after trypsin treatment exhibits fewer but more prominent bands than the prophase/prometaphase chromosomes. Thus the progressive compaction of metaphase chromosomes observed during in vitro aging seems to resemble the condensation processes during the G₂ phase and mitosis.It has been suggested that the chromomeres serve as centers for chromosome condensation during mitosis, probably mediated by a sulphydryl-disulphide transition in chromosomal proteins. The data presented here further suggest that the chromomeres may also serve as centers for chromosomal differentiation, presumable by a mechanism similar to that acting during chromosome condensation in mitosis.     

Dependence of heterochromatin differential staining on the time of its reduplication and the degree of condensation]

A comparison of the chromosomes banding pattern after G-and C-staining with the time of DNA reduplication and the degree of chromosome condensation, was carried out using Chinese hamster metaphase chromosomes. Chromosome condensation was studied under 5-bromodeoxyuridine and 5-bromodeoxycytidine treatment. All the chromosomal segments stained with C-technique are also stainable with G-technique, while only some G-positive segments are capable to be C-bands. C-bands are heterochromatic segments characterized by extremely late replication and great delay in condensation under the analog action, while G-bands are segments with earlier labelling and irregular decondensation. The data obtained suggest a close correlation between the capability of chromosomal region of G- and C- staining and the degree of its heterochromatinization.     

Dynamics of chromosome compaction during mitosis

We have quantitatively studied the space-time dynamics of mitotic chromosome compaction in cultured amphibian cells. After collecting digital phase-contrast images we have done digital image analysis to study spatial correlations in density. We find a characteristic distance at which the strongest correlations occur, which provides a quantitative measure of the size of patches of dense chromatin during interphase and early prophase. Later in mitosis, this length corresponds to the thickness of prophase and metaphase chromosomes. We find that during interphase strong correlations exist at a few-micrometer length; during prophase this correlation length progressively drops as the chromosomes are compacted. Our data are explained by a model based on assembly of chromatin loops onto already fiberlike interphase chromosomes. To test this model we have microinjected cobalt hexamine trichloride into interphase nuclei and have observed the rapid condensation of the interphase chromatin into thick fibers with a spacing similar to the native-state interphase correlation length determined from our image analysis.     

Some factors affecting the action of restriction endonucleases on human metaphase chromosomes

We have investigated whether restriction endonucleases produce bands on human chromosomes by extracting DNA, using staining methods which are stoichiometric for DNA. Restriction enzymes that produce C-band patterns appear to remove DNA extensively from chromosome arms. In general, however, those restriction enzymes that produce G-bands do not extract DNA from chromosomes, and their effects are believed to be due to conformational change in the chromosomal DNA; in these cases, the chromosomal regions affected appear to be determined by the chromosome structure and not by the specificity of the enzyme. DNA loss from chromosomes due to digestion by restriction enzymes may in some cases be uniform, although a G-banding pattern is visible after Giemsa staining.     

Non-apoptotic chromosome condensation induced by stress: delineation of interchromosomal spaces

Chromosomes are known to occupy distinct territories, suggesting the existence of definite borders. Visualization of these borders requires chromatin condensation like that seen in prophase cells. We developed a novel method to induce chromosome condensation in all cells regardless of cell cycle stage using a complex set of stresses. The cells were not apoptotic, as indicated by the absence of DNA damage, maintenance of the intact lamina and scaffold attachment factor A, and by the continuation of metabolic processes as well as proliferative capacity. That the appearance of chromosome condensation did not represent a premature mitotic event was shown by the absence of fibrillarin and Ki67 envelopment of chromosomes, continued protein synthesis and the reversibility of chromosome condensation. That chromosome condensation was achieved was demonstrated by the removal of chromatin from the nuclear envelope and chromosome painting. Specific genetic sites known to be at the surface of chromosomes retained their positions as shown by in situ hybridization. Stress-induced chromosome condensation was used to prove that specific nuclear domains such as ND10 are interchromosomally located and that green fluorescent protein-tagged ND10-associated proteins are useful markers for chromosomal boundaries after adenovirus 5 track formation in vivo. From these observations we conclude that chromosomal territories appear to have boundaries that exclude developing macromolecular aggregates. Received: 26 November 1999 / Accepted: 29 February 2000     

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.     

Trypsin G- and C-banding for interchange analysis and sex identification in the chicken

The trypsin banding methods were applied to feather pulp and embryonic material of the chicken. Two contrasting types of chromosomal banding patterns were obtained by varying the duration of trypsin treatment. A short time treatment shows a G-banding pattern which has characteristic and distinctive bands along the chromosome arms. Prolonging the trypsin treatment causes the G-banding pattern to disappear, and only the centromeres and the W chromosome remained heterochromatic which is characteristic of the C-banding pattern. The application of the G-banding pattern analysis was used to identify regions of chromosomes involved in rearrangements. The simplified trypsin technique which produces the C-banding pattern makes it relatively easy to identify the W sex-chromosome and determine sex in avian species.     

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     

Quantitative chromosome map of the polyploid Saccharum spontaneum by multicolor fluorescence in situ hybridization and imaging methods

Somatic chromosomes of a wild relative of sugarcane (Saccharum spontaneum L.) anther culture-derived clone (AP 85-361, 2n=32) were identified and characterized by computer-aided imaging technology and molecular cytological methods. The presence of four satellite chromosomes and four nearly identical chromosome sets suggests that the clone is a tetrahaploid with the basic number x=8. A quantitative chromosome map, or idiogram, was developed using image analysis of the condensation pattern (CP) at the prometaphase stage of somatic chromosomes. The 45S and 5S ribosomal RNA gene (rDNA) loci were simultaneously visualized by multi-color fluorescence in situ hybridization (McFISH) and precisely localized to the regions of 3p3.1 and 6q1.3 on the idiogram. The simultaneous visualization of two sets of four ribosomal RNA genes confirms tetraploidy of this clone. This conclusion is consistent with results of molecular marker mapping. The quantitative chromosome map produced will become the foundation for genome analyses based on chromosome identity and structure. Previously impossible identification of small chromosomes and untestable hypotheses about the polyploid nature of plants can now be settled with these two approaches of quantitative karyotyping and FISH.     

Experimental condensation inhibition in constitutive and facultative heterochromatin of mammalian chromosomes

What drives the dramatic changes in chromosome structure during the cell cycle is one of the oldest questions in genetics. During mitosis, all chromosomes become highly condensed and, as the cell completes mitosis, most of the chromatin decondenses again. Only chromosome regions containing constitutive or facultative heterochromatin remain in a more condensed state throughout interphase. One approach to understanding chromosome condensation is to experimentally induce condensation defects. 5-Azacytidine (5-aza-C) and 5-azadeoxycytidine (5-aza-dC) drastically inhibit condensation in mammalian constitutive heterochromatin, in particular in human chromosomes 1, 9, 15, 16, and Y, as well as in facultative heterochromatin (inactive X chromosome), when incorporated into late-replicating DNA during the last hours of cell culture. The decondensing effects of 5-aza-C analogs, which do not interfere with normal base pairing in substituted duplex DNA, have been correlated with global DNA hypomethylation. In contrast, decondensation of constitutive heterochromatin by incorporation of 5-iododeoxyuridine (IdU) or other non-demethylating base analogs, or binding of AT-specific DNA ligands, such as berenil and Hoechst 33258, may reflect an altered steric configuration of substituted or minor-groove-bound duplex DNA. Consequently, these compounds exert relatively specific effects on certain subsets of AT-rich constitutive heterochromatin, i.e. IdU on human chromosome 9, berenil on human Y, and Hoechst 33258 on mouse chromosomes, which provide high local concentrations of IdU incorporation sites or DNA-ligand-binding sites. None of these non-demethylating compounds affect the inactive X chromosome condensation. Structural features of chromosomes are largely determined by chromosome-associated proteins. In this light, we propose that both DNA hypomethylation and steric alterations in chromosomal DNA may interfere with the binding of specific proteins or multi-protein complexes that are required for chromosome condensation. The association between chromosome condensation defects, genomic instability, and epigenetic reprogramming is discussed. Chromosome condensation may represent a key ancestral mechanism for modulating chromatin structure that has since been realloted to other nuclear processes.     

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.     

DNA methylation and chromosomal rearrangements in reconstructed karyotypes of Hordeum vulgare L.

Summary. One standard and two reconstructed barley karyotypes were used to study the influence of chromosomal rearrangements on the distribution pattern of DNA methylation detectable at the chromosome level. Data obtained were also compared with Giemsa N-bands and high gene density regions that had been previously described. The effect of chromosomal reconstruction in barley seems to be decidedly prominent in the repositioning of genomic DNA methylation along metaphase chromosomes. In comparison to the standard karyotype, the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complements not subjected to structural alterations. Moreover, differences may occur between corresponding regions of homologues. Some specific chromosomal bands, including the nucleolus-organizing regions, showed a relative constancy in the methylation pattern, but this was not the case when the two satellites were combined by translocation in chromosome 6H^5H of line T-30. Our results suggest that epigenetic changes like DNA methylation may play an important role in the overall genome reorganization following chromosome reconstruction. Correspondence: R. Cremonini, Dipartimento di Biologia, Università di Pisa, Via L. Ghini 5, 56126 Pisa, Italy.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

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.     

Chromosome banding and compaction

Summary We have described a characteristic substructure of mitotic chromosomes, the chromosomal unit fibre, with lengths about five times the length of the corresponding metaphase chromosomes and a uniform diameter of 0.4 m. In order to study the relationship of chromosome banding to chromosome compaction, methods have been devised to obtain banding patterns on chromosomal unit fibres, similar to G-band patterns of intact mitotic chromosomes. The total number of bands plus interbands per haploid human karyotype is estimated at about 3000. The banding pattern of chromosomal unit fibres indicates a certain resemblance to the normal G-banding pattern of human chromosomes even if the details indicate a short-range random distribution.     

Increasing the physical markers of wheat chromosomes using SSRs as FISH probes

In plants the marker sequences used to identify chromosomes are mainly repetitive DNA probes. Simple sequence repeats (SSRs) are major components of many plant genomes and could be good markers for chromosome identification. In a previous work, we reported the physical distribution of 4 oligonucleotides, (AG)12, (CAT)5, (AAC)5, and (AAG)5, on Triticum aestivum L. chromosomes. The distinctive distribution pattern found suggested that SSR in situ hybridization is useful as a diagnostic tool in wheat cytogenetics. To check whether that finding is generally applicable, we analyzed the chromosomal distribution of the rest of the 14 possible classes of di- and tri-nucleotide repeats by FISH. A detailed knowledge of the sequence content of hexaploid wheat chromatin was acquired based on the hybridization signals, which also provide a rich set of chromosome markers for chromosome identification. Except for (AT)10 and (GC)10, for which the chromosomal distribution could not be accurately determined, and (AC)8 and (GCC)5, which were found dispersed throughout the chromosomes, the remaining repeats were observed as clusters on specific chromosome sites. (AGG)5, (CAC)5, (ACG)5, (AAT)5, and (CAG)5 exhibited a preferential distribution in the pericentromeric regions of the B genome chromosomes. The richest patterns of intercalary signals on several A and B genome chromosomes were produced by (ACT)5. A karyotype based on the SSR probes providing the best FISH patterns was constructed for T. aestivum 'Chinese Spring'.     

High-resolution idiogram of Giemsa R-banded human prophase chromosomes

The schematic representation of RHG-banded chromosomes (R-banding was produced by heat denaturation followed by Giemsa staining (RHG) in the 850-band range per haploid set, was prepared showing the relative position, the specific size, and the characteristic staining intensity for each band. To this idiogram was adapted the new International Standard Cytogenetic Nomenclature. Our aim was to produce a realistic idiogram which could help in the preparation of R-banded prophase karyotypes and in the localization of chromosomal rearrangements. A comparative analysis of bands at prophase and metaphase revealed certain aspects of the dynamics involved in chromosome condensation and in R-band organization. The effect of chromosome elongation on the appearance of R-bands within heterochromatic regions has also been discussed.