Three-dimensional structure of G-banded human metaphase chromosomes observed by atomic force microscopy

The structure of G-bands in human metaphase chromosomes was analyzed by comparison between light microscopic and atomic force microscopic (AFM) images of the same chromosomes. G-bands of the chromosomes were made by trypsin treatment followed by staining with a Giemsa solution. The banded chromosomes examined by light microscopy were dried either in air or in a critical point-drier, and observed by non-contact mode AFM. Air-dried chromosomes after G-band staining showed alternating ridges and grooves on their surface, which corresponded to light-microscopically determined G-positive and G-negative bands, respectively. At high magnification, the G-positive ridges were composed of densely packed chromatin fibers, while the fibers were loose in the G-negative grooves. Fibers bridging the gap between sister chromatids of a mitotic pair were often found, especially in the G-positive portions. These findings suggest that the G-banding pattern reflects the high-order structure of human metaphase chromosomes.     

Atomic force microscopy for imaging human metaphase chromosomes

The present study introduces the principle of atomic force microscopy (AFM) and reviews our results of human metaphase chromosomes obtained by AFM. AFM imaging of the chromosomes revealed that the chromatid arm was not uniform in structure but had ridges and grooves along its length, which was most prominent in the late metaphase. The arrangement of these ridges and grooves was roughly symmetrical with the counterpart of the paired sister chromatids. AFM imaging of banded chromosomes also showed that the ridges and grooves were related to the G/Q-positive and G/Q-negative bands, respectively. At high magnification, the chromatid was seen to be produced by the compaction of highly twisted chromatin fiber loops, and its compaction tended to be stronger in the ridged regions of the chromosomes than in the grooved regions. Our AFM studies also showed the presence of catenation of chromatin fibers between the ridged portions of the chromatid in the late metaphase. Thus, AFM is useful for obtaining the three-dimensional surface topography not only in ambient conditions but also in physiological liquid conditions, and is expected to be an attractive tool for investigating the structure of chromosomes.     

Structural analysis of human chromosomes by atomic force and light microscopy in relation to the distribution of topoisomerase IIα

The relationship between the higher-order structure of human metaphase chromosomes and the distribution of topoisomerase IIα was analyzed by a comparison of atomic force microscope (AFM) and fluorescence microscope images of the same chromosome. AFM imaging of chromosomes in liquid revealed the presence of alternating ridges and grooves on the surfaces of the sister chromatids. In contrast, the fluorescence image of the chromosomes stained with the anti-topoisomerase IIα antibody showed that the fluorescence intensity of topoisomerase IIα was not uniform and that there were alternating strong and weak spots along the chromosome axes. A comparison of the AFM image with a fluorescence microscope image of the same chromosome further demonstrated that ridges and grooves corresponded to strong and weak fluorescence intensities of topoisomerase IIα, respectively. These findings suggest that the distribution of topoisomerase IIα has a close connection with the higher-order structure of human metaphase chromosomes.     

Replication Banding Patterns in Human Chromosomes Detected Using 5-ethynyl-2'-deoxyuridine Incorporation

A novel technique using the incorporation of 5-ethynyl-2''-deoxyuridine (EdU) into replicating DNA is described for the analysis of replicating banding patterns of human metaphase chromosomes. Human lymphocytes were synchronized with excess thymidine and treated with EdU during the late S phase of the cell cycle. The incorporated EdU was then detected in metaphase chromosomes using Alexa Fluor® 488 azides, through the 1,3-dipolar cycloaddition reaction of organic azides with the terminal acetylene group of EdU. Chromosomes with incorporated EdU showed a banding pattern similar to G-banding of normal human chromosomes. Imaging by atomic force microscopy (AFM) in liquid conditions showed that the structure of the chromosomes was well preserved even after EdU treatment. Comparison between fluorescence microscopy and AFM images of the same chromosome 1 indicated the presence of ridges and grooves in the chromatid arm, features that have been previously reported in relation to G-banding. These results suggest an intimate relationship between EdU-induced replication bands and G- or R-bands in human chromosomes. This technique is thus useful for analyzing the structure of chromosomes in relation to their banding patterns following DNA replication in the S phase.     

Three-dimensional helical coiling structures and band patterns of hydrous metaphase chromosomes observed by low vacuum scanning electron microscopy

Helical coiling structures and band patterns of hydrous metaphase chromosomes were documented three-dimensionally by low vacuum scanning electron microscopy (SEM). Fixed or unfixed isolated Chinese hamster metaphase chromosomes were stained with platinum blue (Pt blue) and observed in the backscattered electron mode for low vacuum SEM without any hypotonic treatment or drying processes. Fibrous structures were shown both in the fixed and unfixed hydrous chromosomes; helical chromatid coils and their subcoils were clarified especially in the fixed chromosomes having contrasting alternative bands of light and darkness, while the translucent perichromosomal matrix and compact fibrous structures were recognized in the unfixed chromosomes. The helical coils were more clearly represented in a loosened chromatid of metaphase chromosomes. Treatment with a tris-HCl buffer solution and Pt blue staining in a hydrous condition successfully produced banding patterns similar to G-bands on metaphase chromosomes. These banded chromosomes observed by low vacuum SEM were also analyzed stereoscopically by field emission SEM after critical point drying. These findings indicate that: 1) native or unfixed chromosomes maintain the compact arrangement of high-order helical structures covered with the peri-chromosomal matrix; 2) helical coiling appearances of chromatids frequently observed in previous papers might be caused by loosening of the final level of the high-order structure of the metaphase chromosome; and 3) banding patterns might be produced by the rearrangement or reorganization of chromatin fibers at the 30 nm fiber level after the extraction of some chromosomal components including the peri- or intra-chromosomal materials during the banding procedure.     

Imaging of chromosomes at nano-meter scale resolution using scanning near-field optical/atomic force microscopy

Topographic and fluorescent images of whole barley chromosomes stained with YOYO-1 were observed simultaneously by scanning near-field optical/ atomic force microscopy (SNOM/AFM). The chromosome was relatively smooth and flat in the topographic images and no significant difference in height was present between regions of high fluorescent and low fluorescent intensity in the chromosomes. The telomeric region, labeled by fluorescence in situ hybridization (FISH) method, was also observed by SNOM/AFM at high resolution, and fluorescent signals of the telomeric region were clearly defined on the topographic image of chromatin fibers on the chromosome at the nano-meter scale level. Although the telomeric signals were usually visualized as a single fluorescent region at the end of sister chromatids by conventional light microscopy, they were observed separately as two fluorescent regions, less than 100-200 nm distance, using the SNOM/AFM. The SNOM/AFM offers great potential in identifying particular single gene location on chromosomes in the near future.     

The structure of C-banded human metaphase chromosomes as observed by atomic force microscopy

The ultrastructure of C-banded human metaphase chromosomes was studied by the combined use of light microscopy and atomic force microscopy (AFM). Light microscopy of the C-banded chromosomes showed that the centromeric regions of all chromosomes except the Y chromosome were positively stained. AFM further revealed that the C-positive region was higher than the C-negative region. The area of the C-positive region was specific depending on each chromosome; it ranged from the centromere to the proximal end of the long arm in chromosome 1, while it was restricted to the centromere in chromosomes 2 and 3. At higher magnification, chromatin fibers about 50 nm thick were clearly shown in the entire length of the chromosomes. In the C-positive region, these chromatin fibers were densely packed, while chromatin fibers were loosely packed with gentle twisting in the C-negative region. These AFM findings suggest that certain factors related to the chromatin fiber compaction remain in the C-positive region even after successive C-banging treatment.     

Atomic force microscopy of human metaphase chromosomes after differential staining of sister chromatids

Human metaphase chromosomes, in which 5-bromo-deoxyuridine (BrdU) had been incorporated into the DNA, were treated with the fluorescent plus Giemsa (FPG) method. Use of this method distinctly stained one of the paired sister chromatids with the Giemsa solution due to the difference in content of BrdU in the two chromatids. These chromosomes with their differential staining of sister chromatids were observed by atomic force microscopy (AFM). In the air-dried specimens, one of the paired chromatids that was stained strongly with Giemsa solution was about two times higher than the counterpart that was stained faintly with Giemsa solution. In the critical point dried chromosomes, the height of the Giemsa positive chromatid roughly matched that of the Giemsa negative counterpart. These findings imply that the arrangement of the Giemsa negative chromatid after FPG staining is fragile and easily collapses due to the surface tension of water during air-drying. At higher magnifications, the surface structure differed between Giemsa positive and negative chromatids; the Giemsa positive chromatid (i.e., unifilarly BrdU-incorporated chromatid) was composed of fibrous structures while the Giemsa negative chromatid (i.e., bifilarly BrdU-incorporated chromatid) exhibited a fine granular appearance. These structural changes in the sister chromatids are thought to arise from the ultraviolet irradiation and heating of the chromosomes during FPG staining.     

Atomic force microscopy of native human metaphase chromosomes in a liquid

The present study introduces a method for obtaining three-dimensional images of native (i.e., unfixed) chromosomes by atomic force microscopy (AFM) in a liquid. Human metaphase chromosomes were isolated from a human lymphoblast-like cell line, K562, by the hexylene glycol procedure according to Wray and Stubble- field (1970), adsorbed on a silane-coated glass slide, and observed in a dynamic force mode (i.e., intermittent contact mode) of AFM in a hexylene buffer solution. In adequate operating conditions, the shape of chromosomes with paired chromatids was clearly and three-dimensionally observed by AFM. At high magnification, globular or fibrous structures about 50 nm thick could be found on the surface of each chromaid, implying that chromatin fibers were strongly wound or twisted in the chromatid. Thus, AFM imaging enabled the direct visualization of native chromosomes in a liquid at high resolution--which is comparable with that of scanning electron microscopy--and can serve to analyze the mechanism of chromosome condensation and separation in relation to the structure of chromosomes.     

Scanning near field optical/atomic force microscopy of bromodeoxyuridine-incorporated human chromosomes

The present study applied scanning near field optical/atomic force microscopy (SNOM/AFM) to the observation of human chromosomes immunostained with an anti-BrdU antibody after incorporation of BrdU into DNA. Human lymphocytes were cultured in BrdU for 72 h and their chromosomes were prepared with a standard method for light microscopy. After additional fixation with 15% formalin in phosphate buffered saline, the specimens were denatured with 2N HCI with 0.1% Triton-X 100, immunostained with the anti-BrdU antibody, and observed both by fluorescence microscopy and by SNOM/AFM. The preparation technique used in the present study enabled the differential staining of sister chromatids in each chromosome, and sister chromatid exchanges (SCEs) were recognized in some chromosomes of the metaphase spread. Observations of the specimens by SNOM/AFM further provided the simultaneous collection of topographical and fluorescent images of the same portions of BrdU-incorporated chromosomes. The resolution of the fluorescence images by SNOM/AFM was greater than that obtained by fluorescence microscopy. Superimposition of topographical and fluorescent images of the chromosomes is useful for the precise analysis of the fine structure of chromosomes in relation to the SCEs. The application of SNOM/AFM to the BrdU-incorporated chromosomes is thus useful for the analysis of the fine structure of chromosomes in relation to their function.     

The subfibrillar arrangement of corneal and scleral collagen fibrils as revealed by scanning electron and atomic force microscopy

The present study was designed to analyze the subfibrillar structure of corneal and scleral collagen fibrils by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Isolated collagen fibrils of the bovine cornea and sclera were fixed with 1% OsO4, stained with phosphotungstic acid and uranyl acetate, dehydrated in ethanol, critical point-dried, metal-coated, and observed in an in-lens type field emission SEM. Some isolated collagen fibrils were fixed with 1% OsO4, dehydrated, critical point-dried and observed without metal-coating in an AFM. Isolated collagen fibrils treated with acetic acid were also examined by SEM and AFM. SEM and AFM images revealed that corneal and scleral collagen fibrils had periodic transverse grooves and ridges on their surface; the periodicity (i.e., D-periodicity) was about 63 nm in the cornea and about 67 nm in the sclera. Both corneal and scleral collagen fibrils contained subfibrils running helicoidally in a rightward direction to the longitudinal axis of the fibril; the inclination angle was about 15 degrees in the corneal fibrils and 5 degrees in the scleral fibrils. These findings indicate that the different D-periodicity between corneal and scleral fibrils depends on the different inclinations of the subfibrils in each fibril. The present study thus showed that corneal collagen fibrils differ from scleral collagen fibrils not only in diameter but also in substructure.     

Karyotyping chromosomes by electron microscopy. Condensation-inhibition of G bands in human and Chinese hamster chromosomes by a BrdU-Hoechst 33258 treatment

A BrdU-Hoechst 33258 treatment of living cells, which selectively induced condensation-inhibition of G-band chromatin in human and Chinese hamster chromosomes, is presented. As a consequence mitotic chromosomes showed high resolution R-banding patterns when examined by light and electron microscopy. Besides each whole chromosome identification, this procedure also permitted the electron microscopic study of specific structures, such as satellites, secondary constrictions, telomeres, centromeres, as well as G and R bands, some of them not visible by light microscopy. We have also observed that the chromatin of G and R bands behave as blocks of chromatin condensation and that G-band chromatin develops condensation along G₂. Under the BrdU-Hoechst 33258 treatment, chromatin fibers seem to invert their spontaneous pattern of condensation within the chromosomes.     

Analysis by atomic force microscopy of morphological changes in barley chromosomes during FISH treatment

We employed atomic force microscopy (AFM) to examine structural changes in barley chromosomes during the four steps of standard FISH processes. Rehydration and dehydration with alcohol accompanying RNase treatment increased chromosome arm width and decreased chromosome height about 50%. Subsequent heat denaturation reduced chromosome height further. These three-dimensional structural changes of the chromosomes were substantial, but the FISH signal produced by the hybridization of fluorescent probes was clear when observed by a fluorescence microscope. In higher-magnification images, we observed granular structures considered to represent the chromatin fiber on the surface of the chromosomes in each FISH protocol step. These our results indicate that FISH treatments result in severe damage of the three-dimensional higher-order structures of the chromosomes, although nano-structures, such as nucleosome and chromatin fibers, remain intact and relatively unaffected.     

Micromechanical studies of mitotic chromosomes

We review micromechanical experiments on mitotic chromosomes. We focus on work where chromosomes were extracted from prometaphase amphibian cells, and then studied by micromanipulation and microfluidic biochemical techniques. These experiments reveal that chromosomes have well-behaved elastic response over a fivefold range of stretching, with an elastic modulus similar to that of a loosely tethered polymer network. Perturbation by microfluidic ‘spraying’ of various ions reveals that the mitotic chromosome can be rapidly and reversibly decondensed or overcondensed, i.e. that the native state is not maximally compacted. Finally, we discuss microspraying experiments of DNA-cutting enzymes which reveal that the element which gives mitotic chromosomes their mechanical integrity is DNA itself. These experiments indicate that chromatin-condensing proteins are not organized into a mechanically contiguous ‘scaffold’, but instead that the mitotic chromosome is best thought of as a cross-linked network of chromatin. Preliminary results from restriction-enzyme digestion experiments indicate a spacing between chromatin ‘cross-links’ of roughly 15 kb, a size similar to that inferred from classical chromatin-loop-isolation studies. We compare our results to similar experiments done by Houchmandzadeh and Dimitrov (J Cell Biol 145: 215–213 (1999)) on chromatids reconstituted using Xenopus egg extracts. Remarkably, while the stretching elastic response of the reconstituted chromosomes is similar to that observed for chromosomes from cells, the reconstituted chromosomes are far more easily bent. This result suggests that reconstituted chromatids have a large-scale structure which is quite different from chromosomes in somatic cells. More generally our results suggest a strategy for the use of micromanipulation methods for the study of chromosome structure. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed a precise in-situ identification of both chromosomes and free chromatids, and consequently a reliable analysis of chromosomal segregation errors. Of the 104 analysed oocytes, 84 (80.7%) displayed a normal chromosome constitution. Three cases of chromosome non-disjunction (2.8%) were found, whereas seven oocytes (6.7%) presented extra single chromatids. In addition, 12 oocytes (11.5%) showed balanced pre-division of one pair of sister chromatids. Although this phenomenon was not classified as aneuploidy, it could lead to aneuploidy at anaphase II. Abnormalities were observed in all the targetted chromosomes. The present data confirm that both whole chromosome non-disjunction and premature chromatid separation constitute the two major mechanisms of aneuploidy in human female meiosis.     

Centromere spreading and out-of-phase chromatid separation in Burkitt's lymphoma and nasopharyngeal carcinoma

Chromosomes with various degrees of centromere spreading and completely separated chromatids, as well as metaphasic chromosomes, were observed simultaneously in the same Burkitt's lymphoma cell. In another study, a pair of prematurely and completely separated chromatids was found in a metaphase of nasopharyngeal carcinoma cell. Based on these and other observations, the possible significance of centromere spreading and out-of-phase sister chromatid separation in the origin of chromosome nondisjunction in tumor cells is suggested and discussed.     

First-generation physical map of the Culicoides variipennis (Diptera: Ceratopogonidae) genome

Recombinant cosmids labeled with biotin-11-dUTP or digoxigenin by nick translation were used as in situ hybridization probes to metaphase chromosomes of Culicoides variipennis (Coquillett). Paired fluorescent signals were detected on each arm of sister chromatids and were ordered along the 3 chromosomes. Thirty-three unique probes were mapped to the 3 chromosomes of C. variipennis (2n = 6): 7 to chromosome 1, 20 to chromosome 2, and 6 to chromosome 3. This work represents the first stage in generating a physical map of the genome of C. variipennis.     

The telochore: A telomeric differentiation of the chromosome axis

We have analysed by means of silver staining the structure of the chromosome axis at the telomeres of meiotic chromosomes in three different grasshopper species. At metaphase I the chromatid axes run the length of the chromatids although they do not reach the chromosome ends. The axes of sister chromatids are associated and show a round differentiation at their distal ends that we have named the telochore. Telochores never contact the chromosome ends: there is always some chromatin beyond them. In late metaphase I bivalents with a distal chiasma, anaphase I and metaphase II half-bivalents and anaphase II chromatids, the axes clearly possess one telochore in each chromosome end. These results seem to indicate that telochores are differentiations of the distal ends of chromatids. We discuss the possible structural significance of telochores according to the current scaffold/radial loop model of chromatin organization of eukaryotic metaphase chromosomes. Additionally, we suggest the possible functional role of the telochore as a nucleoprotein domain forming a protective cap for telomeric DNA.     

The topology of early- and late-replicating chromatin in differentially decondensed chromosomes

In this study we used a novel technique to reveal both longitudinal and transverse differentiation within mammalian mitotic chromosomes. Structural changes in chromosomes that we term ‘differential decondensation’ were produced in cells that were first incubated in hypotonic medium (15% Hanks’ solution), then adapted to normotonic conditions and thereafter exposed to a second short hypotonic shock. Such a double hypotonic treatment (DHT) is not critical for cell viability, but considerably elongates the G2 phase of the cell cycle. Giemsa staining of differentially decondensed chromosomes corresponds to standard G-banding, but does not need the standard post-fixation treatment. Using ‘dynamic’ BrdU banding, we show that such ‘differential’ staining is a result of differential resistance of the R- and G-bands to DHT. Thus, early-replicating foci, markers of R-bands, are localized in the peripheral chromatin halo, whereas late-replicating foci, corresponding to G-bands, remain associated with the axial regions of chromatids. Remarkably, despite these major changes in the structure of the chromosomal bands, the replication foci still preserve their discrete structure.