Micromechanical studies of mitotic chromosomes

Mitotic chromosomes respond elastically to forces in the nanonewton range, a property important to transduction of stresses used as mechanical regulatory signals during cell division. In addition to being important biologically, chromosome elasticity can be used as a tool for investigating the folding of chromatin. This paper reviews experiments studying stretching and bending stiffness of mitotic chromosomes, plus experiments where changes in chromosome elasticity resulting from chemical and enzyme treatments were used to analyse connectivity of chromatin inside chromosomes. Experiments with nucleases indicate that non-DNA elements constraining mitotic chromatin must be isolated from one another, leading to the conclusion that mitotic chromosomes have a chromatin ‘network’ or ‘gel’ organization, with stretches of chromatin strung between ‘crosslinking’ points. The as-yet unresolved questions of the identities of the putative chromatin crosslinkers and their organization inside mitotic chromosomes are discussed.     

Karyotype diversity and the origin of grapefruit

Grapefruit is a group of citrus of recent origin, probably resulting from a cross between pummelo and sweet-orange. Aiming to investigate this putative origin and the genetic variability among grapefruit cultivars, the karyotype of six grapefruits, two pummelos, and one tangelo cultivar (grapefruit × tangerine) were analyzed using sequential CMA/DAPI double staining and FISH with rDNA probes. The karyotypes of grapefruit ‘Duncan’ and ‘Foster’ differ from those of ‘Flame’, ‘Henderson’, ‘Marsh’ and ‘Rio Red’. The former have two chromosomes with a single CMA⁺ band in both terminal regions (C type chromosome) and six chromosomes with only one CMA⁺ terminal band (D type), whereas the latter have three C and five D type chromosomes. All accessions investigated exhibited two chromosomes with 5S rDNA but a variable number of 45S rDNA. The two former grapefruits displayed four 45S rDNA sites, whereas the remaining grapefruit cultivars had five. The two pummelos showed identical karyotypes, homozygous for CMA⁺ bands and their four rDNA sites. From each pummelo chromosome pair one chromosome seems to be present in grapefruit karyotypes. The different grapefruit karyotypes might result from independent crosses between pummelos of different karyotypic constitution and sweet-oranges. The chromosome markers found in the tangelo ‘Orlando’ and the position of their two 45S rDNA confirm the grapefruit ‘Duncan’ and the tangerine ‘Dancy’ as their parents.     

Meiotic chromosomes and stages of sex chromosome evolution in fish: zebrafish, platyfish and guppy

We describe SC complements and results from comparative genomic hybridization (CGH) on mitotic and meiotic chromosomes of the zebrafish Danio rerio, the platyfish Xiphophorus maculatus and the guppy Poecilia reticulata. The three fish species represent basic steps of sex chromosome differentiation: (1) the zebrafish with an all-autosome karyotype; (2) the platyfish with genetically defined sex chromosomes but no differentiation between X and Y visible in the SC or with CGH in meiotic and mitotic chromosomes; (3) the guppy with genetically and cytogenetically differentiated sex chromosomes. The acrocentric Y chromosomes of the guppy consists of a proximal homologous and a distal differential segment. The proximal segment pairs in early pachytene with the respective X chromosome segment. The differential segment is unpaired in early pachytene but synapses later in an ‘adjustment’ or ‘equalization’ process. The segment includes a postulated sex determining region and a conspicuous variable heterochromatic region whose structure depends on the particular Y chromosome line. CGH differentiates a large block of predominantly male-specific repetitive DNA and a block of common repetitive DNA in that region. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Visualization of Prekinetochore Locus on the Centromeric Region of Highly Extended Chromatin Fibers: Does Kinetochore Autoantigen CENP-C Constitute a Kinetochore Organizing Center?

Kinetochore is morphologically defined as a trilaminated, highly differentiated structure at the primary constriction of mitotic chromosomes. This subcellular organella is assumed to be composed of DNA and proteins. Immunoelectron microscopy has shown that centromere autoantigens CENP-C and CENP-B localize to the kinetochore inner plate and the underlying centromeric region respectively. We previously indicated that both are DNA-binding proteins that constitute centromeric heterochromatin throughout the cell cycle. Here, we tried to elucidate how these molecules are involved in the kinetochore/centromere organization in vivo by analyzing their morphological behavior in nuclei. Using immunofluorescence microscopy, we found that CENP-C remained as round discrete dots, whereas CENP-B displayed larger surrounding materials. To examine the CENP-C-binding locus on the genome, we prepared highly extended chromatin fibers and performed simultaneous immunofluorescence and fluorescence in situ hybridization. We obsreved that centromeric alphoid DNA, targeted by CENP-B, was highly dispersed, whereas the CENP-C antigen persisted as small dots well situated on the fibers. These features reminded us of the ‘ball and cup’ structure that had been presented for ‘prekinetochore’. We propose here that CENP-C constitutes a ‘kinetochore organizing center’ tightly associating with DNA, whereas CENP-B heterochromatin offers the solid support during kinetochore maturation. This revised version was published online in August 2006 with corrections to the Cover Date.     

G-banding and chromosome condensation in the ant,Tapinoma nigerrimum

Well-defined G-bands were obtained on metaphase chromosomes fromTapinoma nigerrimum using trypsin and warm 2×SSC in sequence. The G-banded pattern allowed the identification of all chromosomes. Evidence for asynchronous condensation of the chromosomes of this species is provided. Different banding patterns were obtained when metaphase chromosomes were stained with DA/DAPI alone and with DA/DAPI after a standard G-banding procedure. The G-banding phenomenon is discussed using the result obtained.accepted for publication by H. C. Macgregor     

First trimester chorionic villi sampling and direct chromosome preparations

Chorionic villi sampling was performed on 52 patients prior to elective termination of their pregnancies. Villi were obtained in 42, and direct chromosome preparations were successful in 41 of them. The use of a mixture of 0.075 M potassium chloride and 1% sodium citrate in the ratio of 2:1 for hypotonic treatment and 40% acetic acid for cell dispersal yielded chromosomes with good morphology and G-bands.     

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.     

The role of cytokeratin 19 in the differential diagnosis of true papillary carcinoma of thyroid and papillary carcinoma-like changes in Graves’ disease

Graves’ disease is an autoimmune disease predominantly seen in females. All types of thyroid cancers may co-exist with Graves’ disease but papillary carcinoma is the most frequent. Vesicular nuclei, nuclear grooves, and papillary formations that may be seen in Graves’ disease may lead the pathologist to an overdiagnosis of papillary carcinoma. The differential diagnosis between a true papillary carcinoma and foci mimicking papillary carcinoma in Graves’ disease may be challenging by light microscopic features only. This study is designed to determine whether CK19 is effective in the discrimination between the true papillary carcinoma of thyroid and foci resembling papillary carcinoma in Graves’ disease. Twenty-five cases with papillary carcinoma and 25 cases with Graves’ disease containing foci resembling papillary carcinoma were included in the study. All 25 cases with papillary carcinoma stained positive with CK19, whereas only six of 25 cases with Graves’ disease showed weak staining, and the remaining 19 cases were completely negative. It is known that CK19 may show faint staining in benign thyroid lesions such as adenomas. Staining pattern with CK19 together with histopathological findings may be helpful in the differential diagnosis between foci mimicking papillary carcinoma and true papillary carcinoma in Graves’ disease.     

Membrane protein glycosylation and CD44 content in the adhesion of human ovarian cancer cells to hyaluronan

The adhesion of tumour cells to the hyaluronan (HA) pericellular coat of mesothelial cells is an important step in the peritoneal spread of ovarian cancer. Previously, we have shown that the cell surface molecule CD44 is involved in this process. Paradoxically, the degree of adhesion does not appear to be related to the amount of CD44 expressed. In order to explain this observation we have examined the in vitro adhesion to HA of four high CD44-expressing ovarian cancer lines in relation to their CD44 spliced variant content and the CD44 glycosylation. Adhesion was measured in multiwell plates coated with different concentrations of HA in order to determine both the avidity and the maximum adhesion. Two lines had high adhesion and two lines had low adhesion. The avidity for HA was different for each line, but in all cases this could be totally blocked by treatment with an anti-CD44 antibody. The standard form of CD44 was the major species detected by RT/PCR in all lines and spliced variants were present in low amounts. Neuraminidase treatment increased the adhesion of the ‘low-adhesion’ lines at all HA coating concentrations; but only substantially increased the adhesion of the ‘high-adhesion’ lines at the lower HA coating concentrations. Tunicamycin treatment decreased the adhesion of the ‘high-adhesion lines’ at all HA coating concentrations and only substantially decreased the adhesion of one of the ‘low-adhesion’ lines when the plates were coated with a low concentration of HA. The adhesion of the remaining ‘low-adhesion’ line was slightly increased after tunicamycin treatment. It is concluded that glycosylation and not spliced variant content of CD44 affects the adhesive properties of ovarian tumour cells. This conclusion may have important consequences for developing new therapies in ovarian cancer This revised version was published online in July 2006 with corrections to the Cover Date.     

Evolutionarily conserved cytogenetic changes in hematological malignancies of dogs and humans – man and his best friend share more than companionship

The pathophysiological similarities shared by many forms of human and canine disease, combined with the sophisticated genomic resources now available for the dog, have placed ‘man’s best friend’ in a position of high visibility as a model system for a variety of biomedical concerns, including cancer. The importance of nonrandom cytogenetic abnormalities in human leukemia and lymphoma was recognized over 40 years ago, but the mechanisms of genome reorganization remain incompletely understood. The development of molecular cytogenetics, using fluorescence in situ hybridization (FISH) technology, has played a significant role in our understanding of cancer biology by providing a means for ‘interrogating’ tumor cells for a variety of gross genetic changes in the form of either numerical or structural chromosome aberrations. Here, we have identified cytogenetic abnormalities in naturally occurring canine hematopoietic tumors that are evolutionarily conserved compared with those that are considered characteristic of the corresponding human condition. These data suggest that humans and dogs share an ancestrally retained pathogenetic basis for cancer and that cytogenetic evaluation of canine tumors may provide greater insight into the biology of tumorigenesis.     

X chromosome painting in <Emphasis Type="Italic">Microtus</Emphasis>: Origin and evolution of the giant sex chromosomes

Sex chromosomes in species of the genus Microtus present some characteristic features that make them a very interesting group to study sex chromosome composition and evolution. M. cabrerae and M. agrestis have enlarged sex chromosomes (known as ‘giant sex chromosomes’) due to the presence of large heterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromosome of both species and hybridized on chromosomes from six Microtus and one Arvicola species. Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved, as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probably originated by fast amplification of different sequences, each with an independent origin and evolution in each species. For this reason, the sex heterochromatin in Microtus species is highly heterogeneous within species (with different composition for the Y and X heterochromatic regions in M. cabrerae) and between species (as the composition of M. agrestis and M. cabrerae sex heterochromatin is different). In addition, the X chromosome painting results on autosomes of several species suggest that, during karyotypic evolution of the genus Microtus, some rearrangements have probably occurred between sex chromosomes and autosomes.     

Physical map of two tammar wallaby chromosomes: A strategy for mapping in non-model mammals

Marsupials are especially valuable for comparative genomic studies of mammals. Two distantly related model marsupials have been sequenced: the South American opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii), which last shared a common ancestor about 70 Mya. The six-fold opossum genome sequence has been assembled and assigned to chromosomes with the help of a cytogenetic map. A good cytogenetic map will be even more essential for assembly and anchoring of the two-fold wallaby genome. As a start to generating a physical map of gene locations on wallaby chromosomes, we focused on two chromosomes sharing homology with the human X, wallaby chromosomes X and 5. We devised an efficient strategy for mapping large conserved synteny blocks in non-model mammals, and applied this to generate dense maps of the X and ‘neo-X’ regions and to determine the arrangement of large conserved synteny blocks on chromosome 5. Comparisons between the wallaby and opossum chromosome maps revealed many rearrangements, highlighting the need for comparative gene mapping between South American and Australian marsupials. Frequent rearrangement of the X, along with the absence of a marsupial XIST gene, suggests that inactivation of the marsupial X chromosome does not depend on a whole-chromosome repression by a control locus. Electronic supplementary material  The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63

The organization of the type I interferon (IFN) gene cluster (9p21.3) was studied in a human osteosarcoma cell line (MG63). Array comparative genomic hybridization (aCGH) showed an amplification of ∼6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.3 band. Spectral karyotyping (SKY) combined with fluorescence in-situ hybridization (FISH) identified an arrangement of the gene cluster in a ladder-like array of 5–7 ‘bands’ spanning a single chromosome termed the ‘IFN chromosome’. Chromosome painting revealed that the IFN chromosome is derived from components of chromosomes 4, 8 and 9. Labelling with centromeric probes demonstrated a ladder-like amplification of centromeric 4 and 9 sequences that co-localized with each other and a similar banding pattern of chromosome 4, as well as alternating with the IFN gene clusters. In contrast, centromere 8 was not detected on the IFN chromosome. One of the amplified centromeric 9 bands was identified as the functional centromere based on its location at the chromosome constriction and immunolocalization of the CENP-C protein. A model is presented for the generation of the IFN chromosome that involves breakage–fusion–bridge events. Electronic supplementary material   The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in thermal homeostasis in humans due to repeated cold water immersions

The purpose of this study was to monitor changes in body and skin temperatures, heat production, subjective shivering, cold sensation and body fat content in humans after intermittent cold water immersion. Repeated exposures of young sportsmen to cold water (head out, 14 °C, 1 h, 3 times per week for 4–6 weeks) induced changes in regulation of thermal homeostasis. ‘‘Cold acclimated’’ subjects exhibited an hypothermic type of adaptation. Central and peripheral body temperatures at rest and during cold immersion were lowered. The metabolic response to cold was delayed and subjective shivering was attenuated. The observed hypothermia was due to the shift of the threshold for induction of cold thermogenesis to lower body temperatures. ‘‘Cold acclimated’’ subjects also showed a lowered cold sensation. Because of the observed physiological changes, about 20% of the total heat production was saved during one cold water immersion of ‘‘cold acclimated’’ subjects. Maximal aerobic and anaerobic performances were not altered. No change in the thermosensitivity of the body temperature controller, as assessed from the unchanged slope of the relation between the deep body temperature and total heat production, was observed. Changes in cold sensation and regulation of cold thermogenesis were noticed first after four cold water immersions and persisted for at least 2 weeks after termination of the adaptation procedure. A trend towards a small increase in the body fat content was also observed. This findll as the increased vasoconstriction, evidenced by the lowered skin temperature, indicate that slight changes in body insulation may also occur after ‘‘cold acclimation’’ in humans. Received: 30 December 1995/Received after revision and accepted: 25 March 1996     

A- and B-chromosome pairing and recombination in male meiosis of the silver fox (<Emphasis Type="Italic">Vulpes vulpes</Emphasis> L., 1758, Carnivora,Canidae)

We examined A- and B-chromosome pairing and recombination in 12 males from the farm-bred population of the silver fox (2n = 34 + 0–10 Bs) by means of electron and immunofluorescent microscopy. To detect recombination at A and B chromosomes, we used immunolocalisation of MLH1, a mismatch repair protein of mature recombination nodules, at synaptonemal complexes. The mean total number of MLH1 foci at A-autosomes was 29.6 foci per cell. The XY bivalent had one MLH1 focus at the pairing region. Total recombination length of the male fox genome map was estimated as 1,530 centimorgans. We detected single MLH1 foci at 61% of linear synaptic configurations involving B chromosomes. The distribution of the foci along B- and A-bivalents was the same. This may be considered as a first molecular evidence that meiotic recombination does occur in mammalian B chromosomes. There was no correlation between the number of synaptic configurations involving B chromosomes per cell and the recombination rate of the A-genome.     

Chromosome painting and molecular dating indicate a low rate of chromosomal evolution in golden moles (Mammalia, Chrysochloridae)

Golden moles (Chrysochloridae) are poorly known subterranean mammals endemic to Southern Africa that are part of the superordinal clade Afrotheria. Using G-banding and chromosome painting we provide a comprehensive comparison of the karyotypes of five species representing five of the nine recognized genera: Amblysomus hottentotus, Chrysochloris asiatica, Chrysospalax trevelyani, Cryptochloris zyli and Eremitalpa granti. The species are karyotypically highly conserved. In total, only four changes were detected among them. Eremitalpa granti has the most derived karyotype with 2n = 26 and differs from the remaining species (all of whom have 2n = 30) by one centric and one telomere:telomere fusion. In addition, two intrachromosomal rearrangements were detected in A. hottentotus. The painting probes also suggest the presence of a unique satellite DNA family located on chromosomes 11 and 12 of both C. asiatica and C. zyli. This represents a synapomorphy linking these two sympatric species as sister taxa. A molecular clock was calibrated adopting a relaxed Bayesian approach for multigene data sets comprising publicly available sequences derived from five gene fragments representative of three golden moles and 39 other eutherian species. The data suggest that golden moles diverged from a common ancestor approximately 28.5 mya (95% credibility interval = 21.5–36.5 mya). Based on an inferred chrysochlorid ancestral karyotype of 2n = 30, the estimated rate of 0.7 rearrangements per 10 my (95% Credibility Interval = 0.54–0.93) differs from the ‘default rate’ of mammalian chromosomal evolution which has been estimated at one change per 10 million years, thus placing the Chrysochloridae among the slower-evolving chromosomal lineages thus far recorded. Electronic supplementary material Supplementary material to this paper is available in electronic form at and is accessible for authorized users.     

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.     

Chromosomes lacking kinetochore proteins: their meta-anaphase location suggests potential malsegregation

We have previously reported that a rare chromosome may not carrythe kinetochore protein complex-the CENtromere Proteins or CENPs.These chromosomes should not bind to spindle microtubules and,hence, should be found peripheral to the meta-anaphase arrangementexhibited by the chromsomes which do carry CENPs. This communicationshows that this actually is the case. When 3T3 mouse cells werenot treated with colcemid or hypotonic, the kinetochorelacking(K^–) as well as kinetochore-bearing (K⁺) chromosomes werefound off the spindle zone. When the spindle is disrupted withmild hypotonic treatment or by colcemid, the frequency of K^–chromosomes remains unchanged. However, even mild disruptionof the spindle with hypotonic treatment increases the frequencyof off-lying K⁺ chromosomes significantly. These data indicatethat K^– chromosomes do not bind to the spindle and, hence,are a factor in the genesis of aneuploidy. A considerable proportionof K^– chromosomes carry the putative centromere DNA indicatingthat these are not acentric fragments. Since the CREST serumused recognizes all essential kinetochore proteins, the K^–centromeres must also lack all essential CENPs. ¹To whom correspondence should be addressed